Neritimorpha, six Caenogastropoda, and 28 Heterobranchia). Illustrations and brief taxonomic notes/remarks are provided for every species. We also described Georrisacarinata Sutcharit & Jirapatrasilp, sp. nov. based on some distinct shell morphological characters. Since the first descriptions during the colonial period in the nineteenth century, some land snail species  have not been reported subsequently. This probably reflects a lack of knowledge concerning land snail biodiversity in this country. To our knowledge, this is the first comprehensive survey of land snails in southern Cambodia. A need for more field research and systematic revision of the land snails in this interesting region is also highlighted and demonstrated.Prior to this study, few collections and records were made of the land snails in Cambodia and the historical taxa had never been reviewed. Herein a report on the land snail diversity based on specimens collected recently from karstic and non-karstic areas in southern Cambodia is provided. This checklist presents 36 species of land snails (two  Cambodia forms a part of the Indo-Chinese sub-region of the Indo-Burma biodiversity hotspot . Its terHemiptera  and The Natural History Museum . The placement of each genus within higher order classification followsIFReDI) of the Fisheries Administration, Phnom Penh; Chulalongkorn University Museum of Zoology (CUMZ), Bangkok; Zoological Reference Collection of the Lee Kong Chian Natural History Museum, National University of Singapore (ZRC); The Natural History Museum, London (NHMUK).All the specimens were identified to genus or species level based on shell characteristics by referring to the historical literature including original descriptions, recent catalogues of land snails from Laos by Field surveys were conducted in karstic areas in southwestern Cambodia, Kampot Province. In addition, caves and cave-like chambers which provide appropriate microhabitats for karst-dwelling snails were also surveyed. This area has a monsoonal climate with wet season (May to November) and dry season (December to April). The karst landscape in Kampot is a small, isolated hill rising precipitously from the flat lowlands Fig. . The forLowland habitats of the eastern areas of Kirirom National Park are a conglomerate of hills and a plateau reaching 900 m in elevation, straddling the Kampong Speu and Koh Kong Provinces. The bulk of the plateau is covered with a mosaic of grassland and a reticulated network of pine forest plantations Fig. . The sloThe Preah Monivong Bokor National Park, Kampot Province is in the southeastern portion of the Cardamom Mountain Ranges within a range known as the Elephant Mountains. The plateau reaches an elevation of 1,100 m, and the floral composition of this range is greatly affected by continuous, monsoonal winds arising from the Gulf of Thailand. The climate promotes a mixture of grassland and tropical moist forest that shrouds the upper elevations of the Bokor Plateau in thick fog for much of the year , the conNeritimorpha . The distribution data given under each species was retrieved from the past records.A total of 180 voucher specimen lots was collected over the survey. The total of 36 species . The aperture is round to slightly ovate, with a closed umbilicus. Although the specimens from Perak have denser spiral striation , withoutG.decora Möllendorff, 1900 and G.chrysacme Möllendorff, 1900 both of which were described from “Touranne” , by having a conic shell with ca. ten strong spiral ribs on the last whorl. However, G.decora has an ovate conic shape with fine radial ribs on the last whorl, and G.chrysacme has an elongate conic shape with a deep and narrow suture. In addition, the shell shape of G.monterosatiana approaches the shape of G.insulaeThis species differs from Taxon classificationAnimaliaCycloneritidaHydrocenidaeSutcharit & Jirapatrasilpsp. nov.5B5771A6-B6EC-59E9-BA7E-E23BB4736AE5http://zoobank.org/CA891381-B719-4A88-B4A9-B0E36BAB121ECUMZ-CM094/1  from locality no. 11; CUMZ-CM042 (21 shells), IFReDI (10 shells), ZRC (10 shells) and NHMUK (10 shells) from locality no. 9.Holotype 4/1 Fig.  from loc10°34'1.77"N, 104°28'6.13"E).Phnom Kampong Trach Cave Temple, Kampong Trach District, Kampot Province, Cambodia, Locality no. 11 (CUMZ-CM086 (23 shells). Locality no. 17: CUMZ-CM102 (18 shells).Locality no. 12: Shell minute , conic, solid, translucent, yellowish to pale orange with darker colour on apex. Whorls 4¼, last whorl large ca. two-thirds of shell height. Protoconch ca. one whorl; sculpture nearly smooth and discontinuous spiral appearing immediately after protoconch. Following whorls slightly keeled, sculptured with thin and uneven growth lines; upper periphery with irregular and strong sculpture; below periphery with discontinuous spiral ribs. Sutures angular and impressed. Aperture round to slightly ovate. Umbilicus closed. Operculum unknown.carinata represents its keeled whorls of this new species.The Latin specific name Hypselostoma spp.This new species is found from Kampot and Kep Provinces. The snails were found to live on limestone wall syntopically with other G.bocourti  described from “Eaux douces de Preck-Scholl. Haut Mékong” , by having a conic shell with 4¼ whorls, which are slightly keeled and sculptured with thin and uneven growth lines without conspicuous spiral ribs. However, G.bocourti has a turriform shell with 6–7 whorls and sculptured with conspicuous spiral ribs (Rochebrune 1881a). Georissacarinata sp. nov. differs from G.poirieri Mabille, 1887 and G.conspicua Mabille, 1887 described from “Tonkin” [Vietnam] in that the latter two species are larger  and has a turriform shell. In addition, G.poirieri has very thin, tight, wavy spiral ribs, while G.conspicua has uneven spiral ribs with additional protuberances unequally arranged along the longitudinal rows 262B8A2B-3FD7-5E65-B8DF-40373B15A5F2Cyclostoma (Cyclophorus) amoenum Pfeiffer, 1854[1852]: 62. Type locality: unknown.Cyclophorusamoenus : CUMZ-CM053 (21 shells), CUMZ-CM054 . Locality no. 11: CUMZ-CM071 (2 shells). The empty shells were collected from the ground among leaf litter.Locality no. 10: Cambodia and Thailand . The disCyclophorus is the genus encompassing highly variable shell morphology of both inter- and intraspecific entities. The demarcation among different species is poorly understood. Thus, both intensive and thorough revision and redescription require more effective taxonomic characters, e.g., morphometric analysis of large series of specimens and perhaps molecular phylogeny to clarify the exact species boundaries  [The mountains of Dey-Crahom (red earth)], sur la rive droite du grand fleuve [on the right bank of the great river (Mekong River)]. Cyclophorus (Eucyclophorus) paviei : CUMZ-CM119 (1 shell). Locality no. 6: CUMZ-CM176 (1 shell). Locality no. 9: CUMZ-CM036 (2 shells), CUMZ-C037 . Locality no. 12: CUMZ-CM096 (1 shell). Locality no. 16: CUMZ-CM168 (2 shells), CUMZ-CM169 (1 specimen in ethanol), CUMZ-CM170 (1 specimen in ethanol). The snails were found to live on the ground among leaf litter.Locality no. 13: Cambodia . The disCyclophoruspaviei was described from “Les montagnes de Dey-Crahom”, from Cambodia. It differs from C.cambodgensis Morlet, 1885, which was described from the same area in having a smaller (shell width 32 mm) conical shell, with a white-yellowish apertural lip, while C.cambodgensis has a larger (shell width 42 mm) and turbinate conic shell, with an orange to reddish apertural lip.Taxon classificationAnimaliaArchitaenioglossaCyclophoridaeB24D7F7F-272C-5CAE-BE1A-4ADB522E60F7Rhiostomabernardii Pfeiffer, 1862: 45, 46, pl. 6, fig. 5. Type locality: Siam [Thailand]. Cyclotusbernardii : Opisthoporusbernardii : CUMZ-CM043 (8 shells). Locality no. 13: CUMZ-CM127 (2 shells), CUMZ-CM118 (2 shells). Locality no. 10: CUMZ-CM062 . The snails were found to live on the ground among leaf litter.Locality no. 9: Cambodia, Laos and Thailand .Opisthoporusbernardii was originally described from “Siam” [Thailand], and it has been reported from Cambodia A8BA2B42-822F-538E-889B-28DC9E7AA101Cyclophorusklobukowskii Morlet, 1885[1884]: 391, 392, pl. 12, fig. 1. Type locality: Near the Kamchay rapids, around the Kébal-Réméas cave (Kampot-Hatien road); commonly found on mountains, in forests, up to Compong-Som, and on the banks of Tap-Chéang. Lagocheilusklobukowskii : CUMZ-CM044 (7 shells), CUMZ-CM045 (12 specimens in ethanol). Locality no. 10: CUMZ-CM068 (3 specimens in ethanol). Locality no. 11: CUMZ-CM079 (2 shells). Locality no. 13: CUMZ-CM128 (3 shells), CM129  …”. We collected topotypic specimens that tend to have a variable shell colour from yellowish Fig. . This liLagocheilusklobukowskii was originally placed in the genus Cyclophorus and later was transferred to the genus Lagocheilus , and a thick calcareous, multispiral and plate-like operculum, whereas Cyclophorus has a turbinate shell, a thick and expanded lip, and a corneous multispiral operculum.ilus see . The disTaxon classificationAnimaliaArchitaenioglossaCyclophoridae614B5E69-1CA9-527D-9220-C0A030BE666ECyclophoruslandesi Morlet, 1885[1884]: 392, 393, pl. 11, fig. 5, 5a. Type locality: extrémité de la chaîne de ľÉléphant, non loin de la mer .Cyclophoruslaudesi [sic]: Lagocheiluslandesi : CUMZ-CM080 . Locality no. 12: CUMZ-CM104 (4 shells). Locality no. 14: CUMZ-CM152 , and the thick calcareous, multi-spiral and plate-like operculum characteristic of Pupina Vignard, 1829Taxon classificationAnimaliaArchitaenioglossaPupinidaeMorlet, 1883283DD709-264C-5D8D-A991-BD4F39799F14Pupinacrosseana Morlet, 1883: 108, 109, pl. 4, fig. 5. Type locality: Cambodge [Cambodia]. CUMZ-CM029 (1 shell). Locality no. 9: CUMZ-CM039 (10 shells). Locality no. 10: CUMZ-CM066 (1 shell), CUMZ-CM067 . Locality no. 13: CUMZ-CM133 (1 specimen in ethanol). Locality no. 17: CUMZ-CM142 (1 specimen in ethanol). The snails were found to live on the ground among leaf litter.Locality no. 7: Cambodia .This species was originally described from “Cambodge” [Cambodia]. The diagnostic characters of this porcelain shell are a pupoid shell with varying shell colour from brownish to whitish, having a large, ovate last whorl ca. two-thirds of shell height. The shell has a thickened parietal callus, with a small posterior plica that is located some distance from an angular corner of aperture, which possesses a wide posterior canal. The anterior canal is a narrowly transverse slit overhung by a square and thickened columella plica. The aperture is circular with a white, thickened and slightly expanded lip.Valiguna Grimpe & Hoffmann, 1925Taxon classificationAnimaliaSystellommatophoraVeronicellidae59FA60F6-E456-561B-8B22-19E795F8A8D9Vaginulussiamensis Martens, 1867: 68, pl. 5, fig. 3. Type locality: Petshaburi .Valigunasiamensis : CUMZ-CM116  is thickened, with dark colour and scattered with brownish spots, and without median stripe. The ventral side (hyponotum) is with much lighter, pale creamy colouration, with tiny greyish spots distributed across hyponotum, and a narrow foot sole located in the middle. The foot sole is as long as and slightly narrower than the hyponotum, with pale yellowish brown colour. This slug is different from Succinea Draparnaud, 1801Taxon classificationAnimaliaStylommatophoraSuccineidaeMorelet, 1865EA06A16E-E337-58B7-B934-EF322D27ECB2Succineatenuis Morelet, 1865: 225, 226. Type locality: Cochinchina [South Vietnam]. Succineatenella Morelet, 1875: 244, pl. 12, fig. 5 [unjustified emendation].CUMZ-CM106 (2 shells), CUMZ-CM107 . Locality no. 9: CUMZ-CM087 .Hypselostomacambodjense tends to be abundant and widely distributed in several karstic hills in southern Cambodia and Vietnam B2EDA1FE-87E6-5097-B863-9DCB9ACC6E2FBulimusgracilis (?) Hutton, 1834: 84, 85, 93. Type locality: Mirzapoor; Futtehpoor Sikra; between Agra and Neemuch . (?) Allopeasgracilis [sic]: CUMZ-CM105 . The snails were found to live on the ground among leaf litter.Locality no. 12: Pantropical and subtropical .A.gracile in Cambodia. This species could be found in both natural and transformed anthropogenic habitats. This widespread and pantropical species has been introduced into many countries, including in greenhouses in temperate regions, and occurs throughout Laos, Thailand and Vietnam 3E6BACC4-C635-59E3-824E-AE4521A0DFD0Achatinafulica Bowdich, 1822: pl. 13, fig. 3. Type locality: unknown.Lissachatinafulica : CUMZ-CM065 . The snails were found to live on tree trunks and on the ground among leaf litter.Locality no. 10: Pantropical and subtropical .Angiostrongyluscantonensis . Locality no. 11: CUMZ-CM074 , CUMZ-CM076 , CUMZ-CM099 (1 shell), CUMZ-CM100 (9 specimens in ethanol). Locality no. 13: CUMZ-CM121 (21 shells), CUMZ-CM122 (1 specimen in ethanol). The snails were found to live on the ground among leaf litter in the limestone area.Locality no. 6: H.michaui , but the latter is more ovate and less oblique in shell shape. In addition, this species can be distinguished from H.pellucens , H.porrectus  and H.perlissus Vermeulen et al., 2019 by having strong and prominent radial ridges. For comparison, the latter three species have a smooth to nearly smooth shell surface, H.pellucens has an oblique-ovate shell shape, H.porrectus and H.perlissus have an oblique heliciform shell shape , CUMZ-CM156 (4 shells), CUMZ-CM157 . The snails were found to live on tree trunks and leaves.Locality no. 15: Bertiacambojiensis  by having a brownish shell, with spirally undulated surfaces, while B.cambojiensis has a smooth surface  and D.retrorsa  by having a dark brown shell, with wide angle of peripheral keels. In contrast, D.retrorsa tends to have sharp peripheral keel, D.salangana has round periphery and usually with brownish peripheral band, and both species are pale brownish in shell colour  located on the plateau of Preah Monivong Bokor National Park may be young individuals, as their shell size is relatively small compared to those of other congeners recorded from peninsular Thailand. It differs from l colour .Taxon classificationAnimaliaStylommatophoraDyakiidae9963D788-2D1B-5D3C-883F-9E6D421B1761Helixweinkauffiana Crosse & Fischer, 1863: 350, 351. Type locality: Cochinchine [Southern Vietnam].Quantulaweinkauffiana : CUMZ-CM002 (8 shells). Locality no. 2: CUMZ-CM006 (10 shells). Locality no. 5: CUMZ-CM011 (2 shells). Locality no. 7: CUMZ-CM013 (82 shells), CUMZ-CM014 (1 shell), CUMZ-CM015 , CUMZ-CM035 (3 shells). Locality no. 10 CUMZ-CM052 (1 shell). Locality no. 12: CUMZ-CM093 (5 shells). Locality no. 13: CUMZ-CM120 (5 shells). Locality no. 17: CUMZ-CM135 (3 shells). Locality no. 18: CUMZ-CM143 (3 shells), CUMZ-CM144 (1 shell). Locality no. 16: CUMZ-CM166 (4 shells), CUMZ-CM177 (1 shell). Locality no. 6: CUMZ-CM174 (9 shells), CUMZ-CM175 EDC62306-0411-5BDB-93D2-C99EEEB8A0C5Helixpaviei Morlet, 1885[1884]: 386, 387, pl. 11, fig. 1, 1a. Type locality: dans les forêts, entre Kampot et Phnom-Penh, particulièrement près des rapides de Kamchay (rivière de Kampot), sur les bois pourris et les petite plantes .Trochomorphapaviei : CUMZ-CM153 (3 specimens in ethanol). Locality no. 15: CMZ-CM162 (2 shells), CUMZ-CM163 . The snails were found to live on tree trunks and on the ground among leaf litter.Locality no. 14: Cambodia, Laos and Vietnam .T.paviei closely resembles T.saigonensis  that was described from “Poulo-Condor and Saigon, Cochinchine”. The latter species is slightly smaller (shell width 11 mm), having the last whorl with a wide angled peripheral keel and being slightly convex below the periphery. The type specimens of both species were recently figured in This species was originally described from “Dans les forêts, entre Kampot et Phnom-Penh”. The unique characters are a depressed conic shell (shell width 12 mm) with a very strong and sharp peripheral keel, and a widely opened and deep umbilicus. The shell surface has thin and regular radial ridges, and very thin spiral ridges. Based on shell morphology, Taxon classificationAnimaliaStylommatophoraTrochomorphidaesp.4907C676-3C20-5516-A416-2FE97F57D66CCUMZ-CM057 , CUMZ-CM059 (1 specimen in ethanol). Locality no. 13: CUMZ-CM134 (3 shells). The snail was found to live on a tree trunk.Locality no. 10: T.paviei and T.saigonensis in having a larger shell (shell width 14 mm), an elevated domed spire, more whorls, and being nearly flat below the periphery. However, the identification is provisional, and further evidence from examination of genitalia or DNA will be necessary to elucidate their status.The specimens from Prasat Phnom Totong  have a conic shell with a very strong and sharp peripheral keel, widely opened and deep umbilicus, and slightly convex below the periphery. The shell surface has irregular growth lines and very thin spiral ridges. These specimens tend to differ from Cryptozona Mörch, 1872Taxon classificationAnimaliaStylommatophoraAriophantidaeC459181B-410C-5886-80E0-0BA6909196F3Helixsiamensis Pfeiffer, 1856: 32. Type locality: Siam [Thailand].Hemiplectadichromatica Morlet, 1889: 124, 175, 176, pl. 6, fig. 2. Type locality: de Srakéo à Ang-Son (Siam) .Cryptozonasiamensis : CUMZ-CM147 . The snails were found to live on the ground among leaf litter.Locality no. 4: Cambodia, Laos, Malaysia, Singapore and Thailand .C.siamensis is found throughout the country and the allozyme analysis by C.siamensis probably occupies almost all habitat types through accidental introduction or horticultural trade activities, and this species is especially abundant in human-modified landscapes.This widespread species has recently been recorded from Singapore and Peninsular Malaysia , Laos I and YunnHemiplectadichromatica Morlet, 1889” which was subsequently considered to be conspecific with this species , in which its habitats are associated with human activities. In contrast, Q.weinkauffiana could be found commonly in both natural forest and anthropogenic habitats.The historical record of this species from Cambodia was probably under the name “ species . In thisTaxon classificationAnimaliaStylommatophoraAriophantidae4A498BFE-3341-5F92-8207-B3B3E08EF2DAHelixdistincta Pfeiffer, 1850: 69, 70. Type locality: insulis Moluccis [Molucca Islands].Hemiplectadistincta : CUMZ-CM021 (6 shells), CUMZ-CM022 (1 specimen in ethanol), CUMZ-CM023 (1 specimen in ethanol). Locality no. 11: CUMZ-CM069 (2 shells), CUMZ-CM070 . Locality no. 3: CUMZ-CM123 (1 specimen in ethanol). Locality no. 17: CUMZ-CM136 (4 shells). Locality no. 18: CUMZ-CM145 (1 shell). Locality no. 14: CUMZ-CM149 (1 shell). Locality no. 16: CUMZ-CM171 . Locality no. 6: CUMZ-CM180 (3 specimens in ethanol). The small juveniles were found on tree trunks and leaves, while the adults were found to live on the ground among leaf litter.Locality no. 7: Cambodia, Laos, Thailand and Vietnam .Hemiplectadistincta has a wide distribution from Southern Vietnam, throughout Cambodia, northeastern Thailand, and throughout Laos , CUMZ-CM033 (3 shells). Locality no. 10: CUMZ-CM050 (24 shells), CUMZ-CM051  by having a reddish-brown shell with a wide whitish or creamy area surrounding the umbilicus. Sarikabocourti, which is described from “Battambang, Cambodje”, has a uniform brownish shell . Locality no. 13: CUMZ-CM117 . These specimens differ from M.psyche in having a nearly flattened to slightly elevated spire, with a slightly shouldered last whorl and milky shell colour, while M.psyche has a slightly sunken spire, with a well-rounded last whorl and whitish shell colour , CUMZ-CM092  have a small shell (diameter ca. 10 mm), which is depressed, slightly thick, translucent, shiny, and pale reddish-brown. The shell surface is smooth with obvious irregular growth lines. The shell has 5 to 6 whorls, with wide and shallow suture. The spire is convex, with an elevated apex. The last whorl has a well-rounded periphery, with an ovate-lunate aperture and a simple lip. An umbilicus is widely open and deep.Macrochlamyspsyche, Sarika sp. 1 and sp. 2 by having a small size and slightly elevated spire. In contrast, M.psyche and Sarika sp. 1 have a large, whitish shell and a flatten to slightly shrunken spire, while Sarika sp. 2 has a larger, reddish-brown shell with whitish area surrounding the umbilicus. Live specimens are required so that the anatomical characters can be used to discriminate among the species.These specimens can be distinguished from Cambodiparmarion Kuznetsov & Kuzminykh, 1999Taxon classificationAnimaliaStylommatophoraAriophantidaeKuznetsov & Kuzminykh, 19997F1D24E2-D479-5F7E-96A0-A98EDACC1FA6Cambodiparmariondoroshenkoi Kuznetsov & Kuzminykh, 1999: 113–116, figs 1, 2. Type locality: In tropical forest between Motel Lomherkay and Hotel Koh Pos, SW end of Kompong Som [= Sihanoukville], Kompong Som district, Kampot province, Cambodia.CUMZ-CM108 (4 shells). Locality no. 13: CUMZ-CM130 (2 shells), CUMZ-CM131 . The semi-slug was found to live on tree trunks and leaves in the limestone area.Locality no. 12: Known only from the type locality .Microparmarion Simroth,C.doroshenkoi was described, the authors did not mention the characters used to discriminate this species from Parmarionmartensi Simroth,C.doroshenkoi has a solid, ear-shape shell with ca. 2 whorls, a blackish body and mantle, and a long flagellum, while P.martensi has a thin nail-shape shell with a trace of shell coiling, a greyish to blackish body and a short flagellum , CUMZ-CM151 . The semi-slugs were found to live on tree trunks and leaves.Locality no. 14: Cambodia, Laos, Malaysia and Singapore .Parmarionmartensi has also been reported as an introduced species to Samoa and Hawaii , in which the shell is usually entirely covered with movable mantle lobes. d Hawaii .Taxon classificationAnimaliaStylommatophoraHelicarionidaeD96F0748-53E1-5B2B-845F-79B4D0954B75Vitrinarusseola Morelet, 1865: 225. Type locality: Cochinchina. Megausteniarusseola : CUMZ-CM030  was figured in Durgella. Further additional anatomical examination is necessary since the shell morphology is insufficient for species identification.A syntype of Taxon classificationAnimaliaStylommatophoraAriophantidaesp.456EF9A1-F892-5A09-BB2F-C813B4D198A7CUMZ-CM031 . It is distinguished from  lamella .Amphidromus Albers, 1850Taxon classificationAnimaliaStylommatophoraCamaenidae4392F96C-9762-5E88-9AAE-9603DB4DEBB0Bulimusleucoxanthus Martens, 1864: 526. Type locality: unknown.Amphidromus (Amphidromus) atricallosusleucoxanthus : CUMZ-CM018 41CDD142-CBE9-5E02-A065-AB263656D157Bulimus (Amphidromus) semitessellatus Morlet, 1885[1884]: 387, 388, pl. 11, fig. 2, 2a. Type locality: les montagnes qui bordent le grand fleuve au delà de Stung-Treng. Les forêts et les montagnes de Kampot à Compong-Som .Amphidromus (Syndromus) semitessellatus : Amphidromussemitessellatus : CUMZ-CM040 (2 shells). Locality no. 10: CUMZ-CM055 (1 shell). Locality no. 12: CUMZ-CM101 . The empty shells were collected from the ground, and the living snails probably live on tree trunks and leaves.Locality no. 9: Cambodia, Laos, Thailand and probably in Vietnam , 2019.A.semitessellatus due to the similarity of both brown supra-peripheral and sub-peripheral bands on the penultimate whorls and the geographical proximity. The subgenus Syndromus typically has a small shell which exhibits high variation on shell size, colour, and pattern F49A8378-19A8-5EED-A5E4-A1D43D30DF08Helixnorodomiana Morlet, 1883: 106, 107, pl. 4, fig. 3, 3a, b. Type locality: Khamchay [Cambodia].Chloritisnorodomiana : CUMZ-CM024 (2 shells). Locality no. 9: CUMZ-CM041 (35 shells). Locality no. 10: CUMZ-CM056 (1 shell). Locality no. 11: CUMZ-CM077 . Locality no. 12: CUMZ-CM103 (1 shell). Locality no. 13: CUMZ-CM125 (4 shells), CUMZ-CM126 (1 specimen in ethanol). Locality no. 16: CUMZ-CM173 CC0F820D-5140-5F03-8B7B-20ADBBD3E1F4Helix (Geotrochus) perakensis Crosse, 1879: 199, 200, pl. 8, fig. 4. Type locality: Perak .Ganesellaperakensis : CUMZ-CM159 , CUMZ-CM161  species complex which is composed of 11 nominal species and widely distributed from Western Ghats of India to Indochina and the Greater Sunda Islands  from “Tonquin” and G.vatheleti  from “Van Bu, Tonkin” by having a strong peripheral keel. For comparison, G.subperakensis is convex below periphery, with less strong peripheral keel and without brownish spiral band , which was described from Ha-Giang (Northern Vietnam) and Siam [Thailand]. The description itself was based mainly on the Ha-Giang specimen  in having a smaller shell (shell height up to 13 mm), shallow suture with 6 to 7 convex whorls, while G.lantenoisi performs an elongate trochoid, larger shell (shell height up to 18 mm), suture flattened and smooth 9 to 10 whorls. However, further investigations with both genitalia and DNA analysis will be necessary to elucidate the exact relationship between them.This species is very similar to Taxon classificationAnimaliaStylommatophoraCamaenidaeS. Tumpeesuwan & C. Tumpeesuwan, 20209E87B72C-4CCB-5F6D-86FE-0B4A4924EFC2Ganesella (Giardia)Mollusca: Eupulmonata: Camaenidae). Preoccupied by Künstler, 1882: (Metamonada: Diplomonadida: Hexamitidae). Ancey, 1907: 195, 203 (Pseudobuliminus (Giardia) : Pseudobuliminus (Girardius) Richardson, 1983: 94. [incorrect subsequent spelling]Giardia : Anceyoconcha Tumpeesuwan & Tumpeesuwan in The distinguished shell character of this genus is sinistral, elongate cylindrical to more or less conical, with 6–10 convex whorls. The last whorl is rounded (not keeled), with the aperture ovate to slightly trapezoid and the apertural lip expanded. The columella is vertical, with the umbilicus narrowly opened.Giardia as the subgenus of Ganesella Blanford, 1863 to include two Indochinese sinistral species: Bulimussiamensis Redfield, 1853 and Bulimusrhombostomus Pfeiffer, 1861. Subsequently, this nominal name was used as a subgeneric level of Buliminopsis Heude, 1890 (family Fruticicolidae) by Bradybaenidae as the subgenus of Pseudobuliminus Gredler, 1886, and also designated Bulimussiamensis Redfield, 1853 as the type species. Zilch’s classification was subsequently accepted and used by later authors . authors . Recentlmaenidae . HoweverBradybaenidae, Girardius”, accompanied by diagnostic characters and attributed Bulimussiamensis Redfield, 1853 as the type species. However, this name is considered incorrect subsequent spelling  comb. nov., A.maestratii  comb. nov., A.mantongensis  comb. nov., A.obesa  comb. nov., A.ovoideus  comb. nov., A.pharangensis  comb. nov., A.rhombostomapupoidea  comb. nov., A.rhombostomarhombostoma, A.siamensismaxima  comb. nov., A.siamensisnobilis  comb. nov., A.siamensisobesula  comb. nov., A.siamensispervariabilis  comb. nov., A.siamensissiamensis, A.siamensiszonifera  comb. nov. and A.vignei  comb. nov.Anceyoconcha is probably within the Indochinese region of Cambodia, Laos, Thailand, and Vietnam 773E6D49-5BCA-5F83-8110-6C8F386BBA31Bulimusrhombostomus Pfeiffer, 1861: 194, 195. Type locality: Camboja [Cambodia].Ganesellarhombostoma : Anceyoconcharhombostoma : CUMZ-CM047 (66 shells), CUMZ-CM048 . Locality no. 11: CUMZ-CM085 . Locality no. 13: CUMZ-CM132 (3 shells). Locality no. 17: CUMZ-CM139 (5 shells), CUMZ-CM140 ADF2BB29-F75B-52E1-8FCE-0F006E8D1E31Buliminussiamensis var. obesulaAncey, 1888: 352. Type locality: Saigon, dans le jardin du gouverneur. CUMZ-CM027 (14 shells), CUMZ-CM028 , ovate shell and lower number of whorls. For comparison, A.siamensissiamensis 345FACC2-57F6-5EBD-A592-13F4FB14F3B7Petraeuschaudoensis Rochebrune, 1881a: 70. Type locality: Montagnes de Chaudoe Cambodge .Enachaudocensis [sic]: CUMZ-CM003 . Locality no. 3: CUMZ-CM010 (1 specimen in ethanol). The snails were found to live on tree trunks and leaves.Locality no. 1: Cambodia .This species was originally described from “Montagnes de Chaudoe Cambodge”  probably in the area bordering Cambodia and Vietnam. The original description of this species was brief and without illustration. This species has a sinistral elongate conic to slightly ovate conic shell, with 7 to 9 whorls, which increase regularly; cylindrical, having convex whorl and wide and impressed suture. The shell surface possesses fine growth lines, and the periostracum is thin and brownish. The last whorl is well rounded and without keel near aperture and has a similar diameter to the penultimate whorl. The shell colour is light brownish and translucent (becoming whitish when worn). The aperture is semi-ovate, with expanded and whitish lip and thin or thickened with whitish parietal callus. The columella is straight and dilated, with a rimate umbilicus.A.chaudoensis can be distinguished from A.siamensisobesula in having an elongate cylindrical shell and higher number of whorls, while the latter species has an ovate conic shell and a smaller number of whorls.Based on the original description, Alycaeidae, Clausiliidae, and Plectopylidae. It is possible that the geography of the area without high mountains and other structured habitat types result in comparatively fewer species. In comparison, most of the species Sesarapolita that does not exceed 12 mm in width. However, most land snails collected in our study are large (more than 10 mm) and cover most taxonomic groups, with the exception of the families Assimineidae and Diplommatinidae, both of which have been reported by Cambodia has received the least attention from malacologists for inventorying the land snail fauna, compared to other adjacent countries within the Indo-Chinese region, e.g. Thailand , Laos I and Viet


Methanobrevibacter smithii and Methanobrevibacter oralis DNA sequences, were detected in 21/527 (3.9%) sputum samples, 2/188 (1.06%) bronchoalveolar lavages, and none of 193 tracheo-bronchial aspirations. Further, fluorescence in situ hybridization detected methanogens in three sputum investigated specimens with stick morphology suggesting M. oralis and in another one bronchoalveolar lavage sample investigated, diplococal morphology suggesting M. smithii. These observations extend the known territory of methanogens to the respiratory tract and lay the foundations for further interpretation of their detection as pathogens in any future cases of isolation from bronchoalveolar lavages and the lungs.Methanogens, the sole microbes producing methane, are archaea commonly found in human anaerobic microbiota. Methanogens are emerging as opportunistic pathogens associated with dysbiosis and are also detected and cultured in anaerobic abscesses. Their presence in the respiratory tract is yet unknown. As a preliminary answer, prospective investigation of 908 respiratory tract samples using polyphasic approach combining PCR-sequencing, real-time PCR, fluorescent in situ hybridization (FISH), and methanogens culture was carried out.  Methanobrevibacter oralis, Methanosarcina mazei, Methanobacterium congolense, Methanobrevibacter smithii, and Methanobrevibacter massiliense) [M. smithii [M. smithii, M. oralis, Methanosphaera stadtmanae, Methanomassilicoccus luminyensis, and Methanobrevibacter arboriphilicus) [M. smithii and M. oralis have been associated with dysbiosis affecting the oral cavity, the gut [Methanogenic archaea (referred to in this study as methanogens), the sole organisms known to produce methane, have been characterized in the oral cavity microbiota (iliense) ,2,3, in  smithii , in digehilicus) ,6 and rehilicus)   and blast programme of NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi) were used for sequence analyses. A total of 5μL of distilled water were used as a negative control for sputum samples and bronchial aspirates, and 5μL of 0.9% NaCl saline for bronchoalveolar lavage.All samples were examined by standard polymerase chain reaction (PCR) assays targeting the 16S rRNA gene in an automated 5A PTC-200 thermal cycler . The 16S gene was amplified using broad-range rRNA primers SDArch0333aS15 (5′-TCCAGGCCCTACGGG-3′) and SDArch0958aA19 (5′-YCCGGCGTTGTTGAMTCCAATT-3′)  . The PCRM. oralis cnp602F primer 5-GCTGGTGTAATCGAACCTAAACG-3 (Eurogentec); cnp602R primer 5-CACCCATACCCGGATCCATA-3 (Eurogentec) and the probe cnp602-FAM 5-AGCAGTGCACCTGCTGATATGGAAGG-3 (Eurogentec) [M. smithii ; Smit.16S-862R, 5-CTCCCAGGGTAGAGGTGAAA-3 (Eurogentec) and the probe Smit.16S FAM, 5-CCGTCAGAATCGTTCCAGTCAG-3 (Eurogentec) [The PCR products were sequenced using the same primers as used for PCRs following this program: a 1-min denaturation step at 96 °C, followed by 25 cycles of denaturation of 10 s at 96 °C, a 20-s annealing at 50 °C and a 4-min extension at 60 °C. The MultiScreen 96-well plates Millipore  containing 5% of Sephadex G-50  were used to purify the sequencing products. The sequences were analyzed and edited following the protocol described previously . Furtherogentec) ) speciesBacteroides thetaiotaomicron (104 cells/mL) for hydrogen production [B. thetaiotaomicron in SAB broth were incubated at 37 °C for 7 days. Subculture was seeded on a Petri dish containing SAB medium supplemented with 15 g/L agar (Sigma-Aldrich) and deposited in the upper chamber of a double-chamber box. Methanogen colonies appeared after 9–12 days of incubation [A volume of 250 μL of each sample diluted in PBS was seeded under anaerobic chamber in a sterile Hungate tube  ,21. Eachoduction  and the cubation .Only PCR positive samples were analyzed by FISH using the method previously described ,4. In brWe used a negative methanogen PCR sputum sample and a negative methanogen bronchoalveolar lavage sample as negative controls.M. oralis-positive samples exhibiting 99% sequence similarity with M. oralis strain ZR (GenBank: NR_104878.1) and two M. smithii-positive samples exhibiting 99% sequence similarity with M. smithii ATCC 35,061 (GenBank NR: 074235.1); in 2/188 (1.06%) of bronchoalveolar lavage samples  and one M. smithii-positive sample exhibiting 99.97% similarity with M. smithii strain C2 CSUR P5816 (GenBank: LR590664.1). None of the bronchial aspirate samples were PCR-positive.In the presence of negative control which remained negative, PCR-sequencing disclosed methanogens in 21/527 (3.98%) of the sputum samples including 19 M. smithii 16S rRNA gene yielded a median Ct of 35.7 ± 0.47, indicating a M. smithii load of 1.32 × 104 ± 0.56 × 104/mL in the case of two sputum positive samples and Ct of 36.2 indicative of an M. smithii load of 0.8 × 104/mL in case of one bronchoalveolar lavage positive sample.In the presence of negative control which remained negative, all the positive samples by PCR-sequencing were positive by real-time PCR. Real-time PCR analyses targeting the M. oralis cnp-60 gene yielded a median Ct of 33.2 ± 1.07 indicative of an M. oralis load of 2.61 × 105 ± 1.23 × 105/mL in the case of 19 sputum positive samples and Ct of 35.3 indicative of an M. oralis load of 1.41 × 104 in case of one bronchoalveolar lavage positive sample  in the saliva ,32. Inte


Beilschmiedia erythrophloia, B. robusta, B. yunnanensis, Cryptocarya concinna, C. impressa, C. infectoria, Litsea viridis, Machilus balansa, M. grandifolia, Neolitsea ellipsoidea, and Phoebe angustifolia) have been obtained by hydrodistillation and the chemical compositions analyzed by gas chromatography – mass spectrometry (GC-MS) and gas chromatography with flame ionization detection (GC-FID). The essential oils were screened for larvicidal activity against Aedes aegypti, Ae. albopictus, and Culex quinquefasciatus, and for antimicrobial activity against Enterococcus faecalis, Staphylococcus aureus, Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica, and Candida albicans. The leaf essential oil of N. ellipsoidea, rich in (E)-β-ocimene (87.6%), showed excellent larvicidal activity against Ae. aegypti with a 24 h LC50 of 6.59 μg/mL. The leaf essential oil of C. infectoria, dominated by germacrene D (55.5%) and bicyclogermacrene (11.4%), exhibited remarkable larvicidal activity against Cx. quinquefasciatus (48 h LC50 = 0.40 μg/mL). N. ellipsoidea leaf essential oil also demonstrated notable antibacterial activity against E. faecalis and B. cereus with minimum inhibitory concentration (MIC) values of 16 μg/mL, while the leaf essential oil of C. impressa showed excellent anticandidal with an MIC of 16 μg/mL. Leaf essential oils from the Lauraceae should be considered for utilization as alternative agents for controlling mosquito populations and as antimicrobial agents.The Lauraceae is a family rich in aromatic and medicinal plants. Likewise, essential oils derived from members of this family have demonstrated a myriad of biological activities. It is hypothesized that members of the Lauraceae from Vietnam will yield essential oils that may be useful in controlling mosquito populations and treating microbial infections. In this work, the leaf essential oils of eleven species of Lauraceae ( Persea americana Mill.) for its fruit, bay leaf (Laurus nobilis L.) used in cooking, and the spice cinnamon (Cinnamomum verum J. Presl) [Sassafras albidum (Nutt.) Nees) [Lindera benzoin (L.) Blume) [Aniba rosaeodora Ducke) [Cinnamomum camphora (L.) J. Presl.) [Litsea cubeba (Lour.) Pers.) [The Lauraceae is made up of around 55 genera and 3000 species of tropical and warm temperate trees and shrubs, with Southeast Asia and Brazil serving as species-rich hot spots . Several. Presl) . Several) Blume) . Many spa Ducke) , camphor Presl.) , and aro) Pers.) .Based on the utility and properties of Lauraceae essential oils, it is hypothesized that members of the Lauraceae found in Vietnam have biologically active essential oils that may be useful in controlling mosquito populations or as antimicrobial agents. Eleven species of Lauraceae from north-central Vietnam have been collected, the essential oils obtained by hydrodistillation, chemical compositions analyzed, and the oils screened for mosquito larvicidal activity and for antimicrobial activity.Beilschmiedia Nees is comprised of around 250 species of trees and shrubs [Beilschmiedia has been reviewed [The genus d shrubs  and are d shrubs . The phyreviewed .Beilschmiedia erythrophloia Hayata is a tree found in Taiwan, southern China, Hainan Island, and Ryukyu Islands (Japan) [B. erythrophloia have revealed endiandric acid derivatives from the roots [E)-caryophyllene and α-humulene [ (Japan) ,13. In V (Japan) . Previouhe roots ,16, the humulene .Beilschmiedia robusta C.K. Allen is a tree, 10–15 m tall that is recorded from Guangzi, southwestern Guizhou, Xizang, and Yunnan provinces in China [B. robusta.in China ,18. In Vin China . A perusBeilschmiedia yunnanensis H.H. Hu is a tree, up to 18 m tall and is found in Guangdong, southern Guangxi, and southern Yunnan provinces in China [B. yunnanensis.in China . In Vietin China . A literCryptocarya R. Br. is a pantropical genus of around 300 species [Cryptocarya concinna Hance  is a tree up to 18 m tall, and ranges from southern China  to northern Vietnam [C. concinna have shown the roots to contain cytotoxic cryptocaryone [C. concinna. species . Cryptoc Vietnam ,12. In V Vietnam . Previouocaryone , the leaocaryone , and theocaryone . There hCryptocarya impressa Miq. is native to Vietnam, Laos, the Malay Peninsula, Borneo and Sumatra [C. impressa. Sumatra . In Viet Sumatra . To our Cryptocarya infectoria (Blume) Miq. (syn. Cylicodaphne infectoria Blume) is a tree up to 33 m tall that is native to Indo-China and Malesia [C. infectoria [N-methylisococlaurine, and N-methyllaurotetanine have also been isolated from the bark of C. infectoria [ Malesia ,25,26. I Malesia . The cytfectoria ,28. The fectoria . There hLitsea Lam. consists of around 300 species distributed in tropical and warm subtropical regions of Asia, Australia, and the Americas [Litsea viridis H. Liu is a small tree, 3-6 m tall, found in south-eastern Yunnan province (China) and Cao Bằng, Nghệ An, Đà Nẵng, and Đắk Lắk provinces (Vietnam) [The genus Americas . Litsea Vietnam) ,14. TherMachilus Rumph. ex Nees is comprised of around 100 species distributed in southern and south-eastern Asia [Machilus balansae (Airy Shaw) F.N. Wei & S.C. Tang  is endemic to Vietnam and is generally found at low elevations in north Vietnam [Machilus grandifolia S.K. Lee & F.N. Wei is now regarded as a new synonym of M. balansae [M. balansae or M. grandifolia.The genus ern Asia ,14. Mach Vietnam . Machilubalansae . To our Neolitsea (Benth.) Merr. Contains around 85 species distributed from Indo-Malaysia to East Asia [Neolitsea ellipsoidea K.C. Allen is a tree up to 30 m in height [The genus ast Asia ,14. Neoln height . The spePhoebe Nees [There are around 100 species in the genus ebe Nees , which rebe Nees .Phoebe angustifolia Meisn. is a small shrub found in southeastern Yunnan (China), Myanmar, India, and Vietnam [P. angustifolia from Vietnam has been reported, which showed the major components to be spathulenol (17.0%), palmitic acid (13.0%), sabinene (6.4%), bicyclogermacrene (5.9%), and artemisia triene (5.1%) [ Vietnam . In Viet Vietnam . The leae (5.1%) .The essential oil collection details and yields are summarized in B. erythrophloia, B. robusta, and B. yunnanensis are compiled in Beilschmiedia leaf essential oils were dominated by sesquiterpene hydrocarbons. A preponderance of sesquiterpene hydrocarbons has been previously seen in Beilschmiedia leaf essential oils from Malaysia [The essential oil compositions of Malaysia  and fromMalaysia .B. erythrophloia essential oil were bicyclogermacrene (30.5%), (Z)-β-ocimene (26.1%), and (E)-caryophyllene (18.3%). While qualitatively similar, there are notable differences between the essential oil from Vietnam in this work and that reported by Su and Ho from Taiwan [The major components in m Taiwan ; the samB. robusta and B. yunnanensis leaf oils were rich in (E)-caryophyllene , α-humulene (13.4% and 9.9%), and bicyclogermacrene (8.6% and 8.4%). The leaf oil of B. robusta had a high concentration of germacrene D (20.3%), while B. yunnanensis oil was rich in 9-epi-(E)-caryophyllene (21.2%).Both C. concinna (from two locations), C. impressa, and C. infectoria are listed in C. impressa and C. infectoria leaf essential oils, while oxygenated sesquiterpenoids were abundant in C. concinna essential oil from Nam Dong and monoterpene hydrocarbons dominated the leaf oil of C. concinna from Pu Hoat.The leaf essential compositions of C. concinna from two different collection sites were qualitatively similar, but quantitatively different. That is, the abundant components in the Nam Dong sample were also observed in the Pu Hoat sample, and vice versa. Thus, for example, α-pinene, β-pinene, and myrcene were abundant in the Pu Hoat sample  but were found in lower concentrations in the sample from Nam Dong . Conversely, the sesquiterpenoids, (E)-caryophyllene, spathulenol, and caryophyllene oxide were abundant in the sample from Nam Dong , but less concentrated in the Pu Hoat sample .The leaf essential oils of C. impressa were bicyclogermacrene (18.7%), (E)-caryophyllene (10.8%), dodecanal (10.8%), -α-farnesene (7.9%), and α-humulene (6.3%). Germacrene D (55.5%) dominated the essential oil composition of C. infectoria, which was also composed of bicyclogermacrene (11.4%) and δ-elemene (5.1%) as major components. The major components of the leaf essential oil of L. viridis, M. balansae, M. grandifolia, N. ellisoidea, and P. angustifolia are compiled in The chemical compositions of the leaf essential oils of L. viridis leaf essential oil were bicyclogermacrene (25.5%), decanal (14.4%), α-pinene (11.1%), and β-pinene (8.3%). This is the first report on the essential oil from this plant.The major components in M. balansae and M. grandifolia are considered conspecific, the essential oil compositions showed pronounced differences. The leaf oil of M. balansae was dominated by bicyclogermacrene (41.5%), which was not detected in the essential oil of M. grandifolia. Likewise, the sesquiterpene alcohols (E)-nerolidol and globulol were abundant constituents in M. grandifolia , but (E)-nerolidol was much lower in M. balansae (8.7%) and globulol was not detected. The taxonomy of these two plants deserves closer scrutiny.Although N. ellipsoidea was dominated by (E)-β-ocimene (87.6%). (E)-β-Ocimene was also found to be the dominant compound (85.6%) in the leaf essential oil of N. polycarpa from Vietnam [N. sericea from Korea (13.3%) [N. variabillima from Taiwan (13.4%) [N. aciculata from Korea (9.7%) [E)-β-ocimene was only a minor component in the leaf oils of N. australiensis, N. brassii, or N. dealbata from Australia [N. pallens from India [N. foliosa leaf essential oil from India [The leaf essential oil of  Vietnam , and one (13.3%) , N. vari (13.4%) , and N. a (9.7%) . In contustralia , and N. om India , and wasom India .P. angustifolia from Pù Hoạt Nature Reserve (northern Vietnam) in this study was rich in α-pinene (26.9%), β-pinene (20.8%), spathulenol (5.4%), (E)-caryophyllene (5.3%), and p-cymene (5.0%), which differs markedly from a previous study on the leaf essential oil from Sao La Nature Reserve . The previous work reported spathulenol (17.0%), palmitic acid (13.0%), sabinene (6.4%), bicyclogermacrene (5.9%), and artemisia triene (5.1%) to be the major components [The leaf essential oil of mponents . There iThe 24-h and 48-h larvicidal activities of Lauraceae leaf essential oils from Vietnam are summarized in N. ellipsoidea showed the greatest activity against Ae. aegypti with 24-h and 48-h LC50 values of 6.59 and 4.04 μg/mL, respectively. Similar larvicidal activities were observed against Cx. quinquefasciatus (24-h and 48-h LC50 = 7.47 and 4.65 μg/mL) for this essential oil. Interestingly, although the larvicidal activities of C. infectoria leaf essential oil were not as impressive against Ae. aegypti or Ae. albopictus, the essential oil did show much better activity against Cx. quinquefasciatus (24-h LC50 = 10.8 μg/mL), particularly after 48 h of exposure (48-h LC50 = 0.402 μg/mL). Unfortunately, the limited quantities available for several of the essential oils precluded larvicidal screening. However, the larvicidal activity of the untested essential oils will be investigated in future studies.Of the Lauraceae essential oils screened for larvicidal activity, N. ellipsoidea leaf essential oil, (E)-β-ocimene (87.6%), is not likely responsible for the observed larvicidal activity. The (E)-β-ocimene-rich (94.8%) essential oil of Porophyllum ruderale showed an LC50 of 173.7 μg/mL against Ae. aegypti [Syzygium jambolana, with (Z)-β-ocimene (27.2%) and (E)-β-ocimene (12.2%), was inactive against Ae. aegypti (LC50 = 433 μg/mL) [N. ellipsoidea essential oil can likely be attributed to synergistic effects involving minor components.The major component of  aegypti . Likewis3 μg/mL) . The excC. infectoria was rich in the germacrene sesquiterpenes germacrene D (55.5%) and bicyclogermacrene (11.4%), and these compounds may be responsible for the larvicidal activity. Germacrene D has demonstrated notable larvicidal activity against Ae. aegypti and Cx. quinquefasciatus  [Ae. albopictus and Cx. tritaeniorhynchus  [The leaf essential oil of ctively) , and bicctively) .C. concinna from Nam Dong is consistent with the marginal activities observed for the major components. (E)-Caryophyllene, caryophyllene oxide, and α-pinene have shown modest mosquito larvicidal activities [50 = 65 μg/mL against Cx. quinquefasciatus) [50 = 15.4 μg/mL against Ae. aegypti) [50 = 22.4 μg/mL against Ae. aegypti) [Tephrosia toxicaria (42.3% spathulenol) had an LC50 of 63.1 μg/mL against Ae. aegypti [Guarea sylvatica essential oil from branches (14.3% spathulenol) showed LC50 against Ae. aegypti of 274 μg/mL [The marginal larvicidal activity of tivities . β-Pinensciatus) , (LC50 =aegypti) . Spathul aegypti , while G74 μg/mL .L. viridis and N. ellipsoidea leaf essential oils demonstrated particularly notable activities against E. faecalis and B. cereus with minimum inhibitory concentration (MIC) values of 16 μg/mL. The leaf essential oil of C. impressa also showed excellent anticandidal activity against C. albicans with an MIC of 16 μg/mL.Several of the leaf essential oils of the Lauraceae were screened for antimicrobial activity . All of L. viridis leaf oil, bicyclogermacrene, has shown antibacterial activity against B. cereus [E. faecalis [L. viridis leaf essential oil, bicyclogermacrene, decanal, α-pinene, and β-pinene, can account for the observed antibacterial activity.The major component of . cereus . Likewisfaecalis  as well faecalis . Similarfaecalis ,56. Decafaecalis ,58. ThusE)-β-Ocimene dominated the leaf essential oil of N. ellipsoidea, but this compound has demonstrated relatively marginal antibacterial activity [E)-β-ocimene with minor essential oil components may play a role in the antibacterial activity of N. ellipsoidea leaf oil.-caryophyllene nor α-humulene have shown strong anti-Candida albicans activity [E)-caryophyllene do not exhibit notable activity against Candida spp. [C. albicans with an MIC of 125 μg/mL [The components responsible for the anticandidal activity of activity ,56. The ida spp. ,60. Dode25 μg/mL .Leaves were collected from wild-growing trees in north-central Vietnam. Plants were identified by Do Ngoc Dai and voucher specimens  have bee2 (1 mL/min), injector temperature (PTV: programmable temperature vaporization) 250 °C, detector temperature 260 °C, column temperature programmed from 60 °C (2 min hold) to 220 °C (10 min hold) at 4 °C/min. Samples were injected using a split mode with a split ratio of 10:1. The volume injected was 1.0 μL. Inlet pressure was 6.1 kPa.Gas chromatographic (GC) analysis was performed on an Agilent Technologies HP 7890A Plus Gas chromatograph equipped with a FID and fitted with HP-5ms column . The analytical conditions were: carrier gas HAn Agilent Technologies  HP 7890A Plus Chromatograph fitted with a fused silica capillary HP-5ms column  and interfaced with a mass spectrometer HP 5973 MSD was used for the GC/MS analysis, under the same conditions as those used for GC analysis. The conditions were the same as described above with He (1 mL/min) as carrier gas. The MS conditions were as follows: ionization voltage 70 eV; emission current 40 mA; acquisitions scan mass range of 35–350 amu at a sampling rate of 1.0 scan/s. Compound identification was carried out by comparison of the MS fragmentation patterns and calculated retention indices with those available in the databases ,37 and, Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus were carried out as previously described [50 values, LC90 values, and 95% confidence limits were determined by log-probit analysis using Minitab® 19 .Larvicidal activities against escribed ; LC50 vaEnterococcus faecalis (ATCC299212), Staphylococcus aureus (ATCC25923), Bacillus cereus (ATCC14579), three strains of Gram-negative test bacteria, Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC27853), Salmonella enterica (ATCC13076) and one strain of yeast, Candida albicans (ATCC 10231). Minimum inhibitory concentration (MIC) and median inhibitory concentration (IC50) values were measured by the microdilution broth susceptibility assay as previously described [The bacterial growth inhibition of the essential oils was evaluated using three strains of Gram-positive test bacteria, escribed . Neolitsea ellipsoidea, dominated by (E)-β-ocimene, showed excellent larvicidal activity against Aedes aegypti and antibacterial activity against Enterococcus faecalis and Bacillus cereus; Cryptocarya infectoria leaf essential oil, rich in germacrene D and bicyclogermacrene, showed excellent larvicidal activity on Culex quinquefasciatus and anticandidal activity against Candida albicans. The leaf essential oil of Litsea viridis, which was rich in bicyclogermacrene, also showed good antibacterial properties. The biological properties of these Lauraceae essential oils suggest that they may serve as potential “green” alternatives, as also described for Lamiaceae family plants [Of the eleven species of Lauraceae examined in this work, the leaf essential oil of y plants , for use


Ribosomal protein S6 (RPS6) is the only phosphorylatable protein of the eukaryotic 40S ribosomal subunit.Ribosomes with phosphorylated RPS6 can selectively translate 5’TOP--containing mRNAsthat encode most proteins of the translation apparatus. The study of translational control of 5’TOP-mRNAs, which arepreferentially translated when RPS6 is phosphorylated and cease to be translated when RPS6 is de-phosphorylated,is particularly important. In Arabidopsis thaliana, AtRPS6 is phosphorylated by kinase AtRPS6K2, which should in turnbe phosphorylated by upper level kinases (AtPDK1 – at serine (S) 296, AtTOR – at threonine (T) 455 and S437) for fullactivation. We have cloned AtRPS6K2 cDNA gene and carried out in vitro mutagenesis replacing codons encodingS296, S437 and T455 by triplets of phosphomimetic glutamic acid (E). After the expression of both natural and mutatedcDNAs in Escherichia coli cells, two recombinant proteins were isolated: native AtRPS6K2 and presumably constitutivelyactive AtRPS6K2. The activity of these variants was tested in vitro. Both kinases couldphosphorylate wheat (Triticum aestivum L.) TaRPS6 as part of 40S ribosomal subunits isolated from wheat embryos,though the non-mutated variant had less activity than phosphomimetic one. The ability of recombinant non-mutatedkinase to phosphorylate TaRPS6 can be explained by its phosphorylation by bacterial kinases during the expressionand isolation steps. The phosphomimetically mutated AtRPS6K2 can serve as a tool to investigatepreferential translation of 5’TOP-mRNAs in wheat germ cell-free system, in which most of 40S ribosomal subunits havephosphorylated TaRPS6. Besides, such an approach has a biotechnological application in producing genetically modifiedplants with increased biomass and productivity through stimulation of cell growth and division. Growth and division of cells depending on the availability ofnutrients, energy resources, as well as responding to internaland external stimuli are coordinated by signaling system basedon a multilevel cascade of serine-threonine protein kinases.These kinases transmit signals from internal and externalevents to the protein synthesis apparatus, causing inhibitionor enhancement of protein synthesis . The target of rapamycin (TOR) kinase –is the master signaling integrator, central hub synchronizingcell growth according to the nutrient and energy status as wellas environmental influences . In mammals,TOR forms two functionally distinct protein complexes:mTORC1 containing RAPTOR (regulatory-associated proteinof mTOR), and mTORC2 containing RICTOR (rapamycininsensitivecompanion of mTOR) .In favorable conditions mTORC1 phosphorylates RPS6K. Complete activation of mammalian RPS6K byphosphorylationis dependent on another upper level PDK1kinase . The fully activated RPS6K inturn phosphorylates the S6 ribosomal protein (RPS6) .At transcriptional level, phosphorylation of pRPS6 in nucleolusleads to activation of rRNA gene promoter and ribosomogenesis. In cytosol,RPS6 phosphorylation promotes the selective translationof special group of cellular mRNAs, containing 5′-terminaloligo-pyrimidine tract (5′TOP) in their 5′-untranslated regions(5′UTRs) . The number of these5′TOP-containing mRNAs, according to various estimates,ranges from one hundred to two hundred and forty . They encode almost all theproteins of the translation apparatus -binding proteins, etc.) , as well asother protein families associated with lysosome functions, metabolismand proliferation .As in yeast and animals, TOR kinase is involved in controllingplant growth and cell division . But inplants, only orthologs of genes encoding mTORC1 were found. No clear orthologs of theRICTOR have yet been found in plants . TOR proteins are highly conserved ineukaryotes. For example, in A. thaliana and Homo sapiensthey share 73 % amino acid sequence identity in the kinasedomains .Although functioning of this main regulator of cell processeshas been well studied in other eukaryotes, knowledgeof the regulation of translation and gene expression in plantsis very limited. Most studies of the regulation of cellular processby plant RPS6-kinase were performed on a model objectA. thaliana containing two very similar forms – At RPS6K1and At RPS6K2. It was shown that only At RPS6K2 is able tophosphorylate RPS6 and stimulate an increase in cell size . Forthe complete activation of At RPS6K2, it is necessary that it bephosphorylated by pPDK1 kinase (at Ser296), pTOR kinase (atThr455), as well as by one more, unknown, kinase (at Ser437).Although pTOR→S6K signaling plays multiple roles intranslational control , mechanisms used byTOR kinase to impact global protein synthesis in plants are notwell understood . New data are currently appearing on the involvementof pRPS6K1 in the promotion of translation reinitiationof upstream open reading frame (uORF)-containing viral andcellular mRNAs via phosphorylation of eIF3h  and in regulation of translation initiation underenergy-deficient conditions via formation of the functionaleIF4F complex . Nevertheless, the role ofplant pRPS6K2 and pRPS6 phosphorylation in translationregulation in the cytosol remains unclear .It is practically impossible to control the multiple and simultaneousphosphorylation of At RPS6K2 kinase by the kinases of theupper regulatory level for experimental purposes. Therefore, wedecided to use a different approach to achieve the phosphorylationof plant RPS6 using the mutated form of At RPS6K2,which should be stably active. We have cloned the AtRPS6K2cDNA gene and performed in vitro mutagenesis of this cDNAby replacing codons encoding serines at positions 296 and 437,as well as threonine at position 455 with triplets encoding thephosphomimetic amino acid – glutamic acid. After expressionof non-mutated and mutated cDNA gene in E. coli cells the nativeAt RPS6K2 and the phosphomimetic At RPS6K2 recombinant protein was obtained. The secondone is expected to have stable kinase activity, regardless of theupper-level kinases, that could be used as a unique tool for theartificial phosphorylation of TaRPS6 in a wheat germ cell-freetranslation system. Mutated version of cDNA gene encodingthe constantly active form of AtRPS6K2 may also be used toobtain genetically modified plants with increased productivity,earlier ripening and a higher rate of biomass accumulation.Cloning of AtRPS6K2 cDNA gene. The total RNA was isolatedfrom A. thaliana (Col-0 ecotype) leaves using Tri-reagent(Sigma). The reverse transcription reaction was performedusing Maxima Reverse Transcriptase (Thermo) and ‘AtS6K2-rev-3UTR’ primer (5′-GAATTCGAGAAATAGGTTTCTTCAAACAACCGTTGATTTTG), which allowed to differentiateAt RPS6K2 from At RPS6K1 mRNAs. RT-PCR was performed in 25 μl reaction using Phusion High-fidelity DNApolymerase(Thermo), 0.2 pM primers ‘Nde-AtS6K2-for’(5′- GGGCGAATTGGGTCATATGGTTTCTTCTCAGTG)and ‘AtS6K2-Xho-rev’ (5′-AAACTCGAGCTACAAGTTGGATGTGGTCCGATGA) and 2.5 μl of RT-reaction mixture.Temperature regime: stage 1–5 min at 94 °C, 1 cycle; stage2–10 s at 98 °C, 20 s at 49 °C, 45 s at 72 °С, 4 cycles; stage3–10 s at 98 °C, 20 s at 52 °C, 45 s at 72 °С, 30 cycles; stage4–5 min at 72 °C, 1 cycle. The PCR product (~1425 bp)was digested with NdeI/XhoI and cloned into pET19b vectordigested with the same enzymes resulting ‘Pet19b-His-At RPS6K2’ plasmid.Mutagenesis. In vitro mutagenesis was performed in threesteps using QuikChange II Site-Directed Mutagenesis Kit(Agilent technologies) according to the manufacturer’s protocol.At the first step ‘Pet19b-His-AtRPS6K2’ plasmid wasamplified entirely using Pfu Ultra High-Fidelity DNA polymerase(Thermo) and complementary primers: ‘S296-Glu-dir’(5′-AAACACAAGATCAAACGAAATGTGTGGGACTACGGA) and ‘S296-Glu-rev’ (5′-TCCGTAGTCCCACACATTTCGTTTGATCTTGTGTTT) containing correspondingnucleotide substitutions. Temperature regime: stage 1–30 s at95 °C, 1 cycle; stage 2–30 s at 95 °C, 1 min at 55 °C, 7 min30 s at 68 °C, 18 cycles. The reaction mixture was furthertreated with restriction enzyme DpnI, which cleaves methylatedDNA into fragments at 5′-Gm6ATC-3′ sequences. Sincethe original plasmid was methylated (dam+ E. coli strain DH5was used for plasmid enrichment), the restriction enzymeDpnI had cleaved the original non-mutated plasmid, whereas‘Pet19b-His-AtRPS6K2(S296E)’ plasmid synthesized duringPCR-step remained intact. Subsequently, the competent E. colicells (XL1-Blue strain) were transformed with the reactionmixture. Another two mutagenesis steps for the production of‘Pet19b-His-At RPS6K2’ and ‘Pet19b-His-At RPS6K2’ plasmids were done inthe same manner using ‘S437-Glu-dir’ (5′-ACATGTCTGTTTTGGATGAACCAGCAAGTAGTCCCA)/‘S437-Glu-rev’(5′-TGGGACTACTTGCTGGTTCATCCAAAACAGACATGT) or ‘T455-Glu-dir’ (5′-ACCCTTTTACAAACTTCGAATACGTCAGGCCTCCTCA)/‘T455-Glu-rev’ (5′-TGAGGAGGCCTGACGTATTCGAAGTTTGTAAAAGGGT)primers respectively. Resulting DNA-constructs were usedas templates for the next in vitro mutagenesis step. The insertscloned into the recombinant plasmids were sequencedfrom both ends by the dideoxy chain termination methodusing Big Dye Terminator v.3.1 sequencing kit (Thermo) onthe 310 genetic analyzer (Applied Biosystems) according tomanufacturer’s recommendations.Expression and purification of recombinant proteins.E. coli strain BL21(DE3) cells transformed with recombinant‘Pet19b-His-AtRPS6K2’ or ‘Pet19b-His-At RPS6K2’ plasmid were grown in 100 ml of LB mediumcontaining ampicillin (100 μg/ml) at 30 °C to A600of 0.5 unit. The expression of recombinant proteins wasinduced by 0.8 mM isopropyl β-D-1-thiogalactopyranoside(IPTG) at 30 °C for 4 h. Cells were collected by centrifugation,resuspended in His-buffer  containing 10 mM imidazole, and then lysedby addition of lysozyme (1 mg/ml) and sonication. The celldebris was removed by centrifugation at 10000 g for 20 min at 4 °C. Supernatant was combined with PerfectPro Ni–NTAresin suspension (5-Prime), shaken at 4 °C for 1 h followedby flow throw in column. The resin was washed twice byHis-buffer containing 20 mM imidazole at 4 °C. His-taggedproteins bound to the resin were eluted with His-buffer containing250 mM imidazole and dialyzed against dialysis buffer2, pH 7.6) at4 °C for 12 h. Dialyzed proteins were concentrated by centrifugationin 10,000 MWCO HY columns (Sartorius) accordingto the manufacturer’s instructions. Protein concentration wasestimated by the Bradford protein assay .Gel-electrophoresis. Proteins were separated by standardSDS-PAGE in Tris-Glycine gel system  according toU.K. Laemmli (1970). After electrophoresis, the gels werefixed and stained in PageBlue Protein Staining Solution(Thermo) or subjected to semi-dry blotting in transfer buffer ethanol) for1 h at 0.8 mA/cm2 and 20 V using 0.22 μm pore NitroBindnitrocellulose membranes (GVS).Western blotting. For immunodetection of His-At RPS6K2and His-At RPS6K2 proteins, the blotswere first ‘blocked’ by submerging them in blocking solution Tween 20, pH 7.5) containing 5 % skim milk) for 1 h at25 °C with gentle shaking. The blots were then incubated withPenta-His mouse antibodies (5 Prime) diluted  in theblocking solution for 1 h at 25 °C, thoroughly washed threetimes with TBST buffer, and incubated for 1 h at 25 °C withhorseradish peroxidase-conjugated goat anti-mouse antibodies(Santa Cruz) diluted  in blocking solution. Afterdouble washes in TBST and double washes in TBS, the blotswere chemiluminescence developed using ChemiluminescentPeroxidase Substrate-3 detection reagents (Sigma). Animage of the membrane was then produced on X-ray film.Monoclonal Anti-Phosphoserine Mouse Antibodies (Sigma)and Monoclonal Anti-Phosphothreonine Mouse Antibodies(Sigma) were used as 1st antibodies (at 1:300 dilution in TBSTcontaining 5 % BSA) for the detection of phosphorylationstatus of proteins.260 unit corresponds to 50 pmolof 40S subunits.40S ribosomal subunits isolation. 40S ribosomal subunitswere isolated from wheat  embryos, purified from endosperm, as described previouslyfor ribosomal subunits isolation from human placenta with the ratio of buffer to embryos of6:1. It was considered that 1 AKinase assay. The reaction mixture in 20 μl contained20 mM TrisAc (pH 7.6), 90 mM KAc, 2.5 mM Mg(OAc)2,1 mM DTT, 10 pmol of 40S ribosomal subunits, 0.1 mM ATP.Purified His-At RPS6K2 or His-At RPS6K2 were added in amount of 2.5 ng/μl. The mixtures wereincubated for 20 min at 26 °C.Cloning and mutagenesis of AtRPS6K2 cDNA gene. A totalRNA preparation was isolated from A. thaliana, and reversetranscription was performed using ‘AtS6K2-rev-3UTR’primer, complementary to 3´UTR of AtRPS6K2 mRNA, butnot AtRPS6K1 mRNA, allowing to discriminate between them. Then, AtRPS6K2 cDNA was amplified by RT-PCR andcloned into pET19b vector. According to sequencing analysis,AtRPS6K2 cDNA corresponded to #AT3G08720 (GeneBank)sequence.Thus obtained ‘Pet19b-His-AtRPS6K2’ plasmid was mutatedin vitro in three steps to introduce three phosphomimeticmutations into AtRPS6K2 cDNA. At the first step, theTCC triplet encoding serine at position 296 was replaced bythe GAA triplet, which encodes glutamic acid that imitatesphosphorylated serine. In a second step, the TCT tripletof obtained mutated AtRPS6K2(S296E) cDNA encodingserine at position 437 was mutated to GAA triplet to formAtRPS6K2 cDNA. In the third step, the ACAtriplet of AtRPS6K2 cDNA encoding threonineat position 455 was replaced by the GAA triplet to form themutated AtRPS6K2 cDNA.Expression and purification of recombinant kinases.AtRPS6K2 and AtRPS6K2 cDNAgenes were expressed in E. coli cells, then recombinant Histaggedproteins  respectively) were isolated using immobilizedmetal ion affinity chromatography (IMAC) followed by immunoblottinganalysis .Isolated proteins were purified by dialysis and concentrated.Preparations isolated under native conditions contained acertain amount of impurity polypeptides. Content of recombinantproteins in preparations was corrected according todensitometric analysis data (by ImageJ 1.42). The yield ofpurified and concentrated full-length recombinant proteins His-AtRPS6K2 and His-At RPS6K2was 5.22 mg and 4.52 mg per L of media respectively.Testing the activity of recombinant kinases. Both forms ofkinase (the intact one and that carrying three phosphomimeticsubstitutions) were tested for their ability to phosphorylateTaRPS6 in the composition of 40S ribosomal subunits isolatedfrom wheat embryos. The phosphorylation state of proteinswas tested using monoclonal antibodies against phosphoserine.As can be seen from the data presented in Fig. 2, bothkinases are able to phosphorylate the plant ribosomal proteinS6 (TaRPS6) in composition of 40S ribosomal subunits,although activity of His-At RPS6K2is obviously higher than that of non-mutated His-At RPS6K2. In wheat germ, there are at least two forms of the S6ribosomal protein (A and B); therefore, two bands are observed.Initially, we expected that non-mutated kinase should haveno activity since for its activation in plant cells phosphorylationat three sites is required by upper-level kinases. Thephosphorylation state of purified recombinant kinases waschecked using monoclonal antibodies against phosphoserineand phosphothreonine .As can be seen from the data presented in Fig. 3, thenon-mutated recombinant His-At RPS6K2 kinase producedin E. coli cells was phosphorylated both at serine residues and threonine residues . Thus, some bacterial kinases were able to phosphorylate His-At RPS6K2 protein resulting in its activation.It should be noted that certain non-mutated serine residuesof mutated His-At RPS6K2 recombinantkinase were also phosphorylated ,althoughthis kinase was not phosphorylated at threonineresidues .The interest in studying the mechanisms of TOR-mediatedregulation of mRNA translation in plants is high because othermechanisms of regulation of protein biosynthesis, which arewell described for mammals and yeast, either do not work orfunction within very narrow limits in plants. Indeed, in plantcells eIF4E binding proteins (eIF4E-BPs) were not found, andthere are no genes for these proteins in plant genome . The mechanism of translation suppressionby phosphorylation of peEF2 is not realized in plants. Then,out of four protein-kinases  thatphosphorylate α-subunit of meIF2 in mammalian cells, onlypGCN2-kinase was detected in plants, that can be activatedunder several but not all stresses. Moreover, it was shown,that factor eIF2B is not necessary for cyclic functioning ofplant peIF2 , and neither its biochemicalactivity nor peIF2B-like factor orthologs were detected inplants till now . These circumstancesmake the TOR system one of the few currently known effectiveregulators of protein biosynthesis in plants.Having obtained the constitutively active protein kinaseAt RPS6K2 with phosphomimeticsubstitutions of key amino acids, we acquire a convenienttool that allows to considerably increase phosphorylationof TaRPS6 in the composition of 40S ribosomal subunits in wheat germ cell-free system. This allows studying importantmechanisms of preferential translation of a specific groupof cellular 5′TOP-containing mRNAs, which is preferablytranslated when pRPS6 is phosphorylated and ceases to betranslated when RPS6 is de-phosphorylated . In addition to fundamental interest the use of cDNA encodingconstitutively active RPS6-protein kinase would opennovel routes for increasing crop yield through stimulation ofribosomogenesis and subsequent growth and division of plantcells. It is known that augmented expression of the At TORgene results in a dose-dependent decrease or increase, in organand cell size, seed production . In addition to regulating theprotein synthesis process, TOR acts as a master regulator ofthe cell cycle, coordinator of rRNA transcription, activation ofribosomal protein genes, ribosome assembly and may also regulate long non-coding RNAs (lncRNAs)expression . Therefore, artificial increasingof TOR gene expression in plant cells can lead to seriousundesirable consequences while using of AtRPS6K2 cDNA may help to avoid these complications.We have cloned the AtRPS6K2 cDNA gene encoding kinase 2of ribosomal protein S6 from A. thaliana and performed itsmutagenesis to obtain the AtRPS6K2kinase containing phosphomimetic substitutions. Such mutatedenzyme with constant RPS6-kinase activity may be usedto study specific molecular mechanisms mediating efficienttranslation of 5′TOP-mRNAs depending on phosphorylationof RPS6 in plant cells. 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