Otostigmus (O.) lewisiO. (O.) beroni Lewis, 2001. The latter was also recorded from Jilong County  by O. (O.) beroni from Jilong County with new materials of O. (O.) lewisi from Jiacha County, we reaffirm that O. (O.) lewisi is a valid species. Otostigmus Porat, 1876 currently comprises about 56 species (O. (O.) aculeatus Hasse, 1887, O. (O.) astenus , O. (O.) beroni Lewis, 2001, O. (O.) lewisi Song, Gai, Song & Zhu, 2005, O. (O.) martensi Lewis, 1992, O. (O.) politus Karsch, 1881, O. (O.) scaber Porat, 1876, and O. (O.) xizangensis Niu, Li & Di, 2021.The subgenus  species . Among t species : O. (O.)Otostigmus (O.) lewisi was identified as a synonym of O. (O.) beroni (O. (O.) lewisi has complete paramedian sutures on sternites 4–19, sternite 21 has a shallow medial longitudinal depression, and the coxopleural process is short. However, the sternite paramedian sutures in O. (O.) beroni are incomplete, and the coxopleural process is long. These differences were not shown in the corresponding figures of O.lewisi is a junior subjective synonym of O.beroni”. We would also like to highlight that all type materials used in the O. (O.) lewisi were sub-adults.) beroni . AccordiMHBU .Studied materials were collected by hand and preserved in 75% ethanol. All studied materials in this paper were examined under a stereomicroscope (Motic K700). Photographs and measurements were taken using a Leica Stereomicroscope (M205 A). The standard terminology followed Otostigminae Kraepelin, 1903Subfamily Otostigmus Porat, 1876Genus Taxon classificationAnimaliaLewis, 200160ED176E-3416-5BCE-9AFD-79035CE69070Otostigmusberoni Lewis, 2001: 22; MHBU-SoJL1608050101–Ar.-MHBU-SoJL1608050120: Jilong Town, Jilong (Gyirong) County, Xizang (Tibet), China, 28.4370°N, 85.2568°E, 5/8/2016, leg. Zhiyong Di. Ar.-MHBU-SoJL21080101: Jilong Town, Jilong (Gyirong) County, Xizang (Tibet), China, 28.4350°N, 85.2570°E, 1/8/2021, leg. Zhiyong Di. Housed in MHBU.Ar.-Maximum length 57 mm. Antennae 18 articles, the basal 2.25 to 2.40 glabrous dorsally Fig. . With 4 MHBU-SoJL1608050101). Length.Pigmentation : body color yellow-brown; legs and antennae yellow; cephalic plate, tergites 1–2, tergites 20–21 and penultimate and ultimate legs blue-green : cephalic plate blue-green, tergites blue-brown, penultimate legs and ultimate legs yellow with blue middle part in each segment, the other legs yellow : with complete paramedian sutures from 3 to 20, marginate from 7 to 21 : smooth, with incomplete paramedian sutures from 5 to 19 : L1–16 and L18–19 with 2 tarsal spurs, L1–4, left L5 and right L6 with 1 tibial spur and L1 with 1 femoral spur.Ultimate prefemur with 4 ventro-lateral, 2 ventro-medial, 3 medial, 2 dorso-medial spines and 1 corner spine Fig. .There are multiple differences among individuals as addressed below. The number of teeth of forcipular tooth plates 4+4 (11 specimens), 5+5 (6 specimens), 3+3 (2 specimen) or 3+4 (2 specimens). Coxopleural process with 4–7 spines . One tibial spur on L1–4 (8 specimens), L1–5 (5 specimens), L1–2 (2 specimens), L1–3 (3 specimens), or L1–6 (2 specimens). Two tarsal spurs on 1 to 18 or 19 pairs of legs; 1 tarsal spur on subsequent to penultimate legs. Ultimate legs without tarsal spur. Ultimate legs prefemur with 11–14 spines   and Nepal Fig. .Taxon classificationAnimaliaSong, Gai, Song & Zhu, 2005 80F267B0-9D5B-5FC6-A29B-D11CC060ACBEOtostigmuslewisiMHBU-SoJC1908060301– Ar.-MHBU-SoJC1908060307: Jiacha County (Gyaca County), Xizang (Tibet), China, 29.0857°N, 92.3430°E, 6/8/2019, leg. Zhiyong Di. Ar.-MHBU-SoJC1608DX01: Jiacha County, Xizang, China, 29.1188°N, 92.6969°E, 12/8/2016, leg. Zhiyong Di. Ar.-MHBU-SoJC1608120401: Jiacha County, Xizang, China, 29.1387°N, 92.6880°E, 12/8/2016, leg. Zhiyong Di. Housed in MHBU.Ar.-Maximum length 77 mm. Antennae 17–20 articles, basal 2.2–3 glabrous dorsally Fig. . With 3 O. (O.) lewisi holotype described in The O. (O.) lewisi and O. (O.) beroni; however, the description and figures of O. (O.) lewisi were not consistent in O. (O.) martensi (O. (O.) lewisi (O. (O.) lewisi (O. (O.) martensi (Following O. (O.) lewisi is similar to O. (O.) beroni, and distinguished O. (O.) lewisi from the latter by the length of coxopleural process and the shape of the ultimate sternite. Because O. (O.) lewisi in O. (O.) lewisi is a junior subjective synonym of O. (O.) beroni  beroni .O. (O.) lewisi reported by O. (O.) lewisi described previously were sub-adults.The characteristics of the holotype of MHBU-SoJC1908060301). Length.Pigmentation : cephalic plate and tergites yellow with light green; antennae and legs yellow; penultimate legs and ultimate legs green : antennae light blue mainly, cephalic plate and tergites brownish, penultimate legs and ultimate legs yellow with blue middle part in each segment, the rest of the legs yellow : with complete paramedian sutures from 4 to 20; marginate from 6 to 21 : smooth, with incomplete paramedian sutures from 3 to 4, complete paramedian sutures from 5 to 19 : L2–9, right L11 and left L12 with 2 tarsal spurs; 1 tarsal spur on subsequent to penultimate legs; L1 and left L2 with 1 tibial spur and right L1 with 1 femoral spur.The left prefemur with 1 corner spine, 3 ventro-lateral spines, 1 ventro-medial spines, 2 medial spines, 1 dorso-medial spine; the right prefemur with 2 corner spines, 5 ventro-lateral spines, 2 ventro-medial spines, 7 medial spines, 3 dorso-medial spines Fig. .Adult and juvenile individuals differ primarily in body length and pigmentation Figs , 3A, B. Found under stones in arid mountain bush Fig. .China (Xizang) Fig. .Otostigmus, namely, O. (O.) beroni, O. (O.) lewisi, O. (O.) martensi and O. (O.) xizangensis, were reported in Xizang, China  lewisi can be easily distinguished from O. (O.) beroni using the following characteristics: (1) larger average body length: 73 mm in O. (O.) lewisi (average of 2 adults), versus 48 mm in O. (O.) beroni (average of 5 adults); (2) body color (live): primarily brownish cephalic plate and tergites in O. (O.) lewisi, and blue-green cephalic plate and blue-brown tergites in O. (O.) beroni  lewisi 3+3 in 5 specimens or 4+4 in 4 specimens versus O. (O.) beroni 4+4 in 11 specimens or 5+5 in 6 specimens; (4) the number of tibial spurs on legs: O. (O.) lewisi with one tibial spur on legs 1–2 in 6 specimens or on legs 1 in 3 specimens. In contrast, O. (O.) beroni with one tibial spur on legs 1–4 in 8 specimens or 1–5 in 5 specimens; (5) the number of tarsal spurs on legs: O. (O.) lewisi with 2 tarsal spurs on legs 1(2)–9(13), with no regularity. In O. (O.) beroni, however, 2 tarsal spurs on legs 1–18(19); (6) the length of coxopleural process: coxopleural process of O. (O.) lewisi is shorter than that of O. (O.) beroni. The specimens described by Only four species of the genus ina Fig. , geograponi Fig. ; (3) theO. (O.) beroni and O. (O.) martensi. The main difference between O. (O.) beroni and O. (O.) martensi are as follows: (1) the number of teeth of tooth plates: O. (O.) martensi 3+3, whereas, O. (O.) beroni 4+4 or 5+5; (2) the number of spines on the coxopleural process: O. (O.) martensi coxopleural process with 1 apical spine, 1 lateral spine and 1 dorsal spine. In contrast, O. (O.) beroni coxopleural process with 2–3 apical spines, 1–2 lateral spines, and 1–2 dorsal spines; (3) spurs on legs: O. (O.) martensi L1–4 or 5 with 2 tarsal spurs, whereas O. (O.) beroni L1–19 with 2 tarsal spurs, L20 with 1 tarsal spur (O. (O.) lewisi and O. (O.) martensi are obviously different species.The most geographically close species of this genus in Xizang, China are sal spur . Given tO. (O.) beroni in China) and Zhangmu Town  martensi in China)) is about 78 km, and the distance between Jilong Town and Jiacha County  lewisi in China) is about 714 km. No species of the genus Otostigmus was found in the area between Jilong Town (or Zhangmu Town) and Jiacha County. The habitat of O. (O.) beroni in Jilong Town is more humid than the habitat of O. (O.) lewisi in Jiacha County  lewisi is a valid species.The distance between Jilong Town (the locality of nty Fig. . Based o


Oniscidea) are the most diverse group of troglobionts in caves of continental Portugal. They occur in all karst regions of Portugal, play a major role in decomposition of organic matter in caves and may act as umbrella species for the conservation of all other cave-adapted invertebrates.Terrestrial isopods (We present the IUCN Red List profiles for the cave-adapted terrestrial isopods from continental Portugal, based on recent distribution data from caves. Cave-adapted fauna have a high and global conservationist interest . Cave-adOniscidea) evolved to be the only truly terrestrial crustaceans . The maps were generated in the open source software QGIS 3.14.16, with the layer of the natural protected areas of Portugal (Extent of occurence (EOO) and area of occupancy (AOO) were calculated using the Geospatial Conservation Assessment Tool (GeoCAT) with an approximation to the standard IUCN 2 km × 2 km cells : Suppl. materials 2Basis of EOO and AOO: Known habitat extent2.Basis (narrative): Both the extent of occurrence (EOO) and area of occupancy (AOO) are 4 kmMin Elevation/Depth (m): 380Trichoniscoidesbellesi is known from Algar do Javali Cave in the Montejunto karst massif (Range description: t massif .EOO (km2): 4Trend: UnknownCauses ceased?: UnknownCauses understood?: UnknownCauses reversible?: UnknownExtreme fluctuations?: UnknownTrend: UnknownCauses ceased?: UnknownCauses understood?: UnknownCauses reversible?: UnknownExtreme fluctuations?: UnknownAOO (km2): 4Number of locations: 1Trichoniscoidesbellesi occurs in a single cave : A total of three specimens have been collected in the type locality .Trend: UnknownExtreme fluctuations?: UnknownSevere fragmentation?: UnknownSystem: TerrestrialHabitat specialist: YesTrichoniscoidesbellesi was found in the deepest and most thermally insulated parts of the cave, at 10 m depth (Habitat (narrative):  m depth .Trend in extent, area or quality?: Decline (inferred)Habitat importance: Major ImportanceHabitats: 7.1. Caves and Subterranean Habitats (non-aquatic) - CavesHabitat importance: Major ImportanceHabitats: 7.1. Caves and Subterranean Habitats (non-aquatic) - CavesSize: 1.7 mm , 1.8 mm Generation length (yr): 1Dependency of single sp?: UnknownTrichoniscoidesbellesi is a blind and depigmented troglobiont species  :  species . It shara, 2012)  and Roncra, 2013 ; an undeño, 2010  and a neEucalyptus intensive plantations, which substituted the original native vegetation and is located 50 m from a road, 1.6 km from a quarry and 2.9 km from the closest village.Justification for threats: The cave entrance is surrounded by Threat type: OngoingThreats: 1.1. Residential & commercial development - Housing & urban areas2.2. Agriculture & aquaculture - Wood & pulp plantations3.2. Energy production & mining - Mining & quarrying4. Transportation & service corridorsThreat type: OngoingThreats: 1.1. Residential & commercial development - Housing & urban areas2.2. Agriculture & aquaculture - Wood & pulp plantations3.2. Energy production & mining - Mining & quarrying4. Transportation & service corridorsJustification for conservation actions: Even though this cave is protected under legislation by the “Rede Natura 2000” , this raConservation action type: NeededConservation actions: 1.1. Land/water protection - Site/area protection1.2. Land/water protection - Resource & habitat protection4. Education & awareness5.1.3. Law & policy - Legislation - Sub-national levelConservation action type: NeededConservation actions: 1.1. Land/water protection - Site/area protection1.2. Land/water protection - Resource & habitat protection4. Education & awareness5.1.3. Law & policy - Legislation - Sub-national levelTrichoniscoidesbroteroiScientific name: Species authority: Vandel, 1946AnimaliaKingdom: ArthropodaPhylum: MalacostracaClass: IsopodaOrder: TrichoniscidaeFamily: Region for assessment: EuropeReviewers: ReviewersEditor: EditorReviewers: ReviewersEditor: EditorBiogeographic realm: PalearcticCountries: PortugalMap of records (Google Earth): Suppl. materials 3Basis of EOO and AOO: Known habitat extent2.Basis (narrative): The extent of occurrence (EOO) and area of occupancy (AOO) are both 4 kmMin Elevation/Depth (m): 100Trichoniscoidesbroteroi is a troglobiont isopod known from Alqueves Cave, located in the Sicó karst area (Range description: rst area . This carst area .EOO (km2): 4Trend: UnknownCauses ceased?: UnknownCauses understood?: UnknownCauses reversible?: UnknownExtreme fluctuations?: UnknownTrend: UnknownCauses ceased?: UnknownCauses understood?: UnknownCauses reversible?: UnknownExtreme fluctuations?: UnknownAOO (km2): 4Number of locations: 1Trichoniscoidesbroteroi is known from a single cave : Alqueves Cave is considered a pre- and proto-historic site. It is a natural funerary cave as bone fragments and other human artifacts were there found, testifying to its human occupation at the end of the Neolithic period, beginning of the Chalcolithic . This caTrend in extent, area or quality?: Decline (inferred)Habitat importance: Major ImportanceHabitats: 7.1. Caves and Subterranean Habitats (non-aquatic) - CavesHabitat importance: Major ImportanceHabitats: 7.1. Caves and Subterranean Habitats (non-aquatic) - CavesSize: 4 mm , 4.5 mm Generation length (yr): 1Dependency of single sp?: UnknownTrichoniscoidesbroteroi is a blind and depigmented troglobiont species and a single cave endemic (Porcelliocavernicolus (Ecology and traits (narrative):  endemic . It sharrnicolus .Justification for threats: The entrance to the cave is located within a fully urbanised area, more specifically in the middle of a roundabout . This caThreat type: OngoingThreats: 1.1. Residential & commercial development - Housing & urban areas1.3. Residential & commercial development - Tourism & recreation areas4. Transportation & service corridors9.1. Pollution - Domestic & urban waste water9.5. Pollution - Air-borne pollutantsThreat type: OngoingThreats: 1.1. Residential & commercial development - Housing & urban areas1.3. Residential & commercial development - Tourism & recreation areas4. Transportation & service corridors9.1. Pollution - Domestic & urban waste water9.5. Pollution - Air-borne pollutantsTrichoniscoidesbroteroi : Suppl. materials 4Basis of EOO and AOO: Known habitat extent2 and the area of occupancy (AOO) is 40 km2.Basis (narrative): The extent of occurrence (EOO) is 217.7 kmMin Elevation/Depth (m): 95Max Elevation/Depth (m): 485Trichoniscoidesmeridionalis is recorded from 10 caves distributed along the Estremenho karst massif: Algar do Vale da Pena, Algar do Zé de Braga, Alcobertas, Lapa da Chã de Cima, Moinhos Velhos, Almonda, Papagaio, Algar do Burro, Algar do Pena and Algar do Ladoeiro (Range description: Ladoeiro .EOO (km2): 217.7Trend: UnknownCauses ceased?: UnknownCauses understood?: UnknownCauses reversible?: UnknownExtreme fluctuations?: UnknownTrend: UnknownCauses ceased?: UnknownCauses understood?: UnknownCauses reversible?: UnknownExtreme fluctuations?: UnknownAOO (km2): 40Number of locations: 10Trichoniscoidesmeridionalis occurs in ten caves : A total of 43 specimens have been collected: six in Moinhos Velhos cave, five in Algar do Vale do Pena, five in Algar do Burro, one in Algar do Zé de Braga, fifteen in Algar do Ladoeiro, ten in Almonda and one in Papagaio Caves .Number of subpopulations: 10Trend: Decline (inferred)Extreme fluctuations?: UnknownSevere fragmentation?: UnknownSystem: TerrestrialHabitat specialist: YesHabitat (narrative): The caves are located at an altitude ranging from 95 to 485 m a.s.l. . Amonda Trend in extent, area or quality?: Decline (inferred)Habitat importance: Major ImportanceHabitats: 7.1. Caves and Subterranean Habitats (non-aquatic) - CavesHabitat importance: Major ImportanceHabitats: 7.1. Caves and Subterranean Habitats (non-aquatic) - CavesSize: 2 mm , 2.5 mm Generation length (yr): 1Dependency of single sp?: UnknownTrichoniscoidesmeridionalis is a blind, depigmented troglobiont that is adapted to life in the underground (Ecology and traits (narrative): erground . This sperground .Justification for threats: Almonda Cave is located 50 m from a factory that extracts water from a subterranean river and 420 m from a village, which has many agricultural fields. Algar do Ladoeiro's Cave entrance is 840 m from the closest urbanisation. The Moinhos Velhos Cave is the largest show cave of Portugal with around 140,000 visitors per year. Alcobertas Cave has also been subject to structural alterations with the intention to transform it into a show cave during the last century and it is exploited for tourism by a local association . The subThreat type: OngoingThreats: 1.1. Residential & commercial development - Housing & urban areas1.2. Residential & commercial development - Commercial & industrial areas2.1. Agriculture & aquaculture - Annual & perennial non-timber crops3.2. Energy production & mining - Mining & quarrying3.3. Energy production & mining - Renewable energy6.1. Human intrusions & disturbance - Recreational activities9.1.2. Pollution - Domestic & urban waste water - Run-offThreat type: OngoingThreats: 1.1. Residential & commercial development - Housing & urban areas1.2. Residential & commercial development - Commercial & industrial areas2.1. Agriculture & aquaculture - Annual & perennial non-timber crops3.2. Energy production & mining - Mining & quarrying3.3. Energy production & mining - Renewable energy6.1. Human intrusions & disturbance - Recreational activities9.1.2. Pollution - Domestic & urban waste water - Run-offJustification for conservation actions: Almonda Cave is classified, since 1993, as a Property of Public Interest (IIP) and protected due to its archaeological heritage . The arcConservation action type: NeededConservation actions: 1.1. Land/water protection - Site/area protection1.2. Land/water protection - Resource & habitat protection2.1. Land/water management - Site/area management4. Education & awareness5.1.3. Law & policy - Legislation - Sub-national levelConservation action type: NeededConservation actions: 1.1. Land/water protection - Site/area protection1.2. Land/water protection - Resource & habitat protection2.1. Land/water management - Site/area management4. Education & awareness5.1.3. Law & policy - Legislation - Sub-national levelTrichoniscoidesouremensisScientific name: Species authority: Vandel, 1946AnimaliaKingdom: ArthropodaPhylum: MalacostracaClass: IsopodaOrder: TrichoniscidaeFamily: Region for assessment: EuropeReviewers: ReviewersEditor: EditorReviewers: ReviewersEditor: EditorBiogeographic realm: PalearcticCountries: PortugalMap of records (Google Earth): Suppl. materials 5Basis of EOO and AOO: Known habitat extent2.Basis (narrative): Both the extent of occurrence (EOO) and area of occupancy (AOO) are 4 kmTrichoniscoidesouremensis is only recorded from Lapa da Salgada Cave, located in the Fátima Plateau, in the eastern part of the Estremenho karst massif (Range description: t massif .EOO (km2): 4Trend: UnknownCauses ceased?: UnknownCauses understood?: UnknownCauses reversible?: UnknownExtreme fluctuations?: UnknownTrend: UnknownCauses ceased?: UnknownCauses understood?: UnknownCauses reversible?: UnknownExtreme fluctuations?: UnknownAOO (km2): 4Number of locations: 1Trichoniscoidesouremensis is recorded from a single cave : Lapa da Salgada Cave is composed of a main underground gallery and the floor is mostly covered by flowstone and clay, with a few bat guano deposits.Trend in extent, area or quality?: Decline (inferred)Habitat importance: Major ImportanceHabitats: 7.1. Caves and Subterranean Habitats (non-aquatic) - CavesHabitat importance: Major ImportanceHabitats: 7.1. Caves and Subterranean Habitats (non-aquatic) - CavesSize: 3.5 mm Generation length (yr): 1Dependency of single sp?: UnknownTrichoniscoidesouremensis is classified as a troglobiont species, being blind and depigmented. It is a single cave endemic (Ecology and traits (narrative):  endemic .Justification for threats: The cave entrance is located 270 m from a road in which trucks transport goods from warehouses 600 m away. The cave is also located 1 km away from the closest town.Threat type: OngoingThreats: 1.1. Residential & commercial development - Housing & urban areas1.2. Residential & commercial development - Commercial & industrial areas4. Transportation & service corridors9.1. Pollution - Domestic & urban waste water9.2. Pollution - Industrial & military effluentsThreat type: OngoingThreats: 1.1. Residential & commercial development - Housing & urban areas1.2. Residential & commercial development - Commercial & industrial areas4. Transportation & service corridors9.1. Pollution - Domestic & urban waste water9.2. Pollution - Industrial & military effluentsJustification for conservation actions: Measures need to be put in place in order to protect the habitat and species from the disturbances caused by truck movements and proximity to the urban areas.Conservation action type: NeededConservation actions: 1.1. Land/water protection - Site/area protection1.2. Land/water protection - Resource & habitat protection2.1. Land/water management - Site/area management4. Education & awareness5.1.3. Law & policy - Legislation - Sub-national levelConservation action type: NeededConservation actions: 1.1. Land/water protection - Site/area protection1.2. Land/water protection - Resource & habitat protection2.1. Land/water management - Site/area management4. Education & awareness5.1.3. Law & policy - Legislation - Sub-national levelTrichoniscoidesserraiScientific name: Species authority: Cruz, 1993AnimaliaKingdom: ArthropodaPhylum: MalacostracaClass: IsopodaOrder: TrichoniscidaeFamily: Region for assessment: EuropeReviewers: ReviewersEditor: EditorReviewers: ReviewersEditor: EditorBiogeographic realm: PalearcticCountries: PortugalMap of records (Google Earth): Suppl. materials 6Basis of EOO and AOO: Known habitat extent2.Basis (narrative): Both the extent of occurrence (EOO) and area of occupancy (AOO) are 4 kmMin Elevation/Depth (m): 577Trichoniscoidesserrai is only recorded from Santo Adrião Cave, located in the palaeokarst of Vimioso in north-eastern Portugal : 4Trend: UnknownCauses ceased?: UnknownCauses understood?: YesCauses reversible?: YesExtreme fluctuations?: UnknownTrend: UnknownCauses ceased?: UnknownCauses understood?: UnknownCauses reversible?: UnknownExtreme fluctuations?: UnknownAOO (km2): 4Number of locations: 1Trichoniscoidesserrai occurs in a single cave : Santo Adrião mines are located at 580 m a.s.l. in the Municipality of Miranda do Douro, District of Bragança . The cavTrend in extent, area or quality?: Decline (inferred)Habitat importance: Major ImportanceHabitats: 7.1. Caves and Subterranean Habitats (non-aquatic) - CavesHabitat importance: Major ImportanceHabitats: 7.1. Caves and Subterranean Habitats (non-aquatic) - CavesSize: 3.6 mm ; 4.2 mm Generation length (yr): 1Dependency of single sp?: UnknownTrichoniscoidesserrai is classified as a blind, depigmented troglobiont, adapted to life in the underground (Ecology and traits (narrative): erground .Justification for threats: The cave entrance is in the border of an active quarry, very close to a road .Threat type: OngoingThreats: 3.2. Energy production & mining - Mining & quarrying4.1. Transportation & service corridors - Roads & railroads9.2. Pollution - Industrial & military effluentsThreat type: OngoingThreats: 3.2. Energy production & mining - Mining & quarrying4.1. Transportation & service corridors - Roads & railroads9.2. Pollution - Industrial & military effluentsJustification for conservation actions: Even though this cave is protected under legislation by the “Rede Natura 2000” , this spConservation action type: NeededConservation actions: 1.1. Land/water protection - Site/area protection1.2. Land/water protection - Resource & habitat protection2.1. Land/water management - Site/area management4. Education & awareness5.1.3. Law & policy - Legislation - Sub-national levelConservation action type: NeededConservation actions: 1.1. Land/water protection - Site/area protection1.2. Land/water protection - Resource & habitat protection2.1. Land/water management - Site/area management4. Education & awareness5.1.3. Law & policy - Legislation - Sub-national levelTrichoniscoidessicoensisScientific name: Species authority: Reboleira & Taiti 2015AnimaliaKingdom: ArthropodaPhylum: Class: MalacostraIsopodaOrder: TrichoniscidaeFamily: Trichoniscoidessicoensis is blind and depigmented and it can be distinguished from all species of the genus because the male pereopod 7 merus has a lobe on the mid-sternal margin, the male pleopod 1 exopod has a broadly rounded outer margin and two unequal setae, the endopod has a fusiform distal article with a distinct circular suture in the middle and the male pleopod 2 endopod has a thickset distal article bearing two short triangular lobes and two setae at the apex  or Photo(s): Fig. 1Region for assessment: EuropeReviewers: ReviewersEditor: EditorReviewers: ReviewersEditor: EditorBiogeographic realm: PalearcticCountries: PortugalMap of records (Google Earth): Suppl. materials 7Basis of EOO and AOO: Known habitat extentBasis (narrative): The extent of occurrence (EOO) is 129.2 km² and the maximum estimated area of occupancy (AOO) is 20 km².Min Elevation/Depth (m): 20Max Elevation/Depth (m): 380Trichoniscoidessicoensis is recorded from six caves located in the Sicó karst area: Cerâmica, Santa Maria da Estrela, Soprador do Carvalho, Algarinho, Arrifana and São Simão (Range description: ão Simão .EOO (km2): 129.2Trend: UnknownCauses ceased?: UnknownCauses understood?: UnknownCauses reversible?: UnknownExtreme fluctuations?: UnknownTrend: UnknownCauses ceased?: UnknownCauses understood?: UnknownCauses reversible?: UnknownExtreme fluctuations?: UnknownAOO (km2): 20Number of locations: 6Trichoniscoidessicoensis is known from six caves : The largest population was found in the type locality Cerâmica Cave, where a higher number of specimens has been collected, followed by Santa Maria da Estrela Cave and then Soprador do Carvalho, Algarinho, Arrifana and São Simão Caves .Number of subpopulations: 6Trend: UnknownExtreme fluctuations?: UnknownSevere fragmentation?: UnknownSystem: TerrestrialHabitat specialist: YesTrichoniscoidessicoensis inhabits the deepest and isolated parts of caves of the Sicó karst area, found up to a maximum altitude of 380 m a.s.l. The easternmost locality is Soprador do Carvalho Cave and the westernmost is Santa Maria da Estrela Cave, its distribution being limited at the north by the Arrifana Cave and at the south by the Cerâmica Cave. Cerâmica Cave opens at 355 m a.s.l., it has a horizontal development of 120 m, a depth of 21 m :  of 21 m  and is t of 21 m . Santa M of 21 m . Sopradoalleries .Trend in extent, area or quality?: Decline (inferred)Habitat importance: Major ImportanceHabitats: 7.1. Caves and Subterranean Habitats (non-aquatic) - CavesHabitat importance: Major ImportanceHabitats: 7.1. Caves and Subterranean Habitats (non-aquatic) - CavesSize: 3.5 mm , 3.9 mm Generation length (yr): 1Dependency of single sp?: UnknownTrichoniscoidessicoensis is classified as a troglobiont species, blind and depigmented. It is endemic to the Sicó karst area (Ecology and traits (narrative): rst area . Most ofrst area .Eucalyptus intensive plantations. It is located 270 m from a road, 550 m from an animal farm, 1.6 km from the closest village and 3.6 km from a quarry. Santa Maria da Estrela Cave is located 86 m from a touristic site called Monstro das Bolachas, 230 m from the Nossa Senhora da Estrela viewpoint, 250 m from the closest urbanised area, 80 m from the road, 220 m from agricultural fields and 2.6 km from two quarries. Soprador do Carvalho Cave is surrounded by agricultural lands distances and is located 67 m from the closest house and 1.4 km from a quarry. This cave is also affected by touristic activities, as people who visit the cave walk all over the habitat : Suppl. materials 8Basis of EOO and AOO: Known habitat extent2.Basis (narrative): Both the extent of occurrence (EOO) and area of occupancy (AOO) are 4 kmMin Elevation/Depth (m): 122Trichoniscoidessubterraneus is only recorded from Alta do Cabeço dos Mosqueiros Cave, located in Carvalhal de Aljubarrota, the western part of the Estremenho karst massif (Range description: t massif .EOO (km2): 4Trend: UnknownCauses ceased?: UnknownCauses understood?: UnknownCauses reversible?: UnknownExtreme fluctuations?: UnknownTrend: UnknownCauses ceased?: UnknownCauses understood?: UnknownCauses reversible?: UnknownExtreme fluctuations?: UnknownAOO (km2): 4Number of locations: 1Trichoniscoidessubterraneus occurs in a single cave : This cave is currently part of the Geocaching network; therefore, it is subject to human disturbance without regulation.Trend in extent, area or quality?: Decline (inferred)Habitat importance: Major ImportanceHabitats: 7.1. Caves and Subterranean Habitats (non-aquatic) - CavesHabitat importance: Major ImportanceHabitats: 7.1. Caves and Subterranean Habitats (non-aquatic) - CavesSize: 3 mmGeneration length (yr): 1Dependency of single sp?: UnknownTrichoniscoidessubterraneus is classified as a blind and depigmented troglobiont species (Ecology and traits (narrative):  species .Justification for threats: This cave is frequently visited by geocachers looking for the geocache installed inside the cave.Threat type: OngoingThreats: 6.1. Human intrusions & disturbance - Recreational activitiesThreat type: OngoingThreats: 6.1. Human intrusions & disturbance - Recreational activitiesJustification for conservation actions: Measures to limit the number of visits to the cave should be implemented in order to ensure the integrity of the habitat and the survival of this species.Conservation action type: NeededConservation actions: 1.1. Land/water protection - Site/area protection1.2. Land/water protection - Resource & habitat protection2.1. Land/water management - Site/area management4. Education & awareness5.1.3. Law & policy - Legislation - Sub-national levelConservation action type: NeededConservation actions: 1.1. Land/water protection - Site/area protection1.2. Land/water protection - Resource & habitat protection2.1. Land/water management - Site/area management4. Education & awareness5.1.3. Law & policy - Legislation - Sub-national levelMetatrichoniscoidessalirensisScientific name: Species authority: Reboleira & Taiti 2015AnimaliaKingdom: ArthropodaPhylum: MalacostracaClass: IsopodaOrder: TrichoniscidaeFamily: Metatrichoniscoidessalirensis can be distinguished from all species of the genus because the pleopod 1 exopod of the male has two long distal setae of subequal length and its pleopod 2 endopod has a thickset distal article, ending in a thinner sinuous part with a beak-like small lobe medially directed : Suppl. materials 9Basis of EOO and AOO: Known habitat extentBasis (narrative): Both the extent of occurrence (EOO) and area of occupancy (AOO) are 4 km²Min Elevation/Depth (m): 60Metatrichoniscoidessalirensis is only known from Salir Cave, a single isolated cave located in the western border of Caldas da Rainha Typhonic Valley : 4Trend: UnknownCauses ceased?: UnknownCauses understood?: UnknownCauses reversible?: UnknownExtreme fluctuations?: UnknownTrend: UnknownCauses ceased?: UnknownCauses understood?: UnknownCauses reversible?: UnknownExtreme fluctuations?: UnknownAOO (km2): 4Number of locations: 1Metatrichoniscoidessalirensis is only recorded from one cave : A total of seven specimens have been collected in the type locality .Trend: UnknownExtreme fluctuations?: UnknownSevere fragmentation?: UnknownSystem: TerrestrialHabitat specialist: YesHabitat (narrative): Salir Cave was discovered in the 1960s by the labour force of a quarry. It is located near the sea, at an altitude of 60 m a.s.l. The substrate is mostly composed of flowstone, clay and marine sand that can be found in the deepest parts of the cave .Trend in extent, area or quality?: Decline (inferred)Habitat importance: Major ImportanceHabitats: 7.1. Caves and Subterranean Habitats (non-aquatic) - CavesHabitat importance: Major ImportanceHabitats: 7.1. Caves and Subterranean Habitats (non-aquatic) - CavesSize: 2.2 mm Generation length (yr): 1Dependency of single sp?: UnknownMetatrichoniscoidessalirensis is classified as a troglobiont species, blind and depigmented and is hitherto the only troglobiont species known from this cave and from this karst area (Ecology and traits (narrative): rst area . The meaJustification for threats: The cave entrance is located in a former quarry, at 400 m from the closest house and 1 km from the village centre. Salir Cave has an easy access and mostly horizontal development, which attracts casual visitors. It has suffered recurrent episodes of vandalism, which include paintings inside the first chamber and breakage of many lithological formations. This cave is currently listed in the Geocaching network and subject to human disturbance without regulation.Threat type: OngoingThreats: 1.1. Residential & commercial development - Housing & urban areas9.1.2. Pollution - Domestic & urban waste water - Run-offThreat type: OngoingThreats: 1.1. Residential & commercial development - Housing & urban areas9.1.2. Pollution - Domestic & urban waste water - Run-offJustification for conservation actions: The cave should be a protected site, as it is the only locality known for this species; therefore, a site of maximum priority for biodiversity conservation. The entrance should be closed and the access regulated in order to prevent the recurrent vandalism and human disturbance to the ecosystem.Conservation action type: NeededConservation actions: 1.1. Land/water protection - Site/area protection1.2. Land/water protection - Resource & habitat protection4. Education & awareness5.1.3. Law & policy - Legislation - Sub-national levelConservation action type: NeededConservation actions: 1.1. Land/water protection - Site/area protection1.2. Land/water protection - Resource & habitat protection4. Education & awareness5.1.3. Law & policy - Legislation - Sub-national levelTroglonethesolissipoensisScientific name: Species authority: Reboleira & Taiti 2015AnimaliaKingdom: ArthropodaPhylum: MalacostracaClass: IsopodaOrder: TrichoniscidaeFamily: Taxonomic notes: Antennae have five flagellar articles, the male pleopod 1 exopod is triangular, as wide as long and the male pleopod 2 endopod distal article has a basal and a distal hook-like process .Region for assessment: EuropeReviewers: ReviewersEditor: EditorReviewers: ReviewersEditor: EditorBiogeographic realm: PalearcticCountries: PortugalMap of records (Google Earth): Suppl. materials 10Basis of EOO and AOO: Known habitat extentBasis (narrative): Both the extent of occurrence (EOO) and area of occupancy (AOO) are 4 km².Min Elevation/Depth (m): 42Troglonethesolissipoensis is only recorded from Alvide Cave, located in Cascais Municipality in the Lisbon metropolitan area (Range description: tan area .EOO (km2): 4Trend: UnknownCauses ceased?: UnknownCauses understood?: UnknownCauses reversible?: UnknownExtreme fluctuations?: UnknownTrend: UnknownCauses ceased?: UnknownCauses understood?: UnknownCauses reversible?: UnknownExtreme fluctuations?: UnknownAOO (km2): 4Number of locations: 1Troglonethesolissipoensis occurs in a single cave : A total of 25 specimens have been collected in the type locality .Trend: UnknownExtreme fluctuations?: UnknownSevere fragmentation?: UnknownSystem: TerrestrialHabitat specialist: YesHabitat (narrative): The entrance to Alvide Cave is through a house currently serving as headquarters of Denível Association. Part of the cave's ceiling has concrete that serves as the base of a residential building. This cave is in the margin of a small canyon and it is composed of three levels of horizontal galleries connected by pits, it has a horizontal development of 708 m and total depth of 28 m.Trend in extent, area or quality?: Decline (inferred)Habitat importance: Major ImportanceHabitats: 7.1. Caves and Subterranean Habitats (non-aquatic) - CavesHabitat importance: Major ImportanceHabitats: 7.1. Caves and Subterranean Habitats (non-aquatic) - CavesSize: 3.5 mm , 4.2 mm Generation length (yr): 1Dependency of single sp?: UnknownTroglonethesolissipoensis is a troglobiont species, being blind and depigmented. It is endemic and the only cave-adapted species known from this cave (T.olissipoensis were collected in the deepest parts of the cave (Ecology and traits (narrative): his cave . The avethe cave .Justification for threats: Alvide Cave is located below an over-urbanised area .Threat type: OngoingThreats: 1.1. Residential & commercial development - Housing & urban areas9.1.2. Pollution - Domestic & urban waste water - Run-offThreat type: OngoingThreats: 1.1. Residential & commercial development - Housing & urban areas9.1.2. Pollution - Domestic & urban waste water - Run-offJustification for conservation actions: Measures should be taken to minimise the pernicious effects due to the cave's close proximity to the urbanisation on the habitat.Conservation action type: NeededConservation actions: 1.1. Land/water protection - Site/area protection1.2. Land/water protection - Resource & habitat protection2.1. Land/water management - Site/area management4. Education & awareness5.1.3. Law & policy - Legislation - Sub-national levelConservation action type: NeededConservation actions: 1.1. Land/water protection - Site/area protection1.2. Land/water protection - Resource & habitat protection2.1. Land/water management - Site/area management4. Education & awareness5.1.3. Law & policy - Legislation - Sub-national levelTroglonethesarrabidaensisScientific name: Species authority: Reboleira & Taiti 2015AnimaliaKingdom: ArthropodaPhylum: MalacostracaClass: IsopodaOrder: TrichoniscidaeFamily: Taxonomic notes: Antennae have three flagellar articles, the male pereopod 7 carpus is enlarged in the basal part, the male pleopod 1 exopod is triangular and as wide as long and the male pleopod 2 endopod has the distal article with an apical hook-like process .Region for assessment: EuropeReviewers: ReviewersEditor: EditorReviewers: ReviewersEditor: EditorBiogeographic realm: PalearcticCountries: PortugalMap of records (Google Earth): Suppl. materials 11Basis of EOO and AOO: Known habitat extentBasis (narrative): Both the extent of occurrence (EOO) and area of occupancy (AOO) are 4 km².Min Elevation/Depth (m): 0Troglonethesarrabidaensis is only recorded from Frade Cave, located in the Arrábida karst massif (Range description: t massif .EOO (km2): 4Trend: UnknownCauses ceased?: UnknownCauses understood?: UnknownCauses reversible?: UnknownExtreme fluctuations?: UnknownTrend: UnknownCauses ceased?: UnknownCauses understood?: UnknownCauses reversible?: UnknownExtreme fluctuations?: UnknownAOO (km2): 4Number of locations: 1Troglonethesarrabidaensis occurs in a single cave : A total of 17 specimens have been collected in Frade Cave .Trend: UnknownExtreme fluctuations?: UnknownSevere fragmentation?: UnknownSystem: TerrestrialHabitat specialist: YesHabitat (narrative): Frade Cave is located near the seashore, with several anchialine lakes inside, which are influenced by the sea tides with a slight delay period . The cavTrend in extent, area or quality?: Decline (inferred)Habitat importance: Major ImportanceHabitats: 7.1. Caves and Subterranean Habitats (non-aquatic) - CavesHabitat importance: Major ImportanceHabitats: 7.1. Caves and Subterranean Habitats (non-aquatic) - CavesSize: 2.7 mm Generation length (yr): 1Dependency of single sp?: UnknownTroglonethesarrabidaensis is classified as a troglobiont, depigmented, blind and with an elongated body (Ecology and traits (narrative): ted body . It is aJustification for threats: The cave entrance is located 600 m from a very touristic beach, mainly accessed by boat; therefore, fuel residues brought in by the tidal movement might be a concerning pollutant inside the cave. Contamination of groundwater should be evaluated.Threat type: OngoingThreats: 9. Pollution6.1. Human intrusions & disturbance - Recreational activitiesThreat type: OngoingThreats: 9. Pollution6.1. Human intrusions & disturbance - Recreational activitiesJustification for conservation actions: Even though this cave is protected under legislation by the “Rede Natura 2000” , this siConservation action type: NeededConservation actions: 1.1. Land/water protection - Site/area protection1.2. Land/water protection - Resource & habitat protection2.1. Land/water management - Site/area management4. Education & awareness5.1.3. Law & policy - Legislation - Sub-national levelConservation action type: NeededConservation actions: 1.1. Land/water protection - Site/area protection1.2. Land/water protection - Resource & habitat protection2.1. Land/water management - Site/area management4. Education & awareness5.1.3. Law & policy - Legislation - Sub-national levelMiktoniscuslongispinaScientific name: Species authority: Reboleira & Taiti 2015AnimaliaKingdom: ArthropodaPhylum: MalacostracaClass: IsopodaOrder: TrichoniscidaeFamily: Taxonomic notes: The male pereopod 7 has a long and stout seta on the distal corner of the ischium and a triangular male pleopod 1 exopod . The speFigure(s) or Photo(s): Fig. 2Region for assessment: EuropeReviewers: ReviewersEditor: EditorReviewers: ReviewersEditor: EditorBiogeographic realm: PalearcticCountries: PortugalMap of records (Google Earth): Suppl. materials 12Basis of EOO and AOO: Known habitat extentBasis (narrative): The extent of occurrence (EOO) is 137.96 km² and the area of occupancy (AOO) is 12 km².Min Elevation/Depth (m): 145Max Elevation/Depth (m): 355Miktoniscuslongispina is recorded from three caves with disjunt distribution. Bolhos Cave, also known as Casal da Lebre Cave, located in the Cesaredas Plateau and Ervilha and Cerâmica Caves located in the centre of the Sicó karst area (Range description: rst area .EOO (km2): 137.96Trend: UnknownCauses ceased?: UnknownCauses understood?: UnknownCauses reversible?: UnknownExtreme fluctuations?: UnknownTrend: UnknownCauses ceased?: UnknownCauses understood?: UnknownCauses reversible?: UnknownExtreme fluctuations?: UnknownAOO (km2): 12Number of locations: 3Miktoniscuslongispina can be found in three caves : A total of 12 specimens were collected in the three localities: two in Bolhos Cave, eight in Cerâmica Cave and two in Algar da Ervilha Cave .Number of subpopulations: 3Trend: UnknownExtreme fluctuations?: UnknownSevere fragmentation?: YesSystem: TerrestrialHabitat specialist: YesHabitat (narrative): Bolhos Cave has a horizontal development of 130 m and is located in Cesaredas Plateau, 93 km away from the other localities in the Sicó karst area. Cerâmica Cave has a horizontal development of 120 m and is the richest cave in troglobiont species in central Portugal. Algar da Ervilha Cave has a depth of 52 m and a horizontal development of 150 m . The cavTrend in extent, area or quality?: Decline (inferred)Habitat importance: Major ImportanceHabitats: 7.1. Caves and Subterranean Habitats (non-aquatic) - CavesHabitat importance: Major ImportanceHabitats: 7.1. Caves and Subterranean Habitats (non-aquatic) - CavesSize: 3.5 mm Generation length (yr): 1Dependency of single sp?: UnknownMiktoniscuslongispina is a troglobiont, with a depigmented and elongated body. All specimens, except for the ones collected in Bolhos Cave, are blind.Ecology and traits (narrative): Eucalyptus intensive plantations. It is located 270 m from a road, 550 m from an animal farm, 1.6 km from the closest village and 3.6 km from a quarry. Algar da Ervilha Cave is located in the border of a road in the westernmost access to Ereiras Village, at 90 m from the closest house and 3.5 km from the same quarry as Cerâmica Cave.Justification for threats: Bolhos Cave is located 130 m from an energy windmill and 700 m from the closest village, in an area of extensive agricultural fields. Cerâmica Cave is surrounded by agricultural fields and Threat type: OngoingThreats: 1.1. Residential & commercial development - Housing & urban areas2.2. Agriculture & aquaculture - Wood & pulp plantations2.3. Agriculture & aquaculture - Livestock farming & ranching3.2. Energy production & mining - Mining & quarrying3.3. Energy production & mining - Renewable energy4. Transportation & service corridors9.1.2. Pollution - Domestic & urban waste water - Run-offThreat type: OngoingThreats: 1.1. Residential & commercial development - Housing & urban areas2.2. Agriculture & aquaculture - Wood & pulp plantations2.3. Agriculture & aquaculture - Livestock farming & ranching3.2. Energy production & mining - Mining & quarrying3.3. Energy production & mining - Renewable energy4. Transportation & service corridors9.1.2. Pollution - Domestic & urban waste water - Run-offJustification for conservation actions: The caves located in the Sicó karst area are protected under the “Rede Natura 2000” . Bolhos Conservation action type: NeededConservation actions: 1.1. Land/water protection - Site/area protection1.2. Land/water protection - Resource & habitat protection2.1. Land/water management - Site/area management4. Education & awareness5.1.3. Law & policy - Legislation - Sub-national levelConservation action type: NeededConservation actions: 1.1. Land/water protection - Site/area protection1.2. Land/water protection - Resource & habitat protection2.1. Land/water management - Site/area management4. Education & awareness5.1.3. Law & policy - Legislation - Sub-national levelMoseriusinexpectatusScientific name: Species authority: Reboleira & Taiti 2015AnimaliaKingdom: ArthropodaPhylum: MalacostracaClass: IsopodaOrder: TrichoniscidaeFamily: Moserius species due to the peculiar shape of the male pleopod 1 exopod, with a truncate and sinuous, rather than triangular, distal point : Suppl. materials 13Basis of EOO and AOO: Known habitat extent2.Basis (narrative): Both the extent of occurrence (EOO) and area of occupancy (AOO) are 4 kmMin Elevation/Depth (m): 95Moseriusinexpectatus is only recorded from Almonda Cave, also known as Olho do Moinho da Fonte, located in the easternmost subunit of the Estremenho karst massif (Range description: t massif .EOO (km2): 4Trend: UnknownCauses ceased?: UnknownCauses understood?: UnknownCauses reversible?: UnknownExtreme fluctuations?: UnknownTrend: UnknownCauses ceased?: UnknownCauses understood?: UnknownCauses reversible?: UnknownExtreme fluctuations?: UnknownAOO (km2): 4Number of locations: 1Moseriusinexpectatus is endemic to a single cave : Only one specimen was collected from the type locality .Trend: UnknownExtreme fluctuations?: UnknownSevere fragmentation?: UnknownSystem: TerrestrialHabitat specialist: YesHabitat (narrative): Almonda Cave, the type locality for this species, is the largest cave in Portugal .Trend in extent, area or quality?: Decline (inferred)Habitat importance: Major ImportanceHabitats: 7.1. Caves and Subterranean Habitats (non-aquatic) - CavesHabitat importance: Major ImportanceHabitats: 7.1. Caves and Subterranean Habitats (non-aquatic) - CavesSize: 1.5 mm Generation length (yr): 1Dependency of single sp?: UnknownMoseriusinexpectatus is a troglobiont blind and depigmented isopod. This is the third species described from the genus Moserius, previously known from Slovenia and Italy (Ecology and traits (narrative): nd Italy .Justification for threats: Almonda Cave is located 50 m from a factory that extracts and uses water from a subterranean stream and 420 m from a village, which has many agricultural fields.Threat type: OngoingThreats: 1.1. Residential & commercial development - Housing & urban areas1.2. Residential & commercial development - Commercial & industrial areas2.1. Agriculture & aquaculture - Annual & perennial non-timber crops9.1.2. Pollution - Domestic & urban waste water - Run-offThreat type: OngoingThreats: 1.1. Residential & commercial development - Housing & urban areas1.2. Residential & commercial development - Commercial & industrial areas2.1. Agriculture & aquaculture - Annual & perennial non-timber crops9.1.2. Pollution - Domestic & urban waste water - Run-offJustification for conservation actions: In 1993, Almonda Cave was classified as a Property of Public Interest (IIP) and protected due to its archaeological heritage . The arcConservation action type: NeededConservation actions: 1.1. Land/water protection - Site/area protection1.2. Land/water protection - Resource & habitat protection2.1. Land/water management - Site/area management4. Education & awareness5.1.3. Law & policy - Legislation - Sub-national levelConservation action type: NeededConservation actions: 1.1. Land/water protection - Site/area protection1.2. Land/water protection - Resource & habitat protection2.1. Land/water management - Site/area management4. Education & awareness5.1.3. Law & policy - Legislation - Sub-national levelCordioniscuslusitanicusScientific name: Species authority: Reboleira & Taiti 2015AnimaliaKingdom: ArthropodaPhylum: MalacostracaClass: IsopodaOrder: StyloniscidaeFamily: Taxonomic notes: The male pereopod 7 ischium has a rounded hyaline basal lobe, the triangular male pleopod 1 exopod is as long as the endopod and the male pleopod 2 endopod has a complex apical part .Region for assessment: EuropeReviewers: ReviewersEditor: EditorReviewers: ReviewersEditor: EditorBiogeographic realm: PalearcticCountries: PortugalMap of records (Google Earth): Suppl. materials 14Basis of EOO and AOO: Known habitat extent2 and the area of occupancy (AOO) is 16 km2.Basis (narrative): The extent of occurrence (EOO) is 5,893.93 kmMin Elevation/Depth (m): 10Max Elevation/Depth (m): 370Cordioniscuslusitanicus is recorded from five caves, located in two isolated karst areas: Algar de Santo António from the Estremoz-Cano karst massif and Ibne Ammar, Algarão do Remexido, Vale Telheiro and Senhora Caves from the Algarve karst massif (Range description: t massif .EOO (km2): 5,893.93Trend: UnknownJustification for trend: The four caves, located in the Algarve karst massif, are at 200 km distance from the cave in the Estremoz-Cano karst massif.Causes ceased?: UnknownCauses understood?: UnknownCauses reversible?: UnknownExtreme fluctuations?: UnknownTrend: UnknownCauses ceased?: UnknownCauses understood?: UnknownCauses reversible?: UnknownExtreme fluctuations?: UnknownAOO (km2): 16Number of locations: 5Cordioniscuslusitanicus occurs in five caves : A total of 26 specimens have been collected: nine from Algar de Santo António, six from Ibne Ammar, six from Algarão do Remexido and four from Senhora  and one Number of subpopulations: 5Trend: UnknownExtreme fluctuations?: UnknownSevere fragmentation?: UnknownSystem: TerrestrialHabitat specialist: UnknownCordioniscuslusitanicus was collected in two karst areas, Estremoz-Cano and Algarve, located more than 200 km apart, which are isolated from each other by the dry and flat areas of the Alentejo Province. In Algar de Santo António, the specimens were collected in deep layers of soil at the bottom of an entrance pit of the cave (Habitat (narrative): the cave .Trend in extent, area or quality?: Decline (inferred)Habitat importance: Major ImportanceHabitats: 7.1. Caves and Subterranean Habitats (non-aquatic) - CavesHabitat importance: Major ImportanceHabitats: 7.1. Caves and Subterranean Habitats (non-aquatic) - CavesSize: 3 mm , 5 mm Generation length (yr): 1Dependency of single sp?: UnknownCordioniscuslusitanicus is classified as a troglobiont and endogean species. It is blind, depigmented and has an elongated body (Ecology and traits (narrative): ted body .Justification for threats: Algar de Santo António is located in the middle of an urbanised area of a village. Algarão do Remexido is located under agricultural lands, 370 m from the closest house and 1.7 km from the closest village. Senhora Cave is located 168 m from the closest house and 900 m from an industrial complex. Ibne Ammar Cave is located right in the flooding zone of the Arade river, 380 m from the national road IC4 and 1.4 km from the nearest town. Vale Telheiro Cave is located 50 m from a road and 150 m from the closest house.Threat type: OngoingThreats: 1.1. Residential & commercial development - Housing & urban areas1.2. Residential & commercial development - Commercial & industrial areas2.2. Agriculture & aquaculture - Wood & pulp plantations4. Transportation & service corridors9.1.2. Pollution - Domestic & urban waste water - Run-offThreat type: OngoingThreats: 1.1. Residential & commercial development - Housing & urban areas1.2. Residential & commercial development - Commercial & industrial areas2.2. Agriculture & aquaculture - Wood & pulp plantations4. Transportation & service corridors9.1.2. Pollution - Domestic & urban waste water - Run-offJustification for conservation actions: Of the five caves, only Ibne Ammar Cave is protected under legislation by the “Rede Natura 2000”  and thisConservation action type: NeededConservation actions: 1.1. Land/water protection - Site/area protection1.2. Land/water protection - Resource & habitat protection2.1. Land/water management - Site/area management4. Education & awareness5.1.3. Law & policy - Legislation - Sub-national levelConservation action type: NeededConservation actions: 1.1. Land/water protection - Site/area protection1.2. Land/water protection - Resource & habitat protection2.1. Land/water management - Site/area management4. Education & awareness5.1.3. Law & policy - Legislation - Sub-national levelPorcelliocavernicolusScientific name: Species authority: Vandel, 1946AnimaliaKingdom: ArthropodaPhylum: MalacostracaClass: IsopodaOrder: PorcellionidaeFamily: Figure(s) or Photo(s): Fig. 3Region for assessment: EuropeReviewers: ReviewersEditor: EditorReviewers: ReviewersEditor: EditorBiogeographic realm: PalearcticCountries: PortugalMap of records (Google Earth): Suppl. materials 15Basis of EOO and AOO: Known habitat extent2 and the area of occupancy (AOO) is 24 km2.Basis (narrative): The extent of occurrence (EOO) is 713 kmMin Elevation/Depth (m): 20Max Elevation/Depth (m): 380Porcelliocavernicolus is recorded from seven caves located in two isolated massifs, Gruta d’el Rey in the Cantanhede-Outil karst massif and Santa Maria da Estrela, Soprador do Carvalho, Algarinho, Cerâmica, Abrigo Tomar I and Furjaca caves, located in the Sicó massif (Range description: ó massif .EOO (km2): 713Trend: UnknownCauses ceased?: UnknownCauses understood?: UnknownCauses reversible?: UnknownExtreme fluctuations?: UnknownTrend: UnknownCauses ceased?: UnknownCauses understood?: UnknownCauses reversible?: UnknownExtreme fluctuations?: UnknownAOO (km2): 24Number of locations: 7Porcelliocavernicolus occurs in seven caves : More than 63 specimens have been collected: 28 from Gruta d’el Rey, 16 from Santa Maria da Estrela, seven from Soprador do Carvalho, three from Cerâmica, four from Abrigo Tomar I and five from Furjaca. The specimens, collected from Algarinho Cave, are simply described as “many” .Number of subpopulations: 7Trend: UnknownExtreme fluctuations?: UnknownSevere fragmentation?: UnknownSystem: TerrestrialHabitat specialist: YesPorcelliocavernicolus inhabits the most superficial parts of caves and it occurs on roots that hang from the ceiling. Specimens are easily distinguishable due to their whitish colouration (Habitat (narrative): ouration .Trend in extent, area or quality?: Decline (inferred)Habitat importance: Major ImportanceHabitats: 7.1. Caves and Subterranean Habitats (non-aquatic) - CavesHabitat importance: Major ImportanceHabitats: 7.1. Caves and Subterranean Habitats (non-aquatic) - CavesSize: 10 mm Generation length (yr): 1Dependency of single sp?: UnknownPorcelliocavernicolus is classifid as a troglobiont, endemic to seven caves from central Portugal, distributed in two isolated karst massifs (Ecology and traits (narrative):  massifs .Eucalyptus intensive plantations. It is located 270 m from a road, 550 m from an animal farm, 1.6 km from the closest village and 3.6 km from a quarry. Abrigo Tomar I Cave is located in a quite pristine location and it is a local protected area by the ONG Quercus. Furjaca Cave is located in an abandoned quarry which, after closure, was used as a trash dumping site.Justification for threats: Gruta d‘el Rey is located in the middle of an urbanised area, 1 km from a quarry and 1.2 km from highway A14. Santa Maria da Estrela Cave is located 86 m from a touristic site called Monstro das Bolachas, 230 m from the Nossa Senhora da Estrela viewpoint, 250 m from the closest house, 80 m from the closest road, 220 m from agricultural fields and 2.6 km from two quarries. Soprador do Carvalho is surrounded by agricultural lands and is located 67 m from the closest house and 1.4 km from a quarry. This cave is also affected by touristic activities, as people who visit the cave walk all over the habitat . The subThreat type: OngoingThreats: 1.1. Residential & commercial development - Housing & urban areas1.2. Residential & commercial development - Commercial & industrial areas2.2. Agriculture & aquaculture - Wood & pulp plantations3.2. Energy production & mining - Mining & quarrying4. Transportation & service corridors6.1. Human intrusions & disturbance - Recreational activities9.1. Pollution - Domestic & urban waste water9.4. Pollution - Garbage & solid wasteThreat type: OngoingThreats: 1.1. Residential & commercial development - Housing & urban areas1.2. Residential & commercial development - Commercial & industrial areas2.2. Agriculture & aquaculture - Wood & pulp plantations3.2. Energy production & mining - Mining & quarrying4. Transportation & service corridors6.1. Human intrusions & disturbance - Recreational activities9.1. Pollution - Domestic & urban waste water9.4. Pollution - Garbage & solid wasteJustification for conservation actions: Of the seven locations, only three are protected under legislation by the “Rede Natura 2000” . Almost Conservation action type: NeededConservation actions: 1.1. Land/water protection - Site/area protection1.2. Land/water protection - Resource & habitat protection2.1. Land/water management - Site/area management4. Education & awareness5.1.3. Law & policy - Legislation - Sub-national levelConservation action type: NeededConservation actions: 1.1. Land/water protection - Site/area protection1.2. Land/water protection - Resource & habitat protection2.1. Land/water management - Site/area management4. Education & awareness5.1.3. Law & policy - Legislation - Sub-national levelTroglelumamachadoiScientific name: Species authority: AnimaliaKingdom: ArthropodaPhylum: MalacostracaClass: IsopodaOrder: ArmadillidiidaeFamily: Figure(s) or Photo(s): Fig. 4Region for assessment: EuropeReviewers: ReviewersEditor: EditorReviewers: ReviewersEditor: EditorBiogeographic realm: PalearcticCountries: PortugalMap of records (Google Earth): Suppl. materials 16Basis of EOO and AOO: Known habitat extent2 and the area of occupancy (AOO) is 16 km2.Basis (narrative): The extent of occurrence (EOO) is 356.4 kmMin Elevation/Depth (m): 10Max Elevation/Depth (m): 260Troglelumamachadoi is recorded from six caves, located in the Algarve karst massif: Ibne Ammar, Algarão do Remexido, Vale Telheiro, Senhora, Algarão do Paulino and Abismo Novo (Range description: smo Novo .EOO (km2): 356.4Trend: UnknownCauses ceased?: UnknownCauses understood?: UnknownCauses reversible?: UnknownExtreme fluctuations?: UnknownTrend: UnknownCauses ceased?: UnknownCauses understood?: UnknownCauses reversible?: UnknownExtreme fluctuations?: UnknownAOO (km2): 16Number of locations: 6Troglelumamachadoi occurs in six caves : Trend in extent, area or quality?: Decline (inferred)Habitat importance: Major ImportanceHabitats: 7.1. Caves and Subterranean Habitats (non-aquatic) - CavesHabitat importance: Major ImportanceHabitats: 7.1. Caves and Subterranean Habitats (non-aquatic) - CavesSize: 8 mmGeneration length (yr): 1Dependency of single sp?: UnknownTroglelumamachadoi is a blind and depigmented species, classified as a troglobiont. Specimens are found in the most deep and well isolated parts of of caves, they have the integument covered with clay, while some others were found walking on cave walls and completely clean of clay, probably recently moulted :  moulted . The temicamagna , the relgmaticus , the dyslitoides , the milocalense , the dipamendesi , the giagharbica  and the arvensis .Justification for threats: Ibne Ammar Cave is located right in the flooding zone of the Arade River, 380 m from the national road IC4 and 1.4 km from the nearest town. Algarão do Remexido is located under agricultural lands, 370 m from the closest house and 1.7 km from the closest village. Senhora Cave is located 168 m from the closest house and 900 m from an industrial complex. Abismo Novo Cave is located 100 m from the closest house and 500 m from the village centre and is also located 1 km from Senhora Cave. Algarão do Paulino is located near a road, 90 m from the closest house and 800 m from the closest village. Vale Telheiro Cave is located 50 m from a road and 150 m from the closest house.Threat type: OngoingThreats: 1.1. Residential & commercial development - Housing & urban areas1.2. Residential & commercial development - Commercial & industrial areas2.1. Agriculture & aquaculture - Annual & perennial non-timber crops4. Transportation & service corridors9.1.2. Pollution - Domestic & urban waste water - Run-offThreat type: OngoingThreats: 1.1. Residential & commercial development - Housing & urban areas1.2. Residential & commercial development - Commercial & industrial areas2.1. Agriculture & aquaculture - Annual & perennial non-timber crops4. Transportation & service corridors9.1.2. Pollution - Domestic & urban waste water - Run-offJustification for conservation actions: Of the five locations, only three are protected under legislation by the “Rede Natura 2000” . MeasureConservation action type: NeededConservation actions: 1.1. Land/water protection - Site/area protection1.2. Land/water protection - Resource & habitat protection2.1. Land/water management - Site/area management4. Education & awareness5.1.3. Law & policy - Legislation - Sub-national levelConservation action type: NeededConservation actions: 1.1. Land/water protection - Site/area protection1.2. Land/water protection - Resource & habitat protection2.1. Land/water management - Site/area management4. Education & awareness5.1.3. Law & policy - Legislation - Sub-national levelCave-adapted terrestrial isopods are key species for cave ecosystem conservation: i) they are the most diverse group of cave-adapted species in continental Portugal, ii) they have several single cave endemics that are under threat and require specific protection measures, iii) they are basal in the trophic chains in caves and serve as a food source for several other zoological groups; iv) they play a vital role on the decomposition of organic matter in caves; and v) they are very sensitive to contaminants and climate change .Almost all cave-adapted terrestrial isopod species face direct anthropogenic threats, such as point or diffuse pollution, direct habitat destruction by mining and quarry activities or excess cave visitation . With thOniscidea  . So far,niscidea . Therefod groups .It is a priority to establish concrete protection strategies for cave-adapted species in continental Portugal. We need to improve the knowledge about population size and dynamics, real extent of subterranean distribution, improve our knowledge on the functional ecology, understand species life cycle and evaluate their sensitivity to disturbance. This contribution may be used as a support for decision-making for territory management and to define conservation measures for cave endemic species. Cave-adapted terrestrial isopods have the potential to be used as umbrella species for the conservation of other cave-adapted species sharing the same subterranean habitats.Discussion2F1C3046-2169-5E36-AB53-F8D02E3A9E1410.3897/BDJ.10.e78796.suppl1Supplementary material 1Distribution of cave-adapted terrestrial isopods in continental PortugalData typeSpecies distribution mapBrief description(A) Overview of the distribution of cave-adapted terrestrial isopods in continental Portugal; (B) Sicó karst area distribution detail; (C) Estremenho and Montejunto karst massifs, Caldas da Rainha Typhonic Valley and Cesaredas Plateau distribution detail; (D) Algarve karst massif distribution detail; (E) Lisbon Peninsula and Arrábida karst massif and Estremoz-Cano karst massif distribution detail; and (F) Vimioso paleokarst distribution detail.Species:Trichoniscoidesbroteroi (orange circle), T.ouremensis (pink circle), T.serrai (dark blue circle), T.subterraneus (yellow circle), T.meridionalis (purple circle), T.bellesi (forest green circle), T.sicoensis (light blue circle), Metatrichoniscoidessalirensis (dark blue diamond), Troglonethesolissipoensis (dark blue star), T.arrabidaensis (pink star), Miktoniscuslongispina (yellow cross), Moseriusinexpectatus (light blue hexagon), Cordioniscuslusitanicus (dark blue triangle outline), Porcelliocavernicolus (pink triangle) and Troglelumamachadoi (pink circle outline).File: oo_607434.pnghttps://binary.pensoft.net/file/607434A.S.P.S. Reboleira, R.P. EusébioDD91E314-7C4E-5774-8FF0-6086CE34BF8A10.3897/BDJ.10.e78796.suppl2Supplementary material 2TrichoniscoidesbellesiDistribution of Data typeSpecies distribution mapBrief descriptionTrichoniscoidesbellesi distribution: Algar do Javali Cave, Montejunto karst massif.File: oo_607707.tifhttps://binary.pensoft.net/file/607707A.S.P.S. Reboleira, R.P.Eusébio5914DE8F-D8BC-5B98-9A9F-9D4D7A6E1EE910.3897/BDJ.10.e78796.suppl3Supplementary material 3TrichoniscoidesbroteroiDistribution of Data typeSpecies distribution mapBrief descriptionTrichoniscoidesbroteroi distribution: Alqueves Cave, Sicó karst area.File: oo_607708.tifhttps://binary.pensoft.net/file/607708A.S.P.S. Reboleira, R.P. Eusébio5FB36EF8-919B-5348-86EF-333B2BE58A3510.3897/BDJ.10.e78796.suppl4Supplementary material 4TrichoniscoidesmeridionalisDistribution of Data typeSpecies distribution mapBrief descriptionTrichoniscoidesmeridionalis distribution. (A) Detail of distribution: Algar do Vale do Pena; Alcobertas Cave; Algar do Zé de Braga Cave; Lapa da Chã de Cima Cave; Algar do Ladoeiro Cave; Algar do Pena Cave; Moinhos Velhos Cave; Papagaio Cave; Algar do Burro Cave; and Almonda Cave.File: oo_607711.tifhttps://binary.pensoft.net/file/607711A.S.P.S. Reboleira, R.P. Eusébio597F809C-3090-5016-9767-C3E76CCD0A1610.3897/BDJ.10.e78796.suppl5Supplementary material 5TrichoniscoidesouremensisDistribution of Data typeSpecies distribution mapBrief descriptionTrichoniscoidesouremensis distribution: Lapa da Salgada Cave, Fátima Plateau.File: oo_607712.tifhttps://binary.pensoft.net/file/607712A.S.P.S. Reboleira, R.P. EusébioF92DCDAA-FA35-5326-85D4-F63245509A6C10.3897/BDJ.10.e78796.suppl6Supplementary material 6TrichoniscoidesserraiDistribution of Data typeSpecies distribution mapBrief descriptionTrichoniscoidesserrai distribution: Santo Adrião Cave, Vimioso karst area.File: oo_607713.tifhttps://binary.pensoft.net/file/607713A.S.P.S. Reboleira, R.P. Eusébio48CB91A0-5194-5E31-94A2-A512AA8133E510.3897/BDJ.10.e78796.suppl7Supplementary material 7TrichoniscoidessicoensisDistribution of Data typeSpecies distribution mapBrief descriptionTrichoniscoidessicoensis distribution. (A) Detail of the distribution: Arrifana Cave; Santa Maria da Estrela Cave; Cerâmica Cave; São Simão Cave; Algarinho Cave; and Soprador do Carvalho Cave.File: oo_607714.tifhttps://binary.pensoft.net/file/607714A.S.P.S. Reboleira, R.P.Eusébio774C7847-3F7C-544E-81E9-48B9C9A29AFE10.3897/BDJ.10.e78796.suppl8Supplementary material 8TrichoniscoidessubterraneusDistribution of Data typeSpecies distribution mapBrief descriptionTrichoniscoidessubterraneus distribution: Alta do Cabeço dos Mosqueiros Cave, Aljubarrota Plateau.File: oo_607720.tifhttps://binary.pensoft.net/file/607720A.S.P.S. Reboleira, R.P. Eusébio71845157-2E1F-5082-AAA4-DDC0924AFDBD10.3897/BDJ.10.e78796.suppl9Supplementary material 9MetatrichoniscoidessalirensisDistribution of Data typeSpecies distribution mapBrief descriptionMetatrichoniscoidessalirensis distribution: Salir Cave, Caldas da Rainha.File: oo_607721.tifhttps://binary.pensoft.net/file/607721A.S.P.S. Reboleira, R.P. Eusébio425A22BF-FE18-5FDB-B23D-8937DEED57B610.3897/BDJ.10.e78796.suppl10Supplementary material 10TroglonethesolissipoensisDistribution of Data typeSpecies distribution mapBrief descriptionTroglonethesolissipoensis distribution: Alvide Cave, Lisbon.File: oo_607722.tifhttps://binary.pensoft.net/file/607722A.S.P.S. Reboleira, R.P. Eusébio85C91C9B-5B83-533D-AB14-9F6ABE3F8A6B10.3897/BDJ.10.e78796.suppl11Supplementary material 11TroglonethesarrabidaensisDistribution of Data typeSpecies distribution mapBrief descriptionTroglonethesarrabidaensis distribution: Frade Cave, Arrábida karst massif.File: oo_607723.tifhttps://binary.pensoft.net/file/607723A.S.P.S. Reboleira, R.P.EusébioBC5A499B-29AA-5FBE-B1CE-77CC7E4752A510.3897/BDJ.10.e78796.suppl12Supplementary material 12MiktoniscuslongispinaDistribution of Data typeSpecies distribution mapBrief descriptionMiktoniscuslongispina distribution. (A) Detail of distribution: Casal da Lebre Cave; Algar da Ervilha Cave; and Cerâmica Cave.File: oo_607725.tifhttps://binary.pensoft.net/file/607725A.S.P.S. Reboleira, R.P. EusébioDB2A85A3-79E9-53CC-AC9E-E2C21922CF6F10.3897/BDJ.10.e78796.suppl13Supplementary material 13MoseriusinexpectatusDistribution of Data typeSpecies distribution mapBrief descriptionMoseriusinexpectatus distribution: Almonda Cave, Estremenho karst massif.File: oo_607726.tifhttps://binary.pensoft.net/file/607726A.S.P.S. Reboleira, R.P. EusébioC9060AAB-01F8-5426-86EE-22A80F48CE2110.3897/BDJ.10.e78796.suppl14Supplementary material 14CordioniscuslusitanicusDistribution of Data typeSpecies distribution mapBrief descriptionCordioniscuslusitanicus distribution. (A) Detail of distribution: Ibne Ammar Cave; Algarão do Remexido Cave; Vale Telheiro Cave; Senhora Cave; and Algar de Santo António Cave.File: oo_607729.tifhttps://binary.pensoft.net/file/607729A.S.P.S. Reboleira, R.P. Eusébio9A58FB28-25DF-5F94-842E-3D0690437F8C10.3897/BDJ.10.e78796.suppl15Supplementary material 15PorcelliocavernicolusDistribution of Data typeSpecies distribution mapBrief descriptionPorcelliocavernicolus distribution. (A) Detail of distribution: Gruta d’el Rey; Furjaca Cave; Santa Maria da Estrela Cave; Cerâmica Cave; Algarinho Cave; Soprador do Carvalho Cave; and Abrigo Tomar I Cave.File: oo_607732.tifhttps://binary.pensoft.net/file/607732A.S.P.S. Reboleira, R.P. EusébioF77EA613-7319-514F-BCF6-CF2EFDC4F8E210.3897/BDJ.10.e78796.suppl16Supplementary material 16TroglelumamachadoiDistribution of Data typeSpecies distribution mapBrief descriptionTroglelumamachadoi distribution. (A) Detail of distribution: Ibne Ammar Cave; Algarão do Remexido Cave; Vale Telheiro Cave; Algarão do Paulino Cave; Abismo Novo Cave; and Senhora Cave.File: oo_607736.tifhttps://binary.pensoft.net/file/607736A.S.P.S. Reboleira, R.P. Eusébio


Monocyte derived dendritic cells were generated from these peripheral blood mononuclear cells (PBMC), loaded with SARS-CoV-2 peptides, and used to induce autologous CD4+ and CD8+ T cell activation. The SARS-CoV-2 peptides demonstrated antigenicity against the T-cells from individuals with previous SARS-CoV-2 infection indicating that this approach holds promise as a method to activate anti-SAR-CoV-2 T-cell responses from conserved regions of the virus which are not included in vaccines utilising the Spike protein.The pandemic caused by SARS-CoV-2 has led to the successful development of effective vaccines however the prospect of variants of SARS-CoV-2 and future coronavirus outbreaks necessitates the investigation of other vaccine strategies capable of broadening vaccine mediated T-cell responses and potentially providing cross-immunity. In this study the SARS-CoV-2 proteome was assessed for clusters of immunogenic epitopes restricted to diverse human leucocyte antigen. These regions were then assessed for their conservation amongst other coronaviruses representative of different alpha and beta coronavirus genera. Sixteen highly conserved peptides containing numerous HLA class I and II restricted epitopes were synthesized from these regions and assessed  NC_045512.2, MN996532.2, MT072864.1, MT040333.1, KC881005.1, KY417144.1, KT444582.1, KF367457.1, KC881006.1, KY417152.1, MK211376.1, AY572034.1, AY572035.1, AY572038.1, AY686864.1, AY686863.1, NC_004718.3, AY390556.1, KY352407.1, NC_014470.1, GU190215.1, MG772933.1, MG772934.1, MK211374.1, JX993987.1, MK211377.1, DQ084199.1, GQ153539.1, DQ084200.1, DQ022305.2, KJ473815.1, KJ473813.1, KJ473812.1, KP886808.1, DQ648857.1, DQ412043.1, KY417147.1, DQ648856.1, JX993988.1, FJ588686.1, MK211378.1, KJ473816.1, KJ473811.1, MG596802.1, MG596803.1, NC_009020.1, MG021452.1, MK129253.1, JX869059.2, MT576585.1, NC_038294.1, DQ648794.1, KJ473821.1, KJ473822.1, KX442564.1, EF065505.1, NC_009019.1, KC869678.4, NC_039207.1, KC545386.1, KJ473806.1, NC_026011.1, NC_017083.1, FJ647223.1, KF686343.1, AY391777.1, NC_030886.1, NC_009021.1, HM211098.1, MK211375.1, KY770860.1, KU182964.1, MT430884.1, LC469308.1, NC_048212.1, MF593268.1, NC_034440.1, NC_028814.1, JQ989273.1, NC_018871.1, AF304460.1, JQ410000.1, NC_048216.1, AY567487.2, NC_046964.1, NC_028833.1, KJ473820.1, NC_028811.1, NC_022103.1, EF203064.1, NC_028824.1, DQ648858.1, EU420138.1, KJ473795.1, EU420137.1.The pandemic caused by the novel coronavirus, SARS-CoV-2, has prompted a global response to produce effective vaccines. A number of vaccines have been approved for use having demonstrated varying levels of efficacy in clinical trials . These vin vitro.T-cell responses to SARS-CoV-1 demonstrate greater durability than those of neutralising antibody  and are https://www.ncbi.nlm.nih.gov/labs/virus/vssi/), alignment using Clustal Omega with default settings  and analysis of conservation using Microsoft Excel. Accession numbers of coronaviruses strains can be found in the supplementary material file.Analysis of SARS-CoV-2 and other coronaviruses protein sequences from proteins harbouring epitope rich regions was performed using FASTA sequences deposited in the NCBI database (https://www.iedb.org/).Identification of SARS-CoV-2 HLA restricted epitopes using prediction algorithms and experimentally validated epitopes deposited in the Immune epitope database and by using the NetMHCpan EL 4.1 (2020.09) prediction algorithm against an HLA allele reference set (Selection of conserved immunogenic regions between 15–30 amino acids in length (synthetic long peptides) was determined by assessing their suitability for synthesis based upon the physiochemical properties of the amino acids in the sequence and potential as CPP (cell penetrating peptides) defined by a net positive charge. Selected peptides were synthesized .Peripheral blood mononuclear cells (PBMC) were purchased from the national blood service  prior to the distribution of SARS-CoV-2 vaccines. PBMC demonstrating responses to a SARS-CoV-2 consensus peptide pool and serum antibody responses to the SARS-CoV-2 spike protein were defined as having been previously infected with SARS-CoV-2 whilst PBMC and sera lacking detectable responses were defined as SARS-CoV-2 naïve.−1 IL-4 and 50 ng ml−1 GM-CSF . These mDDC were subsequently co-cultured with the T-cells enriched from the CD14- fraction of PBMC using anti-CD3 magnetic beads  in the presence of SARS-CoV-2 peptides individual and in groups, and control peptides including CEF , CEFT  SARS-CoV-2 reference group  (1 µg ml−1 for each peptide) for antigenicity assays.The generation of mDDC (monocyte-derived dendritic cells) was performed using established protocols. CD14+ cells were isolated by positive selection using anti-CD14 conjugated magnetic beads . The CD14+ cells were cultured for 6 days in RPMI  10% foetal calf serum  and 5% streptomycin/penicillin  of 10 ng ml4 mDDC: 2×105 T-cells) were incubated in IFN-γ ELISPOT plates  for 48 h in order to assess antigen specific T-cell activation. After 48 h ELISPOT plates were processed according to the manufacturers protocol. Briefly, plates were washed in PBS ×3 prior to the incubation with detection anti-IFN-γ antibody for 2 h. Plates were washed ×3 in PBS wash buffer and Strep-Biotin reagent was added for 1 h. Plates were washed ×3 prior to the addition of substrate solution. Spot formation was observed and the plates were washed once in distilled H2O and left to dry before enumeration using a CTL Immunospot entry ELISPOT plate reader. Positive responses compared to the no peptide control were defined as >1.5-fold change and statistical significance using Student’s t-test.IFN-γ ELISPOT (enzyme-linked immunosorbent spot) assays using peptide pulsed mDDC-T-cell co-cultures  since the physiochemical properties of the peptides may make them unsuitable for synthesis or for targeting toward antigen presenting cells or homology between the peptides and human proteins may make them unsuitable for vaccination. Amino acid modifications were made outside of epitope containing regions in order to improve synthesis, stability and internalisation. Each of the peptides was differently conserved between other coronaviruses and contained a different number of epitopes restricted to different HLA. A total of 16 of these candidate SLP were selected as an immunogenic pool for The selected peptides were assessed for their ability to activate T-cell responses. PBMC from seven individuals with previous SARS-CoV-2 infection and four individuals seronegative for SARS-CoV-2, without selection for specific HLA expression, were used to generate monocyte derived dendritic cells , loaded 4895-915 which demonstrated a CD4+ T cell response from the PBMC of one individual but no CD8+ T cell responses. This pattern of responses was consistent with other reports studying anti-SARS T-cell responses [Next a flow cytometry-based activation induced marker (AIM) assay was used to gain a greater insight into the T-cell responses raised by the peptides  and CD8+esponses . Interesesponses .in vitro T-cell antigenicity of SARS-CoV-2 derived SLP containing epitopes restricted to multiple HLA and conserved between SARS-CoV-2 variants and other coronaviruses.Taken together these data demonstrate the SARS-CoV-2 vaccines based upon the Spike protein have demonstrated between 60–95% efficacy in phase three trials and are now in widespread use. Although these vaccines are highly efficacious numerous issues remain unresolved. These include supply, cost and the requirement of some vaccines for a cold chain. From an immunological perspective there remains concern that variation in the Spike protein may evolve against which antibodies raised by vaccination are less effective, demonstrated to some degree by the Gamma  and DeltThe broad therapeutic activation of SARS specific T-cell immune responses may resolve or ameliorate a number of these issues. T-cell responses are more stable than humoral responses  whilst pIn this study, immunogenic regions from SARS-CoV-2 proteins other than the spike were identified and their conservation amongst selected alpha and beta coronaviruses was assessed. The selected peptides contain multiple epitopes restricted to the most common HLA class I molecules and which have previously demonstrated induction of T-cell activation in response to SARS-CoV-2 . ImportaThe peptides are derived from the immunodominant viral proteins other than the spike  so couldin vitro experiments DC-T-cell co-cultures generated from the PBMC of individuals with previous SARS-CoV-2 infection responded with IFN-γ expression to an average of five peptides derived from an average of four proteins. Previous studies have demonstrated that broad T-cell responses against multiple epitopes are more effective than narrow responses targeting fewer epitopes [A recent study showed that CD8+ T cell responses against SARS-CoV-2 were raised against approximately 17 epitopes derived from between 1–6 viral proteins (average 2.7) [epitopes  and lessepitopes  indicatiAnalysis of T-cell responses to the ChAdOx1 spike-based vaccine showed that nearly 30% of unique TCRs raised by the vaccine mapped to a single region of the spike protein which is mutated in the Beta variant of SARS-CoV-2 . This maSynthetic long peptides of the kind studied here have been used in numerous therapeutic vaccines for both infectious disease and cancer and demonstrated an ability to induce efficacious T-cell responses . PeptideClick here for additional data file.


People admitted to a skilled nursing facility (SNF) for post-acute care undergo comprehensive evaluation and rehabilitation, potentially enabling prediction of future functional recovery. We identified the first SNF admission per beneficiary  between 07/01/2014 – 06/30/2016 in a 5% Medicare sample, using the Minimum Data Set (MDS) and the Outcome and Assessment Information Set (OASIS). Episodes were excluded for non-community discharge  or no OASIS admission assessment within 14 days of SNF discharge . A deficit accumulation Frailty Index (FI) was measured on admission MDS assessment and categorized into robust (MDS-FI<0.15), pre-frailty (MDS-FI0.15-0.24), mild frailty (MDS-FI0.25-0.34), and moderate or worse frailty (MDS-FI≥0.35). Outcomes were functional decline obtained from OASIS, readmission, or death after initiation of home care. Functional status was measured by activities of daily living from OASIS assessments. A total of 135,310 SNF episodes were matched to OASIS episodes. Of these, there were 6,472 (4.8%) robust patients, 38,923 (28.8%) pre-frail, 63,727 (47.1%) mildly frail and 26,053 (19.3%) moderately frail or worse. In a logistic regression after adjustment for OASIS admission function, compared to robust status, frailty was associated with hospital readmission or death within 30 days of OASIS admission, (mild frailty OR1.33 [95%CI 1.23-1.45] and moderate or worse OR1.81 [95%CI 1.66-1.97]). Frailty was also associated with functional decline at OASIS discharge, after adjustment for OASIS admission function (mild frailty OR1.50 [95%CI 1.38-1.63] and moderate or worse OR2.30 [95%CI 2.11-2.50]). Among those discharged from SNF with home services, a SNF-based MDS-FI is associated with increased likelihood of poor community outcomes.


Tracheal, bronchus, and lung (TBL) cancer is the leading cause of cancer death globally, but trends in TBL mortality attributable to tobacco, ambient particulate matter pollution (APMP), and household air pollution (HAP) were unequally distributed within global population subgroups over the last three decades. We used data from the Global Burden of Disease 2019 study to quantify the impact of sex, time, sociodemographic development index (SDI), and age for each exposure from 1990–2019. During that interval, tobacco dominated the TBL cancer mortality landscape, with its minimum global age-adjusted death rate of 16.71 deaths/100,000 : 15.27–18.13) outstripping maximums of 3.85 deaths/100,000 (UI: 2.82–4.83) and 2.54 deaths/100,000 (UI: 1.69–3.54) for APMP and HAP, respectively. In 2019, tobacco male TBL death rates exceeded female rates by a factor of 4.4:1. Ratios of 1.9:1 for APMP and 2.1:1 for HAP were seen. Our analysis indicates that both-sex middle SDI and female low, low-middle, and high-middle SDI populations are suffering increasing tobacco TBL burden. Efforts producing successful global reductions in HAP-associated TBL mortality should continue, with attention to low SDI female death rate increases. Finally, except for high SDI populations, global APMP-attributable TBL cancer burden is increasing and represents a major health concern. Lung cancer is the leading cause of cancer death globally , with trRecent studies have shed light on TBL incidence, DALYs, and mortality trends in the GBD 2017 and GBD 2019 data ,11,12. WAnnual TBL cancer deaths and death rate estimates attributable to environmental exposures were extracted from the GBD 2019 dataset . Death rt-tests of the 95% confidence interval of the difference between a given subgroup’s 1990 and 2019 death rates, at a significance level of p < 0.05 . Statistically significant findings are termed “significant” in the Results section; non-significant findings are not preceded by a word modifier. Lastly, using Microsoft Excel software, we generated heat maps for each exposure using death rate stratification by sex, SDI, and age between 1990 and 2019 to highlight granular trends among subpopulations. Other visualizations were generated using the GBD Compare tool [Specific considerations for each stratifying variable were as follows: (1) Time: TBL deaths and age-standardized death rate estimates were available for all years between 1990–2019; (2) SDI: The GBD 2019 authors created SDI as a country’s “composite indicator of socio-demographic development status” utilizing “total fertility rate in those under 25 years old, mean education for those 15 years or older, and lag-distributed income per capita” to give each country an index value from 0 to 100 [are tool .From 1990–2019, tobacco dominated the TBL cancer mortality landscape. Tobacco-attributed global age-adjusted death rates ranged from 16.71 deaths/100,000 (UI: 15.27–18.13) to 19.99 deaths/100,000 (UI: 19.10–20.86), which outstripped interval maximum rates of 3.85 deaths/100,000 (UI: 2.82–4.83) and 2.54 deaths/100,000 (UI: 1.69–3.54) for APMP and HAP, respectively c. Globalp < 0.01). Males in the global population experienced a large, significant decrease (p < 0.01), while the female trend was a non-significant increase (p = 0.80). Exploring these trends by SDI highlighted significant decreases of 30% in HSDI (p < 0.01) and 14% in HMSDI (p = 0.01) both-sex populations (p < 0.01) (p = 0.06) (p = 0.10); MSDI females saw a 31% increase (p = 0.07) (p = 0.02) and LMSDI females a non-significant increase, while male subgroups saw non-significant decreases . The global male rate increased 12% to 5.78 deaths/100,000 (UI: 4.19–7.18), while the female rate increased 60% (p = 0.06) to 2.08 deaths/100,000 (UI: 1.49–2.71) (p = 0.11), with similar decreases in the male and female rates during this period (p = 0.01) and 133% (p < 0.01) for MSDI populations (p = 0.01), 263% in LMSDI (p < 0.01), 178% in MSDI (p < 0.01), and 81% in HMSDI (p = 0.02). LMSDI and MSDI males additionally saw a significant increase of 157% (p = 0.01) and 119% (p = 0.01), respectively, while LSDI and LMSDI males also saw non-significant increases  to 3.78 deaths/100,00 (UI: 2.79–4.86) between 1990–2019 (49–2.71) a–c. HSDIp = 0.01) from 2.54 deaths/100,000 (UI: 1.69–3.54) to 0.97 deaths/100,000 (UI: 0.55–1.93). The decrease in males was larger than that in females, with significant reductions of 66% (p = 0.01) in the former and a 54% reduction (p = 0.03) in the latter  and, more recently, China (HMSDI) do offer some indication that implementing regulations aimed at improving APMP levels can reduce related TBL mortality . HMSDI fHAP trends offered the most universally positive outlook among the three exposures studied. HAP-attributable TBL mortality reductions across almost every global subpopulation can be taken as a sign that international efforts to reduce the burden of disease associated with these exposures, such as indoor stove interventions ,23, haveOur findings further characterize the complex, dynamic, exposure-related trends in TBL cancer mortality from 1990–2019. Our analysis indicates that the most urgent areas for tobacco-related intervention are both-sex MSDI populations and female LSDI, LMSDI, and HMSDI populations suffering from increasing tobacco-attributable TBL burden. Successful efforts that have brought about global reductions in HAP-associated TBL burden should continue, with special attention to LSDI female populations, where death rate increases were seen. Finally, except for both-sex HSDI and younger male HMSDI populations, the TBL cancer burden attributed to APMP is increasing and represents an urgent threat to the health of many global populations. With these targeted insights, global leaders, policy makers, and health care professionals will be better able to address the contribution of TBL cancer to the global burden of disease.