AbstractPageBreakAnillinus Casey, 1918 includes three previously described species (A. affabilis (Brues), 1902, A. depressus (Jeannel), 1963 and A. sinuatus (Jeannel), 1963) and four new species here described: A. acutipennis Sokolov & Reddell, sp. n. ; A. comalensis Sokolov & Kavanaugh, sp. n. ; A. forthoodensis Sokolov & Reddell, sp. n. ; A. wisemanensis Sokolov & Kavanaugh, sp. n. . A key for identification of adults of these species is provided. The fauna includes both soil- and cave-inhabiting species restricted to the Balcones Fault Zone and Lampasas Cut Plain and adjacent areas underlain by the Edwards-Trinity Aquifer. Based on morphological and distributional data, we hypothesize that four lineages of endogean Anillinus species extended their geographical ranges from a source area in the Ouachita-Ozark Mountains to the Balconian region in central Texas. There the cavernous Edwards-Trinity aquifer system provided an excellent refugium as the regional climate in the late Tertiary and early Quaternary became increasingly drier, rendering life at the surface nearly impossible for small, litter-inhabiting arthropods. Isolated within the Edwards-Trinity aquifer system, these anilline lineages subsequently differentiated, accounting for the currently known diversity. The paucity of specimens and difficulty in collecting them suggest that additional undiscovered species remain to be found in the region.The Texas fauna of the genus  Anillina are typically litter- or soil-dwelling carabids, but they are not common in caves. Of the approximately 50 anilline species previously described from North America, only four species of Anillinus  than in the dry regions to the West of the Mississippi River (where only 14 species have been recorded) (Representatives of the subtribe lentinei  and bothecorded) .Coleoptera are represented by Staphylinidae: Pselaphinae   . During PageBreakCarolina (14), Tennessee (12), or Alabama (9)   and possPageBreakAnillus affabilisEciton coecum Latreille [=Labidus coecus], and one from a nest of Solenopsis geminata Fabricius”. The author of the description did not consider the species myrmecophilous, but pointed out that the beetles evidently shared their habitats with ants. After that first description, no new species were described from Texas for more than 60 years and only this species was cited for Texas in the first edition of the Catalogue of the Coleoptera of North America , were incorrectly contrasted with representatives of anillines with the normal aedeagal configurations . We surmise that Jeannel was using members of a Serranillus species, males of which lack the right paramere, leaving just the left one  to Anillinus. Currently, therefore, the Texas anillines are represented by three species in the genus Anillinus: Anillinus affabilis, Anillinus depressus and Anillinus sinuatus.The first anilline species from Texas was described as  America . One morAnillina , he chanBarr see , led himBarr see , p. 48 uleft one , 2012 as Mexico, , Yves BoAnillina collected in caves showed that material at hand could not be identified by reference to the existing original descriptions and, therefore, more thorough taxonomic study was needed. This paper presents the results of our taxonomic study of all the Texas representatives of Anillina known to the authors to date. The question of relationships between Anillinus and Anillodes is beyond the scope of this paper.Our preliminary investigation of the Texas Anillina from Texas, representing seven species, four of which are described as new. Most of the specimens originated from the collection of the Texas Memorial Museum, The University of Texas at Austin, Austin, Texas, USA (TMM). Types and a few additional specimens were borrowed from the U. S. National Museum of Natural History (NMNH), Smithsonian Institution, Washington, DC, and the Museum of Comparative Zoology (MCZ), Harvard University, Cambridge, MA. Specimens from this study, including types, have also been deposited in collections at the California Academy of Sciences, San Francisco (CAS), and the Canadian National Collection, Ottawa, Ontario, Canada (CNC).Verbatim label data are given for type specimens of all newly described taxa, with label breaks indicated by a slash (“\”), and photographs of labels from types of previously described species are also included.This study is based on examination of 39 specimens of  = length of head, measured along midline from anterior margin of labrum to the virtual line, connecting posterior supraorbital setaeLH;  = width of head, at level of anterior supraorbital setaeWH;  = maximal width across pronotumWPm;  = width across anterior angles of pronotumWPa;  = width across posterior angles of pronotumWPp;  = length of pronotum from base to apex along midlineLP;  = width of elytra, at level of 4th umbilicate setaeWE;  = length of the elytra, from apex of scutellum to apex of left elytronLE;  = standardized body length, a sum of LH, LP and LESBL. SBL measurements are given in mm; others are presented as nine ratios: mean widths-WH/WPm and WPm/WE and body parts-WPa/WPp, WPm/WPp, WPm/LP, WE/LE, LE/SBL, WE/SBL and LP/LE. All values are given as mean ± standard deviation.Measurements. All specimens were measured electronically using a Leica M420 microscope equipped with a Syncroscopy AutoMontage Photomicroscopy system . Measurements for various body parts are encoded as follows: Illustrations. Digital photographs of the dorsal habitus of new species were taken with the AutoMontage system using a Leica M420 microscope. Line drawings of selected body parts were made using a camera lucida on an Olympus BX 50 microscope. Scanning electron micrographs were made with coating on an ESEM FEI Quanta 200.PageBreakDissections. Most of the specimens we examined were already dissected by previous investigators, and genitalia of these specimens were preserved in microvials with glycerol, pinned beneath the specimen. Where necessary, dissections were made by present authors using standard technique. Genitalia were dissected from the abdomens of specimens previously softened in boiling water for 20-30 minutes. Contents of the abdomen were cleared using boiling 10% KOH for 2-3 minutes to remove internal tissues, and then washed in hot water before examination. After examination, genitalia were mounted on plastic transparent boards in dimethylhydantoin formaldehyde resin (DMHF) and pinned beneath the specimen.Type material. The authors investigated type material of all three previously described Texan species of anillines.Terms. Terms used in this presentation follow Species ranking. Species recognition is in accordance with our previous approach .Descriptions. The scheme of descriptions follows that of Taxon classificationAnimaliaColeopteraCarabidaeCasey, 1918Anillinus Casey, 1918: 167. For generic synonymy, see Anillinus carolinae Casey, 1918.Anillina are characterized by the following combination of characters: Head totally covered with microsculpture (PageBreaklast two (8th and 9th) pores (eo8 and eo9) of umbilicate series much closer to each other than 7th (eo7) pore is to 8th (not so only in Anillinus depressus (Jeannel)). Abdomen with ventrite 5 of male with two and of female with four setae along the posterior margin. Aedeagi of males of all examined species with two parameres , possess pronota with a rather narrow basal margin (WPa/WPp ≥ 1.00), and are completely covered with microsculpture dorsally .PageBreakHabitus. Body form  subdepreColor. Body rufotestaceous, appendages testaceous.Microsculpture. Distinct over all dorsal surfaces of head, pronotum and elytra, with slightly transverse polygonal meshes of more or less scaly appearance on elytra.Head. Labium . Mental Prothorax. Pronotum  relativeElytra . Widely Male unknown.Female genitalia. Spermatheca with distal part of cornu only slightly dilated, gradually tapered to proximal part. Nodulus short, ramus undifferentiated .This species has been found only in two remote areas of Bell (Lampasas Cut Plain) and Hays  Counties, Texas Anillus affabilis Brues, 1902: 366. Leng 1920Anillinus affabilis (Brues), Bousquet 2012Anillodes affabilis (Brues), Jeannel 1963aPageBreakAnillus affabilis Brues, 1902: 366. Lectotype  By Erwin 78 \. Two paralectotypes: one female, also in NMNH, labeled: \ Austin Texas [printed] \ E. caecum [handwritten] \ type [typed with blue ink] \ TYPE [typed red label] \ ADP 115719 \ Anillodes affabilis Brues R, Jeannel det. 19 \ USNM TM #2067637 \ Paralectotype Anillus affabilis Brues recognized by Sokolov & Kavanaugh 2014 [printed yellow label] \; one male, in MCZ, labeled: \ Austin Tex \ Type [white label] \ H. C. Fall Collection \ Anillus affabilis Brues \ Paralectotype Anillus affabilis Brues recognized by Sokolov & Kavanaugh 2014 [printed yellow label]ectotype , designaectotype : 233, a In his original description, Anillinus affabilis are practically indistinguishable from females of Anillinus wisemanensis, described below. From adults of other Texan species of the genus, those of Anillinus affabilis can be distinguished by the following combination of external characters: large size, comparatively narrow and transverse pronotum with rectilinearly constricted lateral margins, rather wide and long elytra with rounded apices; and males can be further distinguished by the minute spine on the posterior edge of metafemora and shape of the median lobe.Females of Medium-sized for genus .Habitus. Body form , 5A subdColor. Body rufotestaceous or brunneorufous, appendages testaceous.Microsculpture. Distinct over all dorsal surfaces of head, pronotum and elytra, with slightly transverse polygonal meshes of more or less scaly appearance on elytra.Prothorax. Pronotum of normal length (LP/LE 0.39±0.012) and slightly transverse for genus (WPm/LP 1.31±0.016), lateral margins almost rectilinear and moderately constricted posteriorly (WPm/WPp 1.32±0.008). Anterior angles indistinct, posterior angles slightly obtuse (100–110°). Width between anterior and posterior angles of equal length (WPa/WPp 1.00±0.026). Basal margin almost straight.Elytra. Widely depressed along suture, of normal length (LE/SBL 0.58±0.008) and typical width for genus (WE/LE 0.66±0.029), with traces of 4-5 striae. Humeri distinct, rounded, in outline forming right angle with longitudinal axis of body. Lateral margins subparallel, slightly divergent at basal fourth, evenly rounded to apex in apical fourth, without subapical sinuation. Vestiture of elytra short . Apex of elytron rounded.Legs. Male protarsomere 1 markedly dilated apico-laterally with rows of adhesive setae ventrally. Male hind legs modified: metafemora with minute tooth at middle along posteroventral margin .PageBreakAbdomen. Abdominal ventrites 3 and 4 of males each with a pair of protuberances at the places of setal attachments . Last viMale genitalia. Median lobe of aedeagus  with shoPageBreakmatheca .Habitus. Body form  subdeprePageBreakColor. Body rufotestaceous, appendages testaceous.Microsculpture. Distinct over all dorsal surfaces of head, pronotum and elytra, with slightly transverse polygonal meshes of more or less scaly appearance on elytra.Prothorax. Pronotum as in  relativePageBreakElytra as in . Widely Legs. Male protarsomere 1 markedly dilated apico-laterally with adhesive setae ventrally. Male hind legs unmodified .Abdomen. Ventrite 5 of male without depression.Male genitalia. Median lobe of aedeagus  with shoFemale genitalia. Spermatheca with distal part of cornu only slightly dilated, gradually tapered to the proximal part. Nodulus short, ramus undifferentiated .This species is known only from the type locality in the New Braunfels area, Comal County, Texas Micranillodes depressusAnillinus depressus (Jeannel), Bousquet 2012Anillinus depressus are distinguished easily from those of other Texan species of the genus by the following combination of external characters: small size, markedly elongated habitus, laterally oblique pronotal basal margin and simple .Adults of Small-sized for genus (SBL 1.45 mm).PageBreakHabitus. Body form  subdepreColor. Body rufotestaceous, appendages testaceous.Microsculpture. Present over all dorsal surfaces of head, pronotum and elytra, but much finer on head and pronotum than on elytra.Head. Labium with mental tooth present; mentum and submentum separated by suture.PageBreakProthorax. Pronotum relatively long (LP/LE 0.41) and slightly transverse (WPm/LP 1.29), with lateral margins almost rectilinear and strongly constricted posteriorly (WPm/WPp 1.33). Anterior angles indistinct, posterior angles slightly obtuse (100–110°). Width between posterior angles less than between anterior angles (WPa/WPp 1.10). Basal margin oblique laterally, so posterior angles noticeably shifted forward.th and 9th of which not geminate. Apex of elytron rounded with distinct sutural angle.Elytra. Widely depressed along suture, of normal length (LE/SBL 0.57) and narrow for genus (WE/LE 0.61), with traces of 4-5 striae. Humeri distinct, rounded, in outline forming right angle with longitudinal axis of body. Lateral margins subparallel, slightly divergent at basal fourth, evenly rounded to apex in apical third, without subapical sinuation. Umbilicate series with 9 pores, 8Genitalia not examined.The single known specimen of this species was collected in Travis County, Texas .Habitus. Body form  subdepreColor. Body rufotestaceous, appendages testaceous.Microsculpture. Distinct over all dorsal surfaces of head, pronotum and elytra, with slightly transverse polygonal mesh of more or less scaly appearance on elytra.Head. Labium  with menProthorax. Pronotum  relativeElytra . Widely Legs. Male protarsomere 1 markedly dilated apico-laterally with two rows of adhesive setae ventrally . Male hiAbdomen. Ventrite 5 of male with medial depression .PageBreaklength to basal orifice, with numerous poriferous canals. Dorsal sclerite in form of a semicircular blade-like structure with characteristic basal prolongations. Without distinct ventral sclerites. Dorsal membranous field with numerous small spines loPageBreakcated dorsally from dorsal sclerite. Enlarged apical area of median lobe with a dark spine-like structure. Right paramere enlarged, long and wide with numerous (>8) long setae Anillodes sinuatusAnillinus sinuatus (Jeannel), Bousquet 2012Anillinus sinuatus are distinguished from those of other Texan species of the genus by the following combination of external characters: large size, pronotum with long and shallow basilateral sinuations and microsculpture on pronotum much finer than on head and elytra; and males are further distinguished by the minute spine on the posterior edge of metafemora and shape of the median lobe.Adults of Medium-sized for genus .Habitus. Body form , 5B subdColor. Body rufotestaceous, appendages testaceous.Microsculpture. Distinct over all dorsal surfaces of head and elytra. Pronotum with much finer microsculpture, almost indistinct on disc and only at certain angles.PageBreakPageBreakuation before posterior angles, moderately constricted posteriorly (WPm/WPp 1.33±0.024). Anterior angles indistinct, posterior angles nearly rectangular (90–100°). Width between anterior and posterior angles of approximately equal length (WPa/WPp 1.03±0.020). Basal margin almost straight.Prothorax. Pronotum of normal length (LP/LE 0.40±0.004) and slightly transverse for genus (WPm/LP 1.31±0.019), lateral margins with long and shallow sinElytra. Widely depressed along suture, of normal length (LE/SBL 0.58±0.008) and typical width for genus (WE/LE 0.64±0.003), with traces of 5-6 striae. Humeri distinct, rounded, in outline forming right angle with longitudinal axis of body. Lateral margins subparallel, slightly divergent at basal fourth, evenly rounded to apex in apical fourth, without subapical sinuation. Vestiture of elytra short . Apex of elytron rounded.Legs. Male protarsomere 1 markedly dilated apico-lateraly with rows of adhesive setae ventrally. Male hind legs modified: metafemora with minute tooth at middle along posteroventral margin.Abdomen. Abdominal ventrites of males unmodified.Male genitalia. Median lobe of aedeagus  with shoFemale genitalia. Not investigated.Anillinus sinuatus n. sp R. Jeannel det., 19 \ Anillodes sinuatus J. det. T.L.Erwin 96 \; one female  on points, in NMNH, labeled: \ Bexar Co. Tex. Feb. 9. 1938 T-9056 38-2676 \ From soil of peach orchard \ T-9056 \ USNM \.This species is known only from Bexar County, Texas , specimens from Bell County slightly larger .Habitus. Body form  subdepreColor. Body brunneorufous, appendages testaceous.Microsculpture. Distinct over all dorsal surfaces of head, pronotum and elytra, with slightly transverse polygonal meshes of more or less scaly appearance on elytra.Prothorax. Pronotum  of normaElytra . Widely Legs. Male protarsomere 1 markedly dilated apico-lateraly with rows of adhesive setae ventrally. Male hind legs modified: metafemora triangularly dilated along posteroventral margin , and metAbdomen. Ventrite 5 of male unmodified.PageBreakmost all its length, with large protuberance directed backwards and situated before apical orifice. Ventral margin enlarged along all length, but most widely in apical half and then gradually tapered to basal orifice. Numerous poriferous canals on the ventral margin and medial walls of the shaft. Dorsal sclerite in form of a curved blade-like structure with characteristic basal prolongations. Without distinct ventral sclerites or spines. Enlarged apical area of median lobe with a dark spine-like structure. Specimens from Bell County demonstrate slightly different shape of dorsal protuberance and basal prolongations of dorsal sclerite  and Hays  Counties, Texas  labeled: \Zara-3873: TX: Bexar Co., Holy Smoke Cave, 10.XII.2008, P. Sprouse, S. Zappitello \.We are unable to associate definitively the following six specimens, all in TMM, with any of the seven species included above: one female labeled: \ TX: Coryell Co., Lucky Day Cave, Fort Hood, 27.VI.2009, J. Fant, J. Reddell, M. Reyes \ Texas Memorial Museum Invertebrate Zool Coll #70.013 \; one female labeled: \ TX: Bell Co., Sponge Bob Pot, Fort Hood, 17.II.2009, J. Fant, M. Warton \ Texas Memorial Museum Invertebrate Zool Coll #69.724 \; one male labeled (aedeagus lost): \ TX: Williamson Co., Lobo’s Lair, 13.IX.1991, J. Reddell & M. Reyes \ Texas Memorial Museum Invertebrate Zool Coll #27.142 \; one female labeled: \ TX: Williamson Co., Lobo’s Lair, 1.IX.1991, W. Elliot, J. Reddell, M. Reyes, M. Warton \ Texas Memorial Museum Invertebrate Zool Coll #27.126 \; one female  labeled: \ TX: Bexar Co., Up the Creek Cave, 14.XI.1995, J. Cokendolpher, J. Reddell, M. Reyes \ Texas Memorial Museum Invertebrate Zool Coll Females from Coryell and Bell Counties   . PossiblAnillina with the range of Anillinus lescheni in Oklahoma (Anillina species have not yet been reported.Although the Fort Hood Military Installation is separated from the cavernous deposits of the Balcones Fault Zone by about 60 km of non-cavernous deposits, the same genera containing troglobites occur in both areas. This indicates that the ancestral species of troglobites ranged throughout the entire area. Both areas contain the same basic vegetation type and presumably were suitable habitat for the same litter-dwelling and endogean species that gave rise to the troglobites. The Fort Hood region belongs to the Lampasas Cut Plain, a subdivision of the Cross-Timbers ecological region , which mOklahoma , and we Oklahoma , this rePageBreakAnillinus extended their geographical ranges from their source area in the Ouachita-Ozark Mountains to the Balconian region in central Texas. There, they encountered the cavernous Edwards-Trinity aquifer system that provided an excellent refugium as the regional climate in the late Tertiary and early Quaternary became increasingly drier, rendering the near-surface conditions too dry to sustain the life of small litter-inhabiting arthropods. Isolated within the cool damp layers of limestone penetrated by the Edwards-Trinity aquifer system, these anilline lineages differentiated, but to what extent is not known. One of them is represented by four species; each of the others might have remained monobasic, although it seems likely that they too differentiated within their Texan refugium.In summary, we hypothesize that four lineages of endogean, totally microsculptured


M. tuberculosis and other bacterial genomes is a core analysis for studying evolutionary hypotheses, disease outbreaks and transmission events. Whole genome sequencing is providing new insights into the genomic variation underlying intra- and inter-strain diversity, thereby assisting with the classification and molecular barcoding of the bacteria. One roadblock to strain investigation is the lack of user-interactive solutions to interrogate and visualise variation within a phylogenetic tree setting.Phylogenetic-based classification of PhyTB (http://pathogenseq.lshtm.ac.uk/phytblive/index.php) to assist phylogenetic tree visualisation and identification of M. tuberculosis clade-informative polymorphism. Variant Call Format files can be uploaded to determine a sample position within the tree. A map view summarises the geographical distribution of alleles and strain-types. The utility of the PhyTB is demonstrated on sequence data from 1,601 M. tuberculosis isolates.We have developed a web-based tool called PhyTB contextualises M. tuberculosis genomic variation within epidemiological, geographical and phylogenic settings. Further tool utility is possible by incorporating large variants and phenotypic data (e.g. drug-resistance profiles), and an assessment of genotype-phenotype associations. Source code is available to develop similar websites for other organisms (http://sourceforge.net/projects/phylotrack). Mycobacterium tuberculosis complex (MTBC) is an important factor in tuberculosis pathogenesis that may affect virulence, transmissibility, host response and emergence of drug resistance . These SNPs were identified using a combination of bwa-mem alignment software (bio-bwa.sourceforge.net) and the SAMtools/BCFtools suite (samtools.sourceforge.net) complemented by GATK (https://www.broadinstitute.org/gatk/). Variants at Q-score of 30 or more were then selected from the intersection dataset between those obtained from both SAMtools and GATK. SNPs in non-unique regions, including repeat regions in PE/PPE genes were removed (see  and nodes were annotated. Subsequently, the ancestral sequence at all internal nodes was computed using DnaPars from the Phylip package (http://evolution.genetics.washington.edu/phylip/). The main lineage- and sublineage-defining nodes were initially identified from the tree, based on the spoligotypes in each clade. Informative markers at each node in the phylogenetic tree are stored in VCF files and displayed, highlighting clade-defining polymorphism. This functionality has been implemented using the tabix tool [PhyloTrack package. These include scripts to convert a tree in JSON format for use by the D3.js library, produce metadata for each node, and process VCF files containing information for each node and SNP. VCF files containing clade informative and drug resistance markers [bgzip and indexed using tabix to improve computational efficiency, as well as to act as a database. Variants in user uploaded VCF files are compared to those in the database to establish a sample’s position within the tree. Using node-specific SNPs, the possible paths inside the tree are reconstructed, and the one with the most SNP matches is reported. PhyTB’s map view shows allele and strain-type frequencies by geographical location, developed from PolyTB source code [lization  and the entation . The soubix tool  on the sPhyTB uses 1,601 global MTBC whole-genome sequences from 11 studies with representation across all 7 major lineages . The phylogenetic tree constructed using the 91 k SNPs shows the expected clustering by lineage and strain-type  that is fixed in CAS (lineage 3) strains in Malawi . Further tool utility is possible by extending it to incorporate large variants and phenotypic data (e.g. drug-resistance profiles).The Project name:PhyTBProject home page:http://pathogenseq.lshtm.ac.uk/phytblive/index.phpSource code:PhyloTrack - http://sourceforge.net/projects/phylotrackOperating system(s): Platform independent Programming language: JavaScript and Perl Other requirements: None License: None Any restrictions to use by non-academics: None


Varicopsella(Multispinulosa) Li, Dai, and Li, subgen. nov., of the subfamily Macropsinae (Hemiptera: Auchenorrhyncha: Membracoidea: Cicadellidae) is proposed to accommodateVaricopsella(Multispinulosa)hamiltoniLi, Dai, and Li, sp. nov.from Guangxi province of China. The new subgenus and new species are described and illustrated. They can be distinguished mainly by characteristics of the fore wings with two anteapical cells; weak dorsoventrally flattened body; aedeagal shaft with paired apical processes on ventral margin; and the shape of the dorsal connective. An updated checklist and an illustrated key for identification of the species ofVaricopsellaalong with geographical distributions of the species are given.A new monobasic leafhopper subgenus, VaricopsellaMacropsis breakeyiVaricopsella.The oriental leafhopper genusVaricopsella: the fused sclerites between lora and frons on face, and the independently evolved dorsal connectives and pygofer processes in males. These specimens have been identified as a new species ofVaricopsella, and on the basis of the distinctive feature that the fore wings of the new species have two anteapical cells, we propose to place it in a new subgenus. The identification was verified by K. G. A. Hamilton, pers. comm. .Deposited in our collection from Guangxi Province of China , two specimens were found that bear the distinct generic characteristics ofVaricopsellais reported for the first time from China based on the new species. The new taxa (Varicopsella(Multispinulosa)hamiltoniLi, Dai, and Li, subgen. nov.and sp. nov.from Guangxi province (China) are described and illustrated. Photographs of imago and illustrations of the male genitalia are provided, and an illustrated key to the species of the genus based on the original descriptions and illustrations, with a map of distribution of the genus based on the primary, data are given.In this paper, the genusMorphological terminology used in this work followsThis paper and the nomenclature it contains have been registered in ZooBank. The LSID number is:urn:lsid:zoobank.org:pub:F8C13A75-E433-4DA8-8534-1144943E3686VaricopsellaHamilton, 1980Subfamily Macropsinae Evans, 1938 GenusVaricopsella(Varicopsella)Hamilton, 1980SubgenusVaricopsellaHamilton, 1980: 900. Type species:Macropsis breakeyiMerino, 1936.Varicopsellacan be distinguished from other Macropsinae mostly by the following characteristics: fused sclerites between lora and frons on face, and the unique male dorsal connectives each formed by two articulating parts with ventral pair appressed or fused with each other and dorsal ones produced as variously-shaped projections among species. The unique distinctive feature of the nominate subgenericVaricopsella(Varicopsella) is fore wings with three anteapical cells.The genusDistribution.Oriental, distributed in Philippines, Borneo, and India .Varicopsella (Multispinulosa)Li, Dai, and Li, subgen. nov.SubgenusVaricopsella (Multispinulosa) hamiltoniLi, Dai and Li, sp. nov.Type species:Description.Body -4 typicHead , in dorMale genitalia.Pygofer -8 broadDistribution.Oriental, China (new record of genus), Guangxi Province.Varicopsellasubgenus by the fore wings with two anteapical cells; weakly flattened external form of the body; unique male aedeagal shaft with paired apical processes on ventral margin; and shape of the dorsal connective.Remarks.The new subgenus can be easily distinguished from the other“multi-”and“spinu-losus”because of the several spines on the ventral margin projections of the pygofer lobe. The gender of the subgenus is feminine.Etymology.The subgenus name is derived from the Latin wordsVaricopsella (Multispinulosa) hamiltoniLi, Dai, and Li sp. nov. , China .Etymology.This species is named in honor of K. G. A. Hamilton for excellent contributions to the Auchenorrhyncha systematics and invaluable help to the first author.Varicopsella (Multispinulosa) hamiltonisp. nov.Distribution. China .Varicopsella (Varicopsella) basilana(Merino)Macropsis basilanaVaricopsella basilana,Hamilton, 1980: 900. Distribution. Philippines .Varicopsella (Varicopsella) breakeyi(Merino)Macropsis breakeyiVaricopsella breakeyi,Varicopsella (Varicopsella) davaoensis(Merino)Macropsis davaoensisVaricopsella davaoensis,Distribution. Philippines (Mindanao).Varicopsella(Varicopsella)elegansViraktamathVaricopsella elegansVaricopsella(Varicopsella)luzonensis(Merino)Macropsis luzonensisVaricopsella luzonensis,Varicopsella(Varicopsella)obtusaHamiltonVaricopsella obtusaVaricopsella(Varicopsella)otanesi(Merino)Macropsis otanesiVaricopsella otanesi,Varicopsellaspecies are keyed based on the variations of male genitalia, therefore two species only known by the female,V. (V.)basilana(V. (V.)davaoensishamiltonisubgen. nov. and sp. nov.1. Body form not strongly flattened dorsoventrally ; fore wV. (Varicopsella) 2– Body form strongly flattened dorsoventrally; fore wings with 3 anteapical cells; aedeagal shaft without pair of triangular processes apically –20………..V. (Varicopsella)breakeyi2. Pygofer with one small, inturned spine on each lobe ; ventra– Pygofer with two small apical spines or teeth on each lobe –20; venDorsal connective short with tip bifid or trifid , 19…………V. (Varicopsella)obtuse– Dorsal connective with tip bifid (V. (Varicopsella)elegansDorsal connective with tip trifid ; aedeagp bifid ; aedeagp bifid …………………V. (Varicopsella)otanesi– Pygofer with two extremely tiny spines on lobe (V. (Varicopsella)luzonensisPygofer with two small spines on each lobe ; dorsalon lobe ; dorsalon lobe ………………V


TM technology has been employed to analyse transcriptomes from the arm tissues of two European brittle star species, Amphiura filiformis and Ophiopsila aranea. About 48 million Illumina reads were generated and 136,387 total unigenes were predicted from A. filiformis arm tissues. For O. aranea arm tissues, about 47 million reads were generated and 123,324 total unigenes were obtained. Twenty-four percent of the total unigenes from A. filiformis show significant matches with sequences present in reference online databases, whereas, for O. aranea, this percentage amounts to 23%. In both species, around 50% of the predicted annotated unigenes were significantly similar to transcripts from the purple sea urchin, the closest species to date that has undergone complete genome sequencing and annotation. GO, COG and KEGG analyses were performed on predicted brittle star unigenes. We focused our analyses on the phototransduction actors involved in light perception. Firstly, two new echinoderm opsins were identified in O. aranea: one rhabdomeric opsin (homologous to vertebrate melanopsin) and one RGR opsin. The RGR-opsin is supposed to be involved in retinal regeneration while the r-opsin is suspected to play a role in visual-like behaviour. Secondly, potential phototransduction actors were identified in both transcriptomes using the fly (rhabdomeric) and mammal (ciliary) classical phototransduction pathways as references. Finally, the sensitivity of O.aranea to monochromatic light was investigated to complement data available for A. filiformis. The presence of microlens-like structures at the surface of dorsal arm plate of O. aranea could potentially explain phototactic behaviour differences between the two species. The results confirm (i) the ability of these brittle stars to perceive light using opsin-based photoreception, (ii) suggest the co-occurrence of both rhabdomeric and ciliary photoreceptors, and (iii) emphasise the complexity of light perception in this echinoderm class.Next generation sequencing (NGS) technology allows to obtain a deeper and more complete view of transcriptomes. For non-model or emerging model marine organisms, NGS technologies offer a great opportunity for rapid access to genetic information. In this study, paired-end Illumina HiSeq With the publication of the sea-urchin genome acting as a catalyser , 2, the stricto sensu, like echinoderms—have been largely understudied until recently , Oa-opsin 7 [CL2625.Contig1].DNA: DNA fragment size, Pep: peptide size prediction, TM: number of predicted transmembrane helix, MW: predicted molecular weight, Acc.: accession, Q.C.: query coverage, E val: E value, Id: identity. Reference numbers are the following: (XLSX)Click here for additional data file.S5 TableA. filiformis and O. aranea unigene are listed with the E-value of the top blast result. The A. filiformis and O. aranea proteins were then used as query in a reciprocal best BLAST search of the non-redundant protein database (NBCI) and the top result is listed along with the E-value of the blast result. Reference sequence accessions are listed in the A. carolinensis: Anolis carolinensis, B. taurus: Bos taurus, H. sapiens: Homo sapiens, S. kowaleskii: Sacoglossus kowalevski.Homologues to Gq and Gt/o/i phototransduction cascade components and their reciprocal best BLAST hit. For each query protein, the corresponding (XLSX)Click here for additional data file.


Phytophthora cinnamomi Rands remains an important concern on forest tree species. The pathogen causes root and collar rot, stem cankers, and dieback of various economically important Eucalyptus spp. In South Africa, susceptible cold tolerant Eucalyptus plantations have been affected by various Phytophthora spp. with P. cinnamomi considered one of the most virulent. The molecular basis of this compatible interaction is poorly understood. In this study, susceptible Eucalyptus nitens plants were stem inoculated with P. cinnamomi and tissue was harvested five days post inoculation. Dual RNA-sequencing, a technique which allows the concurrent detection of both pathogen and host transcripts during infection, was performed. Approximately 1% of the reads mapped to the draft genome of P. cinnamomi while 78% of the reads mapped to the Eucalyptus grandis genome. The highest expressed P. cinnamomi gene in planta was a putative crinkler effector (CRN1). Phylogenetic analysis indicated the high similarity of this P. cinnamomi CRN1 to that of Phytophthora infestans. Some CRN effectors are known to target host nuclei to suppress defense. In the host, over 1400 genes were significantly differentially expressed in comparison to mock inoculated trees, including suites of pathogenesis related (PR) genes. In particular, a PR-9 peroxidase gene with a high similarity to a Carica papaya PR-9 ortholog previously shown to be suppressed upon infection by Phytophthora palmivora was down-regulated two-fold. This PR-9 gene may represent a cross-species effector target during P. cinnamomi infection. This study identified pathogenicity factors, potential manipulation targets, and attempted host defense mechanisms activated by E. nitens that contributed to the susceptible outcome of the interaction.Damage caused by  Phytophthora are the most economically important pathogens of plants worldwide  has contributed knowledge towards several host responses during pathogen challenge . This is an invaluable resource that provides insight into pathogenicity determinants in this species. This has been demonstrated for other Phytophthora species sequenced genomes  in potato during its biotrophic phase, then manipulating the induction of HR during the necrotrophic phase  gene classes are differentially regulated against Phytophthora and are thought to be important for successful defense  discover pathogenicity factors produced by P. cinnamomi (ii) to determine the E. nitens defense response to the pathogen, and (iii) to identify host genes that could potentially be suppressed by the pathogen to promote susceptibility. We observed, among other responses, high expression of a putative P. cinnamomi crinkler effector in planta and the down-regulation of a PR-9 gene, which may represent a common host effector target in E. nitens; two factors possibly contributing to the susceptible outcome of the interaction.The E. nitens seedlings were obtained from parents that were part of a third generation commercial breeding program  The criterion for selection was based on wood density gain. An MLRelate analysis, using the microsatellite markers developed by Faria et al. . Previous sampling in E. nitens stands in South Africa have shown root rot due to P. cinnamomi , then stem inoculated with P. cinnamomi . A 4 mm cork borer and mycelial plug on cV8 agar  was used for inoculations. Mock-inoculated plants were treated identically to infected trees, with a sterile cV8 agar plug. Inoculation sites were covered with damp sterile cheesecloth, tinfoil, and Parafilm .The  Ribeiro : 200 ml/P. cinnamomi in commercially grown E. nitens, for both the mock-inoculated control and inoculated trees, stem tissue was harvested from 18 trees  at 5 days post inoculation (dpi). Three centimeters of stem tissue was harvested per inoculation site, with 1.5 cm of stem tissue below and above the center of the site. Harvested material was immediately frozen in liquid nitrogen.Stems were inoculated at two sites, 10 cm apart. In the attempt to represent the typical responses to Since sampling was destructive, nine extra trees were used to observe symptom development for 6 weeks following inoculation. Beneath-bark lesions were measured in other trials to statistically validate the effectivity of inoculation. For these lesions, a Shapiro-Wilk test was performed in GraphPad Prism 6 , 48 hpi, 96 hpi, and 1 week post inoculation (wpi). The harvested tissue was fixed in formalin-acetic acid-alcohol . For confocal microscopy, thin longitudinal- and cross-sections were made of stem tissue away from the immediate vicinity of the inoculation site. These sections were stained in 0.01% Calcofluor white fluorescent brightener 28 (Sigma-Aldrich®).Approximately 1.5–2 cm of stem tissue surrounding the inoculation site of Representative stem tissue samples from each time-point were visualized under a Stemi SV6 stereo microscope . Images were captured using an AxioCamMRc digital camera (Zeiss) and Axiovision 4.7 software (Zeiss). The presence of hyphae inside the sampled tissue at various time-points was verified through use of a confocal laser scanning microscope . To view Calcofluor-stained tissue, a wavelength of 405 nm was used, and a 543 nm wavelength was used to visualize autofluorescence. LSM Image Browser v4.2.0.121 (Zeiss) was used to view images generated from the confocal microscopy.RNA was isolated using a modified cetyl-trimethyl-ammonium-bromide (CTAB) method . Sequencing of mRNA was performed with random fragmentation of the mRNA and adapter ligation. Fifty bp paired end reads were obtained using an Illumina HiSeq 2000 .http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) were used to format reads and to assess read quality.Adaptors, low quality reads, and reads with more than 10% unknown nucleotides were removed from the dataset by BGI. The Galaxy platform  was performed by Cufflinks . The genome assembly, based on Illumina, 454 and Sanger sequencing, is 77.97 Mbp and the sequence read coverage depth is 69.6x . The database was downloaded in July 2014 and hits with the lowest e-value were considered. Selected genes were also subjected to Blast2GO® analysis to annotate the gene models.Reads from the three uninoculated samples and the three inoculated samples were mapped to the E. grandis v1.1 gene and transcript models to TAIR10 and TAIR9 identifiers based on a reciprocal BLAST analysis. The significantly differentially expressed genes  with TAIR10 putative orthologs were divided into up- and down-regulated datasets, which were analyzed for gene ontology (GO) over-representation in BiNGO v2.44  was used to match significantly differentially expressed Genomic contamination in total RNA samples was removed by treating extracted total RNA samples with RNase-free DNaseI enzyme . Total RNA samples were then purified using the RNeasy® MinElute Kit (Qiagen Inc.) and subsequently analyzed using a Bio-Rad Experion automated electrophoresis system , to determine RNA integrity. The Improm-IITM Reverse Transcription System  was used to synthesize first strand cDNA from purified RNA samples. Primers were designed using Primer Designer 4 v4.20 . Primer pairs are indicated in Table t-test in Microsoft® Office Excel 2010.Real-time quantitative reverse transcriptase PCR (RT-qPCR) was conducted according to the Minimum Information for Publication of RT-qPCR Experiments (MIQE) guidelines  was observed in the control (mock inoculated) samples, which were considered conserved eukaryotic gene sequences. On average, 1% of the reads mapped to the P. cinnamomi genome in the inoculated samples. Approximately 78% of the transcripts derived from both inoculated and mock-inoculated E. nitens samples mapped to the E. grandis genome  to the plant host interactions database described as loss in pathogenicity, avirulence determinants, or reduced virulence are indicated in Table P. cinnamomi putative CRN (JGI: Phyci261170) was aligned with other putative and described CRN family members and a maximum likelihood phylogenetic tree describing the relationship was produced. A congruent topology was recovered from Bayesian inference. The closest relationship was to a putative CRN protein from P. ramorum. Both the P. ramorum and P. cinammomi putative CRN proteins are closely related to the better characterized P. infestans CRN proteins  of 0.05. The expression of a sub-set of differentially expressed genes EgrWRKY75, EgrPR-3, EgrNRT2.5, EgrMLO, and EgrLRR-RLK7 were validated using RT-qPCR , and PR-5 (thaumatin-like and osmotin). These were not only consistently highly up-regulated in inoculated tissue, but there were multiple E. nitens putative orthologs per A. thaliana gene that are all regulated at similar fold-change levels. Putative E. nitens orthologs of PR-4 (chitin-binding), PR-8 (chitinase class III), and PR-12 (defensins) were up-regulated. The PR-9 (peroxidase) and PR-10 (ribonuclease-like) classes contained a mix of up- and down-regulated putative orthologs. PR-14 (lipid transfer proteins) and PR-15  putative orthologs were all down-regulated.One of the noteworthy aspects of the RNA-seq data obtained was that several putative E. nitens putative PR-9 ortholog (Eucgr.E04056) was down regulated by a 1.85 fold-change on a log2 scale in the P. cinnamomi-inoculated tissue (indicated in bold in Table Carica papaya (EL784270) and A. thaliana. The amino acid alignment is indicated in Figure E. nitens PR-9 sequence shared 80% amino acid identity with the C. papaya PR-9 sequence.The  Eucgr.E0056 was dE. nitens and P. cinnamomi provided a model system to study the compatible interaction between Eucalyptus spp. and Phytophthora spp. One year old E. nitens plants were stem inoculated using P. cinnamomi and pronounced lesions were obtained  transcript, which elicits necrosis and defense gene expression in hosts and is necessary for attachment onto plant surfaces , chitin-binding protein (PR-4), and thaumatin-like protein/osmotin (PR-5) putative orthologs appears to be highly up-regulated in E. nitens  and its putative orthologs have been suppressed in different hosts upon inoculation with P. sojae, P. palmivora, and P. infestans  are potential cross-species P. cinnamomi pathogenicity and virulence factors and E. nitens defense mechanisms utilized against P. cinnamomi. Several factors may contribute to the compatibility however, we have used further sequence and functional genetics support to motivate that the P. cinnamomi CRN and the E. nitens PR-9 genes are important contributors to the susceptible outcome. Future work involving comparison with a resistant interaction over a time-course is required to provide an indication of host targets manipulated by P. cinnamomi and to enhance understanding of the defense pathways required for resistance.The outcomes of this dual RNA-seq study provided valuable insights into FM, LS, SiN, and TM performed the experimental work, conducted and interpreted data analyses. SaN conceived the study, obtained funding to support the research. AM provided input into the experimental design and technical aspects of RNA-sequencing and assisted with critical evaluation of the manuscript. FM, LS, and SaN wrote the manuscript with input from DB and NV, who supervised aspects of this research.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Actinobacillus pleuropneumoniae is the pathogen of porcine contagious pleuropneumoniae, a highly contagious respiratory disease of swine. Although the genome of A. pleuropneumoniae was sequenced several years ago, limited information is available on the genome-wide transcriptional analysis to accurately annotate the gene structures and regulatory elements. High-throughput RNA sequencing (RNA-seq) has been applied to study the transcriptional landscape of bacteria, which can efficiently and accurately identify gene expression regions and unknown transcriptional units, especially small non-coding RNAs (sRNAs), UTRs and regulatory regions. The aim of this study is to comprehensively analyze the transcriptome of A. pleuropneumoniae by RNA-seq in order to improve the existing genome annotation and promote our understanding of A. pleuropneumoniae gene structures and RNA-based regulation. In this study, we utilized RNA-seq to construct a single nucleotide resolution transcriptome map of A. pleuropneumoniae. More than 3.8 million high-quality reads (average length ~90 bp) from a cDNA library were generated and aligned to the reference genome. We identified 32 open reading frames encoding novel proteins that were mis-annotated in the previous genome annotations. The start sites for 35 genes based on the current genome annotation were corrected. Furthermore, 51 sRNAs in the A. pleuropneumoniae genome were discovered, of which 40 sRNAs were never reported in previous studies. The transcriptome map also enabled visualization of 5'- and 3'-UTR regions, in which contained 11 sRNAs. In addition, 351 operons covering 1230 genes throughout the whole genome were identified. The RNA-Seq based transcriptome map validated annotated genes and corrected annotations of open reading frames in the genome, and led to the identification of many functional elements . The transcriptional units described in this study provide a foundation for future studies concerning the gene functions and the transcriptional regulatory architectures of this pathogen. During the past decade, a large number of bacterial complete genomes have been finished, providing the sequence basis for transcriptomics and proteomics. The genome sequence contains all the information of the functional elements including genes, non-coding RNAs, and UTRs, which are required for a system-level understanding of the survival mechanisms and pathogenesis of microorganism . HoweverS. Typhi has identified 40 novel non-coding RNAs and re-annotated a number of hypothetical genes  . The 2.2th phase . After rdatabase  Fig 1)..A. pleurA. pleuropneumoniae genomes.Overall, 32 novel protein coding regions were identified according to the transcriptome map . The aveYrhG”   are short (30–500 bp) untranslated transcripts and often act as regulators of gene expression through a variety of mechanisms –33. The 3 genome . The avecle RNA) . The othA. pleuropneumoniae, the sequences of all predicted sRNA were searched against the non-redundant, nucleotide database at NCBI. Thirty of the sRNAs were highly conserved (>95% identity with 100% coverage) only in A. pleuropneumoniae. A set of 20 sRNAs were conserved in the related Pasteurellaceae family including M. varigena, M. haemolytica, A. suis, B. trehalosi and A. lignieresii. Only 1 sRNA was also present in the species far from A. pleuropneumoniae, such as in Mycoplasma pulmonis and Vibrio.To study the conservation of identified sRNAs of A. pleuropneumoniae JL03  contain regulatory elements to control gene expression , 38. Theiae JL03 . One 5’UA. pleuropneumoniae JL03 . With the accumulation of sequence information and the improvements in bioinformatic techniques, original genome annotations need to be revised to make them more accurate and useful to explore gene functions and regulations. To acquire complete and well described genome annotation, plenty of studies have worked on genome re-annotation with various methods in eukaryotes and prokaryotes –43. Bothca PHL213.A. pleuropneumoniae JL03 genome was sequenced and published in 2008 which provided the foundation for all subsequent analysis of the A. pleuropneumoniae genome and gene functions [A. pleuropneumoniae JL03 genome at the transcriptome level. A single resolution transcription map of the A. pleuropneumoniae JL03 genome was generated which enabled us to re-annotate the existing genome. Ninety-four percent of the RNA-seq reads mapped to the A. pleuropneumoniae JL03 genome, which is comparable with the other RNA-seq studies. Ninety-four percent cDNA reads were assignable to the genome of Bacillus anthracis [Burkholderia cenocepacia [Histophilus somni [The unctions –58. But nthracis , 95% in ocepacia  and 94% us somni , which iA. pleuropneumoniae. Because of the small size and low expression level, sRNAs are hard to detect using traditional screening strategy. On account of lack of defined sequence features, computational approach for sRNA prediction is not powerful enough. Previous studies have shown the power of RNA-seq in bacterial sRNA identification [A. pleuropneumoniae. We found that 10 of the 51 sRNAs were homologous to characterized sRNAs. For instance, APP-S17 was classified as T44 RNA, which has been identified in several species of enteric bacteria [E. coli, 6S RNA alters the transcription of most genes in stationary phase. During this phase, 6S RNA accumulates in cells; loss of 6S RNA leads to decreased survival compared to wild-type cells [E. coli suggest 6S RNA may contribute significantly to transcriptional regulation even when levels are not maximal, such as in exponential phase when 6S RNA is only one-tenth as abundant as in stationary phase [S-dependent genes in Gram-negative bacteria [A. pleuropneumoniae. We also identified APP-S9 which was classified as GcvB RNA. The GcvB RNA has been found in a range of bacteria such as E. coli [Y. pestis [H. somni [Salmonella [E. coli and Salmonella species, the GcvB RNA has been shown to regulate a large number of genes which are involved in the amino acid transport systems and amino acid biosynthesis [E. coli, transcription of the GcvB RNA is activated by the adjacent gcvA gene and regulates genes by acting as an antisense binding partner of the mRNAs for each regulated gene [gcvA (APJL_0132) which was annotated as DNA-binding transcriptional activator. Taken together, we hypothesize that APP-S9 may be activated by gcvA (APJL_0132) and have regulatory activity in the amino acid transport systems and amino acid biosynthetic genes in A. pleuropneumoniae. The other 41 novel identified sRNAs may play regulatory roles in basic life and/or virulence as described in other bacteria [A. pleuropneumoniae implying these sRNA are A. pleuropneumoniae-specific. Only 1 sRNA is present in the species far from Pasteurellceae family. This sRNA may be responsible for the basic survival of many species. The sRNAs present in different species may be functional in adaptation to different environmental conditions.In bacteria, small regulatory RNAs (sRNAs) play a pivotal role in regulation of the gene expression, controlling a variety of adaptation processes in response to changes in the environment. Although the genome has been sequenced several years ago, no information about sRNAs is available in fication , 60. In bacteria . The funpe cells . Howeverpe cells . Recent ry phase . In addibacteria . Taken t E. coli , Y. pest. pestis , H. somnlmonella . In E. cynthesis , 65, 66.ted gene . APP-S9 bacteria , 68. AmoA. pleuropneumoniae which can discover regulatory elements in these regions such as riboswitches, RNA thermometers or small leader peptides. We identified 5’-UTR of 715 annotated genes and 3’-UTR of 384 annotated genes in A. pleuropneumoniae JL03. Most of the identified 5’-UTRs (72%) were very short with the size less than 100 bp, which is similar to other bacterial, such as 75% of the 5’-UTRs for annotated genes were shorter than 100 bp in C. glutamicum ATCC 13032 [E. coli, the riboswitches upstream of moaA gene regulate translation of the moaABCDE operon by blocking ribosome-binding sites [moaA (APJL_0688) which was annotated as molybdenum cofactor biosynthesis protein A, implying its regulatory role in molybdenum cofactor biosynthesis. Additionally, one RNA thermometer (APP-S8) was observed within the 5’-UTR of cspC (APJL_0119). The gene cspC encodes a cold shock-like protein. Cold shock proteins are stress induced regulators which is functional in response to low temperature and also in normal conditions [cspC was repressed after acute infection of A. pleuropneumoniae [A. pleuropneumoniae. In E. coli, the cspA thermosensor acts as an RNA thermometer, regulating the expression of cspA in response to temperature [cspC. A recent study has documented that Gram-negative bacteria contain a conserved motif in their 5’-leader sequences [A. pleuropneumoniae, we found a small leader peptide (APP-S51) within 5’-UTRs of the operon involved in histidine synthesis . These leader peptides are possibly involved in transcription attenuation of the target genes dependent on the amino acid level [A. pleuropneumoniae.RNA-Seq data provides information to identify the UTRs of annotated genes. Until now, the mapping of UTRs has not been performed on CC 13032 , 69. WitCC 13032 . In E. cng sites . APP-S20 5’-UTRs % were veeumoniae  and afteeumoniae , suggestequences . In A. pid level . In concA. pleuropneumoniae. The majority of the identified operons contain 2–9 genes, which is a general trend in other previously reported bacteria [pgaA, pgaB, pgaC, pgaD) has been characterized as the operon involved in biofilm formation of A. pleuropneumoniae [A. pleuropneumoniae were also identified, such as cps3ABCD (APJL1611-1614) encoding CPS biosynthetic enzymes, cpxDCBA (APJL1615-1618) encoding proteins involved in the export of CPS and apxIIICABD (APJL1344-1347) encoding the ApxIII structure, activation and secretion proteins [apxII operon which has been reported to consist of apxIICA and a partial apxIIB’ without apxIID [putA and putP, annotated as bifunctional proline dehydrogenase/pyrroline-5-carboxylate dehydrogenase and proline symporter/permease was not predicted by DOOR. The functions of putAP gene products are quite conserved among different bacteria [E. coli and S. typhimurium, the orthologs of these genes are well known to form the proline utilization operon [A. pleuropneumoniae and also demonstrated RNA-Seq can improve operon identification in bacterial genomes.For prokaryotic organisms, identification of operon structures at the genome scale is significant in studying the gene function and the regulatory networks. Based on the RNA-seq data, we identified 351 expressed operons covering 1,230 genes of bacteria , 54. Fureumoniae . Operonsproteins . Remarkat apxIID . The probacteria , 81. In n operon , 83. OurA. pleuropneumoniae at single nucleotide resolution using deep RNA-seq was presented in this work. This study confirmed previously annotated open reading frames and provided a more accurate map of gene boundaries. The analysis also identified 32 novel protein-coding genes and 51 sRNAs, defined UTRs regions and generated complete operon structures throughout the genome. The analysis results indicated high complexity of the A. pleuropneumoniae transcriptome. The functional elements on a whole genome level identified in this study provide a useful reference transcriptome to comprehensively understand the A. pleuropneumoniae survival and infection process.In summary, a global transcriptome profiling of S1 Fig(TIF)Click here for additional data file.S2 Fig(A) The time of cell harvest for RNA sequencing is at mid-log phase . (B) Total RNA was extracted from cells and verified for integrity on a 1% agarose gel.(TIF)Click here for additional data file.S1 TableList of the UTRs and their genome location identified in this study.(XLS)Click here for additional data file.S2 TableLine A has a list of the co-expressed gene pairs predicted by DOOR program. Line B has a list of the co-expressed gene pairs predicted by RNA-seq. Line C has a list of the co-expressed gene pairs common to both DOOR and RNA-Seq. Line D has a list of the co-expressed gene pairs unique in RNA-Seq experiment.(XLS)Click here for additional data file.S3 TableOperons identified by RNA-seq were compared with the operons predicted by DOOR database. The operons along with their ID and genes are listed. Operon type: A- operons common to both DOOR and RNA-Seq; B- operons based on RNA-seq data and not predicted by DOOR; C-operon having different compositions.(XLS)Click here for additional data file.


Testudo graeca tortoises are distributed in the south-eastern part (Dobrogea region) of Romania. T. graeca is a potential host for the three-host ticks, Hyalomma aegyptium. H. aegyptium ticks are important from epidemiological point of view as they constitute potential reservoirs for numerous zoonotic bacterial pathogens . However, H. aegyptium was reported to host less studied bacteria, non-Lyme members of genus Borrelia. Despite its relatively wide distribution range, the extent of co-distribution of ticks with these bacteria was not investigated in detail. The aim of the present study was to evaluate H. aegyptium engorged ticks collected from tortoises in south-eastern Romania for the presence of non-Lyme Borrelia. Between 2008 and 2013, 448 H. aegyptium ticks were collected from 45 T graeca tortoises located in Dobrogea region in Romania. DNA extraction was performed individually from each tick using a commercial kit. For the total 78 (17.4%) Borrelia spp. positive ticks, PCR analysis targeting the intergenic spacer 5S-23S region, glpQ, respectively gyrB genes, and further sequencing was performed for the further identification. Sequences of gyrB and glpQ genes showed 99%-100% similarities with reptile-associated Borrelia turcica. The most frequently infected stages were males  followed by females  and nymphs . This is the first report of Borrelia turcica in Romania.This research was supported by grant CNCSIS IDEI PCCE 7/2010 and IDEI PCE 236/2011.


Rhabdastrella globostellata, Callyspongia sp., Rhaphoxya sp., and Acanthella cavernosa, were collected from exposed shallow reef slopes and a deep reef drop-off. Based on 16S rRNA gene pyrosequencing profiles, beta diversity analyses revealed that each sponge species possessed a specific microbiota that was significantly different to those of the other species and exhibited attributes that are characteristic of high- and/or low-microbial-abundance sponges. These findings emphasize the influence of host identity on the associated microbiota. Dominant sponge- and seawater-associated bacterial phyla were Chloroflexi, Cyanobacteria, and Proteobacteria. Comparison of individual sponge taxa and seawater samples between shallow and deep reef sites revealed no significant variation in alpha diversity estimates, while differences in microbial beta diversity (variation in community composition) were significant for Callyspongia sp. sponges and seawater samples. Overall, the sponge-associated microbiota is significantly shaped by host identity across all samples, while the effect of habitat differentiation seems to be less predominant in tropical reef sponges.Sponges (phylum Porifera) are important members of almost all aquatic ecosystems, and are renowned for hosting often dense and diverse microbial communities. While the specificity of the sponge microbiota seems to be closely related to host phylogeny, the environmental factors that could shape differences within local sponge-specific communities remain less understood. On tropical coral reefs, sponge habitats can span from shallow areas to deeper, mesophotic sites. These habitats differ in terms of environmental factors such as light, temperature, and food availability, as well as anthropogenic impact. In order to study the host specificity and potential influence of varying habitats on the sponge microbiota within a local area, four tropical reef sponges,  In marine ecosystems, sponges represent common and versatile members of the benthos, with distribution ranges along large environmental gradients and across various habitats including deep sea benthos, seamounts, polar regions, and temperate and tropical coral reefs . Many spWhile sponges occupy a range of different depths, knowledge about the influence of depth on the composition of the sponge microbiota still remains rather limited . Spatialin situ sponge-associated communities along an MCE depth gradient suggested host-specific local variations in community structure, which are possibly influenced by prevailing biotic and abiotic factors  based on all available samples we investigate the degree of host specificity of microbial communities among several tropical sponge species, and (2) based on a habitat-specific subset of samples we estimate the influence of local habitat variation on sponge- and seawater-associated microbial community patterns. We sampled two closely related but environmentally differentiating habitats; a deep drop-off (Guam Blue Hole) for the collection of the mesophotic reef samples and nearby shallow reef slope sites for comparison.In this study, we apply high-throughput 16S rRNA gene amplicon pyrosequencing to profile four demosponge speciesR. globostellata (n = 6 specimens), Callyspongia sp. (n = 7), A. cavernosa (n = 3), Rhaphoxya sp. (n = 5) and seawater (n = 5) were collected from closely connected Guam reef sites and depths  via snorkelling and technical diving  and A2 & A3  (see http://cdip.ucsd.edu/themes/). SST averaged 29.8 °C in June and 29.7 °C in July 2010. Temperatures at depth (90 m) averaged 25 °C (taken with Dive rite-Intel HE and VR3-trimix dive computers). All samples were frozen, freeze-dried, and then stored at −20 °C prior to further processing. Sponges were identified by Dr Nicole J. de Voogd and vouchers of each species were preserved in 70% ethanol at the Naturalis Biodiversity Center, Leiden, Netherlands. Genomic DNA was extracted from sponge tissue and water filters  by bead-beating in an ammonium acetate buffer, as previously described . After chimera-checking with UCHIME (http://www.mothur.org/wiki/Greengenes-formatted_databases). Non-target sequences  were removed.Sequences were processed using mothur v.1.33.0 . Pyroseqh UCHIME , unique summary.single & subsample=T; 1,000 iterations). For visualization and interpretation of the microbial community data, we used standardized 97%-OTU abundance information . To estimate the variance of beta diversity, two hypothetical treatments were applied to the dataset: (a) ‘habitat’  and (b) ‘group’  (betadisper (followed by pairwise Tukey’s Honestly Significant Difference tests) and permutest function from the vegan package in R (v. 3.0.2) (adonis function (1000 permutations) from the vegan package to estimate the variances in beta diversity for both treatment groups  were assessed with multivariate non-metric multidimensional scaling (nMDS) using the metaMDS function from the vegan package. Hypothesis-based treatments were added as dispersion ellipses to the ordination plots with the vegan function ordieellipse . All multivariate analyses were performed with relative abundance and presence/absence data. The contribution of OTUs to average overall pairwise sample dissimilarity in R. globostellata, Callyspongia sp. and seawater specific datasets (‘habitat’ treatment) was assessed using the vegan function simper for similarity percentages (SIMPER). Relative abundance of the 30 most abundant OTUs (each ≥0.5% relative abundance) and relative phylum abundance were visualized with JColorGrid (get.oturep command in mothur and BLAST searched against the NCBI Nucleotide collection (discontiguous megablast & Models excluded). The best hits and representative sequences for the most abundant OTUs can be accessed via the Figshare online repository (https://dx.doi.org/10.6084/m9.figshare.2366827). Hierarchical clustering based on all OTUs was performed using the vegan package in R via the function vegdist (Bray-Curtis dissimilarity) and hclust (method = average) and subsequently added onto the phylum fingerprint. As described above, all samples were included in the group-based  multivariate community (adonis and betadisper) and ordination (nMDS) analysis. In the subsequent habitat-based  comparison, A. cavernosaand Rhaphoxya sp. samples were omitted from all analyses, and seawater samples from multivariate community analysis and ordination analyses, due to insufficient numbers of replicates in the dataset for the particular habitats. The 97% OTU abundance table combined with the Greengenes classification for each individual OTU can be accessed via the Figshare online repository (https://dx.doi.org/10.6084/m9.figshare.2063280).After raw data processing, mothur was used to group the obtained high quality sequences into 97% average neighbour sequence-similarity threshold operational taxonomic units , for calculation of Shannon & inverse Simpson diversity, and rarefaction curves. Reads were evenly subsampled to 2,387 sequences per sample for all alpha diversity calculations (mothur command eawater) . These teawater)  with the. 3.0.2) . We usedt groups . VisualiolorGrid . RepreseA. cavernosa and R. globostellata compared to seawater and Callyspongia sp. samples , number of sequences, observed and average 97%-OTUs were higher overall within the seawater group in comparison to the sponge groups . Coverag samples . These g samples . Across  samples . Pooled bacteria . Howeverrchaeota . Almost rchaeota . Hierarcrchaeota . In addirchaeota –S2E, whi samples .n = 30) included members of the Proteobacteria , Cyanobacteria, Acidobacteria, Chloroflexi, “Poribacteria”, Actinobacteria, Nitrospirae and PAUC34f  was mostly limited to Callyspongia sp. and seawater samples. Among the abundant R. globostellata OTUs were representatives of Chloroflexi class SAR202, the candidate phylum “Poribacteria,” various Acidobacteria and Proteobacteria, and the uncultured sponge symbiont PAUC34f. Interestingly, while most abundant OTUs in R. globostellata and Rhaphoxya sp. were largely shared, and evenly distributed overall, the abundant OTU0016 (Synechococcus) in R. globostellata was not detected in Rhaphoxya sp. On the contrary, the latter sponge species featured an Acidobacteria OTU (OTU0033) that was predominantly shared with A. cavernosa instead of R. globostellata , OTU0007 , and OTU0027  (The most abundant OTUs ( PAUC34f . The disstellata . Additioeraceae) .Callyspongia sp. samples to the differences between the groups  sponge taxa  in the shallow reef slope habitats. In the deep drop-off Callyspongia sp. and seawater samples a second cyanobacterium, genus Prochlorococcus (OTU0003), was most dominant. The dominant Synechococcus OTUs were also separated in R. globostellata (OTU0016) and Callyspongia sp. and seawater . Additionally, compared to Callyspongia sp. and seawater, which exhibit dominant OTUs either in shallow reef slopes or deep habitats, the main contributing OTUs in R. globostellata dominate completely the specimens from shallow habitat  with the highest average abundance in SIMPER in each of the sample groups presents three distinct microbial communities, with only slight variations between the two habitats . For exae groups . The mos habitat . The con habitat –5C; R. gCallyspongia sp. and seawater samples appear to have an intrinsic microbial community composition, which is variable enough to significantly separate the intra-species communities by their shallow reef slope or deep drop-off habitats. On the other hand R. globostellata exhibits an observable difference in community variation that is not significant. In addition, we could also observe significant microbial specificity across all analyzed sponge taxa independent of habitat. Since the shallow collection sites for Callyspongia sp. and R. globostellata inside Apra Harbor represent the same shallow water fore reef slope habitat dominated by Porites rus corals, it is unlikely that the observed differences in beta diversity between shallow reef slope or deep drop-off habitats are due to collection of Callyspongia sp. from the two shallow Apra Harbor sites.In the present study divergent patterns between two different habitats were visible in the tropical sponge and seawater microbiota. R. globostellata and Rhaphoxya sp. microbial communities. Culture-dependent approaches reported only Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria (Alpha & Gamma) from R. globostellata  or low microbial abundance (LMA) sponges is lacking in the literature, the associated microbial phylotypes are congruent with the microbial phyla commonly found in association with HMA sponges   .A. cavernosa and Callyspongia sp. to the LMA group, with abundant Proteobacteria  and Cyanobacteria (Synechococcus) microbial community members , Actinobacteria, Bacteroidetes and Proteobacteria (Beta & Gamma). Within the Betaproteobacteria, the order EC94 exhibited the highest overall abundance in our Callyspongia sp. samples and among all OTUs. Reports on this order are rare, but recent community analyses on different demosponges found this microbial taxon to be dominant in the deep sea sponge Inflatella pellicula and several shallow water sponges from Korea . In addition, the observed microbial community patterns in Callyspongia sp. appear to be more closely related to the seawater samples (see OTU and phylum composition). This high similarity with seawater communities is a well-known feature of LMA sponges  have been sampled at different points in time. However, a temporal effect on the community structure is not evident, which is in accordance with recent research showing the low seasonal variability of the sponge-microbiota , which leads to the conclusion that in this particular case the observed habitat-specific nMDS and hierarchical clustering patterns are random variations of the microbial community composition. In order to sample in two environmentally very distinct but closely related habitats, we focused sampling on “shallow” and “very deep” depths as categorized by In the present study, Callyspongia sp. and seawater samples, either Cyanobacteria or Proteobacteria OTUs were mainly responsible for the differences observed by SIMPER. In particular, cyanobacterial Synechococcus OTUs were among the main contributors to the shallow microbiota, whereas only one Prochlorococcus OTU was a dominant cyanobacterium contributing to the deep group from mesophotic depths. The predominance of photoautotrophs in tropical filter-feeding sponges is intuitive given the widespread prevalence of cyanobacteria in the ocean. LMA sponges in particular are known to harbour cyanobacteria in high abundance (Synechococcus spongarium)  . Moreovengarium) . Therefonges see . Besidesnges see . Given tncertain . A recenncertain . Conversncertain , differe hystrix  and Montavernosa . Recent avernosa .R. globostellata, one cyanobacterial OTU (OTU0016) is predominant within the deep sheltered communities, but also present at high abundance in the shallow specimens of this species  yielded different BLAST results . This corresponds to the theory that LMA sponges generally acquire their microbial symbionts via horizontal transmission from the surrounding environment, while HMA sponges possess a more individual microbial community that does not mirror the surrounding seawater microbiome as closely as their low abundance counterparts  are known to contain (bacterio)chlorophyll-based phototrophic lineages  is large enough to observe significant differences. However, the actual environmental factors contributing to the observed habitat-dependent variances remain uncertain, although we speculate that temperature may be less likely to have caused the variations in the sponge microbiota between shallow and deep specimens. While sponge-microbe communities show an overall stability along large geographic and temporal gradients, local environmental factors may have an effect on the small-scale patterns observed here. To further test the hypothesis of differential sponge-associated microbial communities along local depth gradients, functional and temporal aspects should be considered in future in situ studies. Moreover, since temporal turnover of phytoplankton is faster in the tropics  R. globostellata, (D) seawater, (E) A. cavernosa, (F) Rhaphoxya sp. sponge specimens based on 97%-OTU amplicon subsets.Dendrograms showing the Bray-Curtis dissimilarity of microbial communities of (A) the complete sample dataset, (B) Click here for additional data file.10.7717/peerj.1936/supp-3Table S1Habitat file used for grouping of samples in multivariate analyses (nMDS & adonis).Click here for additional data file.10.7717/peerj.1936/supp-4Table S2p adj giving the p-value after adjustment for the multiple comparisons.Tukey multiple comparisons of means for the group based betadisper analysis. 95% family-wise confidence level—diff giving the difference in the observed means, lwr giving the lower end point of the interval, upr giving the upper end point and Click here for additional data file.10.7717/peerj.1936/supp-5Table S3p-value below diagonal, permuted p-value above diagonal.Pairwise comparisons of the group based permutation test for homogeneity of multivariate dispersions. Observed Click here for additional data file.


Hoeflea may be examples of such bacteria; however, data describing their metabolisms are scarce. Here, we report the draft genome sequence of Hoeflea sp. strain BAL378, a putative producer of bacteriocins, polyketides, and auxins, as demonstrated by genome mining.Some phytoplankton-associated marine bacteria produce bioactive compounds. Members of the genus  Hoeflea belongs to the Phyllobacteriaceae family of the Alphaproteobacteria. It was established in 2005  into the novel Pseudorhodobacter genus  and promote phytoplankton growth (auxins) . Hence, we report here the draft genome sequence of Hoeflea sp. BAL378, a strain isolated from Baltic Sea surface water  , 10. Whece water .600) of ≈1 in ZoBell medium  resource version 2.0 (Genomic DNA was extracted from a culture growing to an optical density at 600 nm (ODl medium  using thl medium , and seqsion 2.0 . Secondasion 2.0  and NaPDHoeflea sp. BAL378 harbors two bacteriocin gene clusters and two terpene-encoding gene clusters. The RAST annotation suggested that bacteriocins produced by the strain are similar to colicin V and the broad-spectrum antibacterial protein marinocine from the marine bacterium Marinomonas mediterranea (According to antiSMASH, terranea . The annJRJG00000000. The version described in this paper is the first version.This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession no.


Prevotella (P-type) or Bifidobacterium/Bacteroides (BB-type), respectively. Majority in China, Japan and Taiwan harbored BB-type, whereas those from Indonesia and Khon Kaen in Thailand mainly harbored P-type. The P-type microbiota was characterized by a more conserved bacterial community sharing a greater number of type-specific phylotypes. Predictive metagenomics suggests higher and lower activity of carbohydrate digestion and bile acid biosynthesis, respectively, in P-type subjects, reflecting their high intake of diets rich in resistant starch. Random-forest analysis classified their fecal species community as mirroring location of resident country, suggesting eco-geographical factors shaping gut microbiota. In particular, children living in Japan harbored a less diversified microbiota with high abundance of Bifidobacterium and less number of potentially pathogenic bacteria, which may reflect their living environment and unique diet.Asia differs substantially among and within its regions populated by diverse ethnic groups, which maintain their own respective cultures and dietary habits. To address the diversity in their gut microbiota, we characterized the bacterial community in fecal samples obtained from 303 school-age children living in urban or rural regions in five countries spanning temperate and tropical areas of Asia. The microbiota profiled for the 303 subjects were classified into two enterotype-like clusters, each driven by  Bacteroides, Prevotella, and RuminococcusPrevotella or Bacteroides, within or across cohorts, although the typing depends on the clustering model used and boundaries between the enterotype clusters become less clear-cut with increase of sample sizes456789Several hundred microbial species inhabit the human gastrointestinal (GI) tract and constitute a complex ecological community that influences the host's normal physiology and susceptibility to disease through their collective metabolic activities of the community members and their interactions with the host1Bacteroides enterotypeMultiple intrinsic and extrinsic factors influence the structure of gastrointestinal (GI)-tract microbiota. Those identified include microbes acquired at birth, diet, host genetics and physiology, drug intake, and disease1112141516181920Prevotella and Xylanibacter  living in urban and rural cities in China, Japan, Taiwan, Thailand, and Indonesia that span temperate and tropical areas of Asia. Children aged 7 to 11 years old were chosen due to the following two reasons: i) gut microbiota of this age was reported to associate with adult-like configuration deviating from infant microbiota625To investigate the bacterial composition of the GI tracts of healthy Asian children, we collected fresh stool samples from 303 children aged 7 to 11 years old who were living in 10 cities in 5 countries, together with their physiological characteristics, dietary intake, and health condition . The V6–Prevotellaceae, whereas those of children in the other cities are highly abundant in Bacteroidaceae and Bifidobacteriaceae. These results were further supported by the qPCR data on the number of each bacterial group  in the majority of children residing in other cities. Notably, in Japan, Prevotella was detected in only approximately 10% of the children. In contrast, Bacteroides fragilis group and the genus Bifidobacterium were detected at >108 cells/g feces in most children  residing in the five countries, although they were considerably less populated in Thailand and Indonesia.The bacterial composition of 303 samples was determined in each taxonomic level according to the read counts of the 3,003 taxonomically-annotated OTUs in each sample. The 303 individual data were statistically compared according to residential place . The fecal group . In KhonPrevotellaceae and negatively loaded with the rest of the predominant families, Bacteroidaceae, Bifidobacteriaceae, Lachnospiraceae, and Ruminococcaceae. Principal component 2 (PC2) is heavily loaded with Bifidobacteriaceae and negatively loaded with Lachnospiraceae and Ruminococcaceae. In Prevotellaceae or Bacteroidaceae/Bifidobacteriaceae along with the common dominant layer of Lachnospiraceae and Ruminococcaceae. In contrast, the PC2-ordered graph shows a continuous gradient from Bifidobacteriaceae to Lachnospiraceae. As shown in the box plot under the bacterial composition graph in Using principal component analysis (PCA), family-level bacterial-composition data of all 303 Asian children were decomposed into two factors that explained 64.4% of the variance . PrincipPrevotellaceae-defined microbiota of PC1-positive group is referred to as “P-type”, whereas the Bifidobacteriaceae/Bacteroidaceae-defined microbiota of the PC1-negative group is termed as “BB-type”. The ratio of P-type and BB-type in each city is graphed in Clustering of the 303 Asian samples was attempted on each taxonomic level , some of which are affiliated to the P-type genera shown in Eubacterium biforme, Catenibacterium mitsuokai, and Desulfovibrio piger.Community complexities of BB- and P-type microbiota were compared according to alpha-  and beta-  diversities. Rarefaction curves of the number of phylotypes observed in each sample indicates that the P-type individuals harbor a greater number of phylotypes than BB-type subjects . The numInter-individual similarity of fecal microbiota was calculated as the Morisita-Horn index and is displayed as a heat map diagram . This heTo investigate the local variations in the fecal bacterial community among Asian children rather than the two enterotype-like variations, we employed random forest analysis, which is an ensemble classifier based on a machine-learning algorithm. Bifidobacterium and Bacteroides species, which are abundant in the six cities from China, Japan, and Taiwan. Phascolarctobacterium faecium in Cluster I exhibits a unique distribution profile and is notably abundant in China and Taiwan. Cluster III comprises P-type species such as P. copri and Desulfovibrio piger. Clusters II and IV display a unique distribution profile independent of the two microbiota types. Cluster II species, including two Dorea species, are abundant in China as well as in the P-type cities. Cluster IV species are particularly frequent in Japanese children compared to the other countries. Notably, Dialister invisus was detected from 67% of children in Japan but only 18% from other cities. It is known that decrease of Dialister invisus is associated with dysbiosis of the fecal microbiota in patients with Crohn's diseaseThe abundance of species with the 30 highest Gini scores in the random forest analysis is represented in a heat map . The selAlpha-diversities of the microbiota were compared among cities . The alpThe pairwise similarities between samples presented in Enterobacteriaceae is remarkably low in children in Japan compared to the other countries' children . Two alpha-amylases (K01176 and K07405) were mainly involved in this orthology group. These two enzyme genes were enriched in the P-type group, although more than 50% of these genes were encoded by non-Prevotellaceae family  analysise family . In contP = 2.98 × 10−30 and P = 9.81 × 10−30, respectively, in Welch t-test). The abundance of primary and secondary bile acid syntheses inversely correlated with the number of observed OTUs, which may explain the lower bacterial diversity in BB-type microbiota . In particular, most children in Yogyakarta eat rice three times daily. Using the food intake frequency data, we performed logistic regression analysis to correlate diet and the type of gut microbiota (p < 0.001). A distinct gradient of rice intake frequency was observed according to PC1, in which subjects who depend heavily on rice are distributed at higher frequency in the P-type cluster of the PC1-positive region (As shown in the summarized result of food frequency questionnaire (FFQ) , rice accrobiota . The rice region . FurtherPrevotella in intestine as suggested by David et alLogistic regression analysis also indicated that soybeans, eggs, and chicken correlated positively with P-type , whereasIt is noted that the obtained FFQ data do not necessarily explain microbiota of all subjects. For example, P-type subjects in China and Japan did not necessarily follow the P-type carbohydrate-rich diet or the other way around, suggesting that non-investigated factors, maybe including host genetics, interact with gut microbiota. Furthermore, quantitative survey will be required to compare a tremendous variety of Asian diet at nutritional and matrix level. We should also pay attention to the varieties of cultivars and livestock, such as Indica rice and Japonica rice. Cooking method including the amount and type of cooking oils or seasonings, may also be taken into account.Bifidobacteriaceae in BB-type and Ruminococcaceae in P-type slightly decreased with increase of RI .According to Rohrer index (RI), which represents body leanness-fatness in children, approximately 50% of the subjects in this study were classified in the normal weight and 4.7% and 10.7% were classified in severe underweight and obese, respectively . These sse of RI  and negaPrevotella and Bacteroides/Bifidobacterium. This agrees with the findings of a meta-analysis performed using a large Human Microbiome Project (HMP) datasetPrevotella-enterotype (ET2) in MetaHIT  consortium studyBacteroides-enterotype (ET1) and Ruminococcus-enterotype (ET3) observed in the MetaHIT study were not apparent in our samples. Instead, the BB-type cluster diverged widely from the positive to negative range in PC2 with the gradient of the ratio of Bifidobacteriaceae to Clostridiales (Lachnospiraceae and Ruminococcaceae). The BB-type cluster appears to contain three subtypes of microbiota, each enriched by Bifidobacterium, Bacteroides, and Clostridiales. The latter two appear to correspond to ET1 and ET3 of the MetaHIT study, respectivelyBifidobacterium-rich community is unique to our present study.The present study indicates that the variation in the gut microbiota of Asian children clusters into two enterotype-like groups that are driven by trade-off between rotype ET observedBifidobacterium is a particular feature of Asian children. The average relative abundance of the genus Bifidobacterium was 13% among subjects living in the five countries and 5.5% even among the three P-type cities, whereas none of the data obtained from school-age children in Western cohorts showed a relative abundance of >5% on average for the Bifidobacterium population616Bifidobacterium accounted for 20%. Interestingly, a study on the diversity of gut microbiota of the Russian population showed that a rural population residing in Tyva in central Asia exhibits a high abundance of Bifidobacterium (average approximately 10%), whereas other rural populations, in Tatarstan and Omsk, do not. The carbohydrate-based Asian diet could be a possible factor that drives the colonization of Bifidobacteria.The high abundance of Prevotella-enterotype strongly depends on carbohydrates in diets15Prevotella and Bacteroides remain relatively constant, whereas those of Faecalibacterium and Eubacterium vary with the seasonal dietThe two microbiota types were strongly associated with country with some exceptions, and were independent of the host physiology represented by Rohrer index. It has been demonstrated that the P. copri and P. stercorea, the two dominant Prevotella species in the P-type subjects, are sensitive to bile acid40Predictive metagenomics indicated a higher abundance of genes involved in primary and secondary bile acid syntheses in the BB-type bacterial community. This coincides with the finding by David et al.36The urbanization trend is seen in the gut microbiota profile of Thai children. The contrast between urban and rural population is remarkable in the distribution of microbiota type in Thailand. Chidren living in Bangkok tended to harbor the BB-type while children living in Khon Kaen children harbor the P-type. The fact that children in Bangkok eat vegetables and fruits much less frequently compared with Khon Kaen children  suggestsBifidobacterium and Enterobacteriaceae, respectively, and low collective and individual diversity. They are particularly less colonized by potentially pathogenic species, such as Escherichia coli, Clostridium perfringens, and Lactococcus garvieae  and were stored at room temperature. The samples were transferred to the laboratory within 12 h, stored at 4°C, and used for extracting DNA within four weeks. Preliminary experiments confirmed that the bacterial composition data did not change in four weeks under the storage conditions used.A total 303 children, aged 7–11-years old, who had not contracted any infectious disease that required medical attention in the two weeks preceding the sampling, provided a single stool sample. All participants were born and grew up in the sampling country. The parents/guardians answered a questionnaire that addressed their physiological characteristics, dietary intake in the past two weeks before stool sampling, and healthy condition. The details regarding these study participants are summarized in DNA and RNA were extracted from stool samples by using the bead-beating method as previously describedWe performed qPCR to quantify six major bacteria groupsBacteroidetes44http://qiime.org/)48The V6–V8 region of the bacterial 16S rRNA gene was PCR amplified using the barcode-tag universal primer sets Q-968F-# and Q-1390R-# (where # indicates a series of 128 barcode sequence tags underlined in the sequence)http://rdp.cme.msu.edu/seqmatch/seqmatch_intro.jsp), in which the lower threshold of the S_ab score was set at 0.84. If more than two species showed the same highest score, the one with the highest count among the top 20 matches was selected for annotating the species by using a Microsoft Excel macro file named Seqmatch Q400The taxonomy of each OTU was assigned using the RDP classifier with the confidence threshold of 80% (assign_taxonomy.py) in the QIIME pipeline based on the Greengenes taxonomy (97_otu_taxonomy) and a Greengenes reference database (97_otus.fasta)http://grenegens.lbl.gov/Download/Sequence_Data/Fasta_data_files/core_set_aligned.fasta.imputed) using the align.seqs program with the suffix-tree search of Mothur software Ver. 1.29.2 (http://www.mothur.org/)http://evolution.genetics.washington.edu/phylip.html). The phylogenetic tree was displayed using the iTOL online tool (http://itol.embl.de/)Representative sequences of phylotypes were aligned with an alignment core set of 16S rRNA gene sequences  . At phylotype level, the OTU table was rarified at 1,000 sequences per sample from five random iterations and the weighted UniFrac distancehttp://qiime.org/scripts/beta_diversity.html). At the other taxonomic levels from phylum to genus, the JSDhttp://enterotype.embl.de/enterotypes.html). Based on the obtained distance matrix, the 303 samples were clustered using PAM clustering by using the “pam” function in the R library “cluster” . The optimal number of clusters was chosen by maximizing the Calinski–Harabasz index (“index.G1” function in the R library “clusterSim”)(http://cran.r-project.org/web/packages/clusterSim/index.html)http://cran.r-project.org/web/packages/ade4/index.html)http://cran.r-project.org/web/packages/fpc/index.html)Clustering was performed according to the enterotyping tutorial provided in the R environment by the EMBL (http://qiime.org/scripts/multiple_rarefactions.html). Indices of alpha-diversity including observed_species (the number of OTUs), PD_whole_treehttp://qiime.org/scripts/alpha_diversity.html). Results from the two random iterations were averaged. The Morisita-Horn indexhttp://chao.stat.nthu.edu.tw/blog/software-download/) and the OTU table rarified to 1,000 reads from five random iterations.The OTU table was rarefied by using the multiple_rarefactions.py script in QIIME (http://cran.r-project.org/web/packages/randomForest/index.html). Calculated proximity matrices were plotted in two dimensions by using the MDSplot function in the randomForest package. Hierarchical analysis was performed using the R-package amap (http://cran.r-project.org/web/packages/amap/index.html). Distances based on Pearson correlations were calculated for input into an agglomerative algorithm through complete linkage, by using the Bioconductor hclust function in the R-package amap. The population densities of species scaled by color are displayed together with dendrogram of bacterial species in a heat map generated using the R-package amap.Random-forest analysis was performed using the R-package randomForesthttp://huttenhower.sph.harvard.edu/galaxy/root, version 1.0.0). 97% OTUs are picked using a closed-reference OTU picking protocol (QIIME 1.8.0) against the Greengenes database pre-clustered at 97% identify (GG 13.5). The obtained OTU table was normalized by 16S rRNA copy number and metagenomes were predicted from the KEGG cataloguePICRUSt analysishttp://cran.r-project.org) for relative abundance of each bacteria group determined by pyrosequencing of 16S rRNA, Tukey'test in SAS System version 5.0  for the number of each bacterial group determined by qPCR, chi-square test in Excel 2013 (Microsoft Corporation) to examine the localization of phylotypes between groups, and Welch t-test in Excel 2013 for relative abundance of each KEGG Orthology group. For comparative analysis of alpha-diversity between cities, pairwise Wilcoxon rank sum test was performed with Bonferroni correction in R 2.13.1. Logistic regression analysis and linear regression analysis were performed in Stata SE12 (Stata Corporation).To assess the significance of differences between groups, we performed Mann-Whitney U-tests in R 2.13.1 (Bifidobacterium (-1.75%/y) and two Lachnospiraceae genera, Blautia (+1.22%/y) and Roseburia (+0.72%/y), significantly associated with age but the correlation between bacterial population and sampling city did not changed in the age-adjusted multivariate analysis. Therefore, we concluded that age was not a significant confounding factor to see differences of bacteria population across residential places in our dataset.To examine confounding effect of age bias among sampling places, we performed multivariate analysis using age and city as the independent variable. The result indicated that the abundance of genus Conceived and designed the experiments: J.N., K.W., M.C., V.L., C.L., S.N., E.S.R., F.R., Y.T., Y.L. Performed the experiments: J.N., K.W., K.C., J.J., K.M., S.C., P.H., L.O., M.S., I.N.S., L.Z., Y.C., H.C., N.H., C.K., T.K., N.S., K.S., K.T., H.T., T.H. Analyzed the data: J.N., K.W., Y.L. Wrote the paper: J.N., K.W., Y.L.Accession codes: Sequence data from this article were deposited in the DNA Data Bank of Japan (DDBJ) database under BioProject no. PRJDB1664, which contains links and access to stool sampling data under BioSample SAMD00000022 to SAMD00000324 and pyrotag sequence data designated in the DDBJ sequence read archive (DRA001863 to DRA001872).Supplementary InformationSupplementary Tables S1, S2, S4, S5, S6, S7, S8, S10, S11, S12