Scrupocellaria comprises about 80 species in the family Candidae. We propose a hypothesis for the phylogenetic relationships among species assigned to Scrupocellaria to serve as framework for a phylogenetic classification using 35 morphological characters. Our results suggest that the genus Scrupocellaria is polyphyletic. Scrupocellaria s. str. is redefined according to four morphological features: vibracular chamber with a curved setal groove, ooecium with a single ectooecial fenestra, two axillary vibracula, and a membranous operculum with a distinct distal rim. Thus, the genus includes only 11 species: Scrupocellaria aegeensis, Scrupocellaria delilii, Scrupocellaria harmeri, Scrupocellaria incurvata, Scrupocellaria inermis, Scrupocellaria intermedia, Scrupocellaria jullieni, Scrupocellaria minuta, Scrupocellaria puelcha, Scrupocellaria scrupea, and Scrupocellaria scruposa. The monophyly of Cradoscrupocellaria is supported and five new genera are erected: Aquiloniella n. gen., Aspiscellaria n. gen., Paralicornia n. gen., Pomocellaria n. gen. and Scrupocaberea n. gen. Two other new genera, Bathycellaria n. gen. and Sinocellaria n. gen., are erected to accommodate two poorly known species, Scrupocellaria profundis Osburn and Scrupocellaria uniseriata Liu, respectively. Scrupocellaria congesta is tentatively assigned to Tricellaria. Fifteen species are reassigned to Licornia: Licornia cookie n. comb., Licornia micheli n. comb., Licornia milneri n. comb., Licornia curvata n. comb., Licornia diegensis n. comb., Licornia drachi n. comb., Licornia mexicana n. comb., Licornia pugnax n. comb., Licornia raigadensis n. comb., Licornia regularis n. comb., Licornia resseri n. comb., Licornia securifera n. comb., Licornia spinigera n. comb., Licornia tridentata n. comb., and Licornia wasinensis n. comb. Notoplites americanus n. name is proposed as a replacement name for Scrupocellaria clausa Canu & Bassler. Three fossil species are reassigned to Canda: Canda rathbuni n. comb., Canda triangulata n. comb. and Canda williardi n. comb. A species is reassigned to Notoplites, Notoplites elegantissima n. comb. The generic assignment of eleven species of Scrupocellaria, including Scrupocellaria macandrei, remains uncertain.The bryozoan genus  Scrupocellaria van Beneden, 1845 Scrupocellaria have also been reported on artificial substrates The bryozoan genus Scrupocellaria was erected to include Sertularia scruposa Linnaeus, 1758 Scrupocellaria van Beneden, 1845 grew in size and morphological diversity over time. In its broad sense Scrupocellaria has been defined as having the following characteristics: erect, biserial, branching colonies anchored to the substratum by rhizoids; rhombic autozooids with partially membranous frontal walls, with spines, including a modified lateral spine (the scutum); zooid polymorphs often including lateral and/or frontal avicularia, almost always including baso-lateral vibracula, and subglobular hyperstomial ooecia. At least three genera were synonymized under Scrupocellaria, viz. Cellarina van Beneden, 1848 Crisina van Beneden, 1850 Licornia van Beneden, 1850 Licornia, has since been treated as a distinct taxon and its generic status restored et al.Cradoscrupocellaria, for some additional species previously assigned to Scrupocellaria and described 18 new species.The genus ScrupocellariaScrupocellaria sensu lato, finding previously unrecognized homologies of the character states in order to (ii) propose a hypothesis of the phylogenetic relationships among Scrupocellaria to (iii) serve as framework for a phylogenetic classification of the group and the validation of new nomenclatural decisions.Neither a morphological nor a molecular phylogenetic hypothesis has been published for any taxa of the family Candidae, despite the molecular evidence for non-monophyletic status of http://zoobank.org/”. The LSID for this publication is: urn:lsid:zoobank.org:pub:8A2439E2-4B08-419C-BDA2-804A675F8B7B. The electronic edition of this work was published in a journal with an ISSN, and has been archived and is available from the following digital repositories: PubMed Central and LOCKSS.The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature, and hence the new names contained herein are available under that Code from the electronic edition of this article. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix “viz. Notoplites Harmer, 1923, Tricellaria Fleming, 1828, Canda Lamouroux, 1816 and Caberea Lamouroux, 1816. Incorporating these taxa was also important in order to test the monophyly of Scrupocellaria. We chose Notoplites marsupiatus  to root the unrooted cladogram of the analysis because of its distinct scutum shape (when compared with other Candidae species) and the presence of abfrontal avicularia rather than abfrontal vibracula  are deposited in the following collections:AMNH, American Museum of Natural History (USA)LSL, Linnean Society of London (United Kingdom)MCZ, Museum of Comparative Zoology, Harvard University (USA)MM, Manchester Museum (United Kingdom)MNHN, Muséum national d'Histoire naturelle (France)MOM, Musée océanographique de Monaco (Monaco)MTQ, Museum of Tropical Queensland MZUSP, Museu de Zoologia da Universidade de São Paulo (Brazil)NMV, Museum Victoria NHMUK, Natural History Museum, London (United Kingdom)NCB, Nederlands Centrum voor Biodiversiteit Naturalis SBMNH, Santa Barbara Museum of Natural History (USA)USNM, National Museum of Natural History, Smithsonian Institution (USA)VMNH, Virginia Museum of Natural History (USA)All necessary permits were obtained for the described field studies in Brazil (collecting permit numbers 10186 and 19936 SISBIO/Instituto Chico Mendes de Conservação da Biodiversidade). The reported localities do not include protected areas and did not involve endangered or protected species. Permissions from all museums were obtained to access and study their collections.e.g. absence of scutum and rhizoids with hooks). All characters were treated as unordered and equally weighted. Autapomorphies of terminal taxa, which do not provide evidence to support monophyly at supraspecific levels, were excluded from the analysis. Unknown states were indicated as “?” in the data matrix, inapplicable states were indicated as (“-”). Whenever necessary, polymorphisms were explicitly considered in the coding and are marked in the data matrix. The list and discussion of characters used are given below.All specimens were examined under the stereomicroscope. Selected specimens were mounted for examination in a scanning electron microscope (SEM)  for description and selection of the characters. We scored 35 characters for all terminal taxa; some of these characters show homoplasies among the family members : (0) absent, (1) present  colonies may not have the connecting tubes, but they are present in more developed colonies . present . RemarksRhizoid surface : (0) smooth (Scrupocellaria species ) smooth , (1) wit) smooth , (2) rin) smooth . RemarksRhizoids adjacent to the abfrontal surface of the colony : (0) absent (e.g. Notoplites clausus and Notoplites marsupiatus) absent , (1) pre) absent . RemarksPosition of the joints at bifurcation : (0) passing across zooids FD and GC . In Canda spp., the joints are often seen in older branches; and in a few Licornia species  the joints are inconspicuous due to heavier calcification in all parts of the colonies of those species. The bifurcation pattern of the colony and position of the joints have been adopted to differentiate some genera of branching, erect bryozoans with membranous frontal walls Aspiscellaria bellula because of the absence of bifurcations in the colonies studied; the joints are apparently rare, present in a single zooid at the base of the colony 4. D and GC , (1) pasD and GC . RemarksPosition of the joints in relation to zooids C and D : (0) passing across the gymnocysts  pasmnocysts .Position of the joints in relation to zooids J and K : (0) passing across zooid gymnocysts, (1) passing across the zooid opesiae.6. Adjacent zooids along the axis : (0) placed side by side in the same plane or slightly inclined in relation to the axis .7. the axis , (1) arethe axis . RemarksAperture area : (0) continuous and in the same plane as the frontal membrane  plamembrane . RemarksDistal edge of autozooid : (0) smooth  smooth , (1) too) smooth , (2) too) smooth . RemarksMorphology of operculum : (0) membranous and only rim distinctly chitinous  opehitinous . RemarksCryptocyst : (0) vestigial, as a very tiny rim around the opesia  fore opesia .Bugula species viz. inner, outer and median spines. Both the presence of polymorphic data and the lack of topographic correspondence suggest that not all spines are homologous, but may be a product of serial homology. Hence, it is only possible to homologize the states between the spines of the same nature, as indicated by topographical correspondence. For example, the most proximal outer spines of different taxa are comparable with each other but not with the proximal inner spines of different taxa or even those in the same individual. Thus, we coded them as three separate characters (Characters 12 and 13 and 14), i.e. proximal-most outer, the proximal-most inner and distal-most oral spines.In Candidae the oral spine is characterized by the presence of a jointed base : (0) absent (12. ) absent , (1) pre) absent , (2) pre) absent , (3) pre) absent .Proximal-most inner spine : (0) absent (13. ) absent , (1) pre) absent , (2) pre) absent , (3) preDistal-most spines : (0) absent (14. ) absent , (1) pre) absent , (2) preScutal spine at the inner edge of opesia : (0) absent (15. ) absent , (1) pre) absent , (2) pre) absent .Shape of scutum arising at the median region of opesia : (0) spine-like and unbranched .16. branched , (1) forbranched , (2) brabranched , (3) brabranched , (4) forbranched , (5) forShape of scutum arising at the distal third of opesia : (0) slender base, i.e. as wide as distal spines, with unbranched asymmetrical enlarged portion in which the distal region is less developed than the proximal one  stout base, i.e. two or more times wider than distal spines, enlarged portion developed proximally  sleoximally , (3) stooximally , (4) stor margin .Development of the unbranched asymmetrical portion of the scutum at the distal third of opesia : (0) narrow paddle-shaped, curved towards the proximal region of opesia  enlf opesia .Sessile distolateral avicularium : (0) absent (19. ) absent , (1) pre) absent .Direction of rostrum of sessile lateral avicularium : (0) lateral  obl lateral , (2) oblRostrum of lateral avicularium : (0) serrated lateral edge, straight to slightly curved at its tips  smoits tips , (2) serits tips .Giant lateral avicularium : (0) absent, (1) present, with triangular to elongate mandible  premandible .Dimorphic frontal avicularium : (0) absent, (1) present, with triangular mandible  preSurface of ectooecium : (0) with a single proximal fenestra .24. fenestra , (1) witfenestra , (2) porfenestra . Remarksscruposa  and one Sessile avicularium associated with ooecium : (0) absent (25. ) absent , (1) pre) absent .Heterozooids on abfrontal surface of the colony : (0) absent  26. ) absent , (1) pre) absent , (2) pre) absent . RemarksLateral rhizoidal (rootlets) chamber associated with outer zooids at the bifurcation : (0) absent, (1) present : (0) present, complete, without foramen  pre foramen , (2) abs foramen . Remarks foramen .Position of the setal groove on vibracular chamber  in relation to the internodal axis : (0) transverse  oblansverse .Shape of oblique setal groove in vibracular chamber : (0) curved, of medium length, occupying at maximum two thirds of the inner margin of the vibracular chamber length; setal groove developed up to half of the width of the zooid  curhe zooid , (2) strhe zooid , (3) strhe zooid , (4) strhe zooid , (5) strNumber of axial vibracula : (0) one (31.  (0) one , (1) two (0) one .Setal groove in a single axial vibraculum : (0) lateral  lon lateral .Setal morphology : (0) delicate, translucent white, (1) hard, chitinous, yellowish-gold.33. Surface of setae : (0) smooth, (1) barbate.34. Length of setae : (0) short, as long as internode width, shorter than zooid length  lond length .Scrupocellaria s. l. (Canda) has Bremer support of 5; Node 1 has Bremer support of 4; and the Nodes 35 (Caberea) and 37 (Scrupocaberea n. gen.) have Bremer support of 2 . The semi-strict consensus tree  shows a polyphyly of the genus ia s. l. . Most clort of 2 .Tricellaria Fleming, 1828, viz. Tricellaria arctica n. comb.  and Tricellaria elongata n. comb. , instead of creating new generic names to accommodate these species. Our decision is based on some shared characters, such as (i) absence of abfrontal vibracula/avicularia, (ii) presence of a lateral rhizoidal chamber associated with the outer zooids of the bifurcation, (iii) a scutum arising from the distal third of the inner opesial rim and (iv) joints passing across the gymnocyst of outer zooids in the zooids C and D. The species described as Scrupocellaria congesta is distinguishable from other Tricellaria in having joints passing across the opesia of outer zooids in the zooids C and D (assigned in the tree with question mark). Tricellaria is the sister group of ?Tricellaria congesta (genus incertae sedis) + Group A + Clade B.The basal part of the tree of the Candidae has a low resolution, probably because of the high number of characters coded as inapplicable in those basal taxa causing unstable relationships. Taxonomically, we decided to consider two species of the basal polytomies to be part of the genus Aquiloniella n. gen. All species of Aquiloniella n. gen. are endemic to Arctic and sub-Arctic waters, and may be characterized by colonies with chitinous joints passing across the gymnocyst of outer zooids of the bifurcation, as do those of Tricellaria, but with abfrontal avicularia. Thus, they are distinct from Tricellaria in the absence of the lateral rhizoidal chamber associated with the outer zooids of the bifurcation, in the presence of abfrontal avicularia, and a scutum arising at the median region of the inner part of the opesia.A polytomy of four species is here considered to be part of Group A and a large Clade B . The unresolved polytomy would allow a monophyletic Group A to be tested in future analysis. Nomenclaturally, it is convenient to consider the species of Group A as belonging to a unique genus, described below as The analysis supports the monophyly of the species of Candidae with an abfrontal vibracular chamber (Clade B). The switch from smooth lateral edge to serrated lateral edge of the rostrum of the lateral avicularium is another synapomorphy for this clade. This clade comprises four monophyletic groups (Clade C + Clade D + Clade E + Clade F). The topology of Clade B shows an interesting history concerning the development of setal groove of the vibracular chamber along the evolution of the species of Candidae. The possession of a vibracular chamber with transversal setal groove would be primitive (plesiomorphic), as it is present in three of the included clades , and the change to an oblique setal groove would be synapomorphic for Clade F.Pomocellaria californica n. comb. and Pomocellaria varians n. comb., are absent in Pomocellaria inarmata n. comb. and Pomocellaria talonis n. comb. In the basalmost taxon, Pomocellaria inarmata, the scutum and oral spines are absent.Clade C seems to be endemic to the Eastern Pacific. It has a basal phylogenetic position in Clade B and it is characterized by (i) an abfrontal vibracular chamber with a transversal setal groove, (ii) ooecium with a large ectooecial fenestra, and (iii) a single axial vibracula. Presence of abfrontal vibracula with a transverse setal groove is a character also present in Clade D and Clade E, but the two clades are distinguished from Clade C by the presence of an ooecium with some ectooecial pores in Clade D and E. The dimorphic lateral avicularia, characteristics of Aspiscellaria bellula n. comb. and in Cradoscrupocellaria, may be considered a homoplasy.Members of Clade D and Clade E have a circumtropical distribution in shallow waters. Clade D has an unambiguous synapomorphy, the outer spines are branched three or more times (cervicorn); this clade also includes species with an oval scutum with internal channels (ornamentation), whereas the branched scutum with a planar frontal surface, present in Cradoscrupocellaria is supported here (Clade E). This genus is characterized by the presence of (i) articulate distal unbranched spines, (ii) a branched scutum arising from midline of the inner edge of the opesia, (iii) a trapezoidal vibracular chamber, (iv) single axial vibraculum, and (v) an ooecium with some ectooecial pores The monophyly of the genus Clade F has an unambiguous synapomorphy, a vibracular chamber with an oblique setal groove, derived from a primitive condition of a vibracular chamber with transversal setal groove which becomes a short to long oblique setal groove.Paralicornia hamata n. comb. + Paralicornia obtecta n. comb. + Paralicornia sinuosa n. comb.) defined by a dimorphic lateral avicularium with trifoliate rostrum, and two other species, Paralicornia limatula n. comb. and Paralicornia pusilla n. comb. The phylogeny suggests that the polytomy may comprise a distinct clade with circumtropical distribution  due to the (i) absence of bifurcated oral spines (characteristic of Licornia species), (ii) the presence of joints passing across the gymnocysts of outer zooids at the branch, and (iii) the presence of a shorter setal grove than those of Licornia species.The basal part of the Clade F has a polytomy including one monophyletic clade  n. comb., Licornia diegensis  n. comb., Licornia drachi  n. comb., Licornia regularis  n. comb., Licornia securifera  n. comb., and Licornia tridentata  n. comb. . According to the phylogeny, the taxonomic position of Licornia macropora remains uncertain.The monophyly of Scrupocellaria s. str. according to four morphological features: (i) vibracular chamber with curved setal groove, (ii) ooecium with a single and small ectooecial fenestra, (iii) two axillary vibracula, and (iv) a membranous operculum with a distinct distal rim. Thus, the genus Scrupocellaria encompasses only nine species from the polytomy: Scrupocellaria harmeri, Scrupocellaria minuta, Scrupocellaria delilii, Scrupocellaria incurvata, Scrupocellaria inermis, Scrupocellaria intermedia, Scrupocellaria scrupea, Scrupocellaria scruposa, and Scrupocellaria aegeensis. Although these species do not form a unique group, future studies may test the monophyly of the genus.Despite the low resolution of the semi-strict tree, we use a part of the polytomous taxa of Clade H—characterized by presence of an avicularium at the outer wall of the ooecium—to redefine the genus Scrupocaberea n. gen., comprises four species distinct from Scrupocellaria s. str. in having a (i) well chitinized operculum placed in an obliquely truncate distal area, and a (ii) scutum with a stout base arising at the distal third of opesia and with an enlarged portion developed proximally. Hence, Scrupocaberea has a mixture of the morphological features of Caberea  and Scrupocellaria . Caberea, however, has been morphologically well defined due to the well-developed vibracular chamber with a very long setal groove and barbate seta Caberea is supported by these two unambiguous synapomorphies. The genus Canda, previously characterized by the presence of rhizoids forming cross connections between branches and zooids in two series with their frontal surfaces facing obliquely outwards from the axis viz. (i) joints passing across opesiae of the zooids J and K at the bifurcation, (ii) adjacent zooids abruptly inclined in relation to the axis, (iii) scutum arising at the median region of opesia and forming an asymmetrical plate, without internal channels.The monophyletic Clade I, here described as ; Text S3Scrupocellaria van Beneden, 1845: 26 Sertularia scruposa Linnaeus, 1758  Sl. harmeri Osburn, 1947 Sl. incurvata Waters, 1897 Scrupocellaria aquitanica Jullien & Calvet, 1903 Sl. inermis Norman, 1867 Scrupocellaria grimaldii Jullien & Calvet, 1903 Sl. intermedia Norman, 1893 Sl. jullieni Hayward, 1978 Sl. minuta Kirkpatrick, 1888 Sl. puelcha  n. sta.Sl. scrupea Busk, 1851 Sl. scruposa  Scrupocellaria macandrei Busk, 1852 Scrupocellaria s. str. was not confirmed because the morphological characteristics of the species cannot be recognized in the type specimen (NHMUK 1854.11.14.78).Tilbrook and Vieira Aquiloniella n. gen., Aspiscellaria n. gen., Paralicornia n. gen., Pomocellaria n. gen. and Scrupocaberea n. gen. (see below). Two other new genera, Bathycellaria n. gen. and Sinocellaria n. gen. are erected to accommodate two additional species . The diagnosis of Licorniaor more of the vibracular chamber length; thus, in addition to the six species included in the phylogenetic analysis (Licornia (see below).Five new genera are erected to accommodate species included in the present phylogeny: analysis , anotherScrupocellaria s. str. resembles those of Canda and Scrupocaberea n. gen.; the genus Canda is distinct in the shape of the internodes, position of the joints in relation to the bifurcation, the presence of interconnective rhizoids, and a well-developed cryptocyst. The genus Scrupocaberea n. gen. is distinguishable from Scrupocellaria s. str. by the presence of a well-chitinized operculum placed at the distal truncate area of the zooid (see below).The curved setal groove of the vibraculum of members of the genus Scrupocellaria inermis is distinguishable from the other species of the genus by the absence of oral spines and scutum. The scutum is also absent in the type of the genus, Sl. scruposa. Scrupocellaria aegeensis, described from Mediterranean, has a scutum with a convex distal edge and cuspidate projections at the proximal and distal inner rim. Scrupocellaria minuta has a distinctly different scutum with a stout base three times wider than the distal spines and an enlarged portion more developed distally than proximally. Small differences are seen in scuta shape of Sl. delilii, Sl. harmeri and Sl. scrupea; they can be clearly distinguished by the position of the joints across the bifurcation, and the shape and size of abfrontal vibracula. Scrupocellaria intermedia and Sl. jullieni are distinct among Scrupocellaria species in having a scutum arising from the median region of the inner part of the opesia; Scrupocellaria intermedia is also distinct in having dimorphic lateral avicularia with trifoliate rostra. Other species with trifoliate lateral avicularia are assigned to Paralicornia n. gen. (see below). Scrupocellaria incurvata has a large scutum, as wide as the opesia; this species is also characterized by the presence of dimorphic lateral avicularia with a triangular, laterally directed mandible.urn:lsid:zoobank.org:act:EF783DC7-CA4B-4399-8B91-56F211F913B1; Text S4Scrupocellaria americana Packard, 1863 , little, in allusion to its short distal spines.The genus name refers to the Roman god of the north wind, Aquiloniella n. gen. is erected to include five species reported in Arctic and sub-Arctic waters: Aq. americana  n. comb. Aq. aviculareae  n. comb. Aq. orientalis  n. comb. Aq. paenulata  n. comb. Aq. scabra  n. comb. Scrupocellaria scabra var. paenulata forma minor Kluge, 1915 has abfrontal avicularia, like those of Aquiloniella species, but it is considered a nomen nudumAquiloniella n. gen. is distinguished from Tricellaria by absence of the lateral rhizoidal chamber associated to the outer zooids of the bifurcation, presence of abfrontal avicularia and a scutum arising at the median region of the inner part of the opesia. Aquiloniella n. gen. is easily set apart from Scrupocellaria s. str. in having abfrontal avicularia  and in the shape of lateral avicularia.urn:lsid:zoobank.org:act:648B9E0D-6793-4190-85A4-EFA4B4652E50; Text S5Scrupocellaria piscaderaensis Fransen, 1986 . Frontal scuta symmetrical, oval, arising at or slightly below the median region of the inner part of the opesia. Lateral avicularia often present, monomorphic, with a slightly serrated rostrum and straight tip. Frontal avicularia often present, small, monomorphic. Vibracular chamber trapezoidal, with a rhizoidal foramen; setal groove straight, directed transversely; the setal groove is placed distally to the rhizoidal pore; 1 axillary vibraculum with lateral setal groove present. Ooecium with some ectooecial pores.aspis (Greek), the generic term for shield, in allusion to its rounded scutum, + cellaria, used for some bryozoan genera.The genus names is formed from the word Cradoscrupocellaria and Pomocellaria n. gen. Aspiscellaria n. gen. is easily distinguished from these two genera by its proximal-most spine which is cervicorn in shape. Pomocellaria n. gen. is distinct in the position of the scutum, arising from the distal third of the inner edge of the opesia, and in having an ooecium with a single frontal fenestra.The vibracular chamber with transverse setal groove resembles those of Aspiscellaria: Ap. bellula  n. comb. Ap. carmabi  n. comb. Ap. cornigera  n. comb. Ap. frondis  n. comb. Ap. hildae  n. comb. Ap. panamensis  n. comb. Ap. piscaderaensis  n. comb. Ap. unicornis  n. comb. We reassigned eight species to Aspiscellaria n. gen. are characterized by the absence of a lateral avicularium: Ap. frondis, Ap. hildae, and Ap. unicornis; these three species are distinguished from each other by the shape of the scutum, the shape and size of the frontal avicularium and the shape of ectooecial pores. Aspicellaria frondis has a rounded scutum covering the majority of the opesia, with a narrow cryptocyst, distinct from Ap. hildae and Ap. unicornis; the two later species are distinct in the size of the zooids and in having shorter frontal avicularia than Ap. unicornis. Aspiscellaria carmabi is distinguishable from Ap. piscaderaensis by the position of joints at the outer zooids at the bifurcation, and shape of frontal avicularium. Aspiscellaria cornigera is characterized by a well-developed scutum with projections at its proximal and distal inner edge. The examination of museum specimens of Aspiscellaria also revealed that additional new species await description.Three species of urn:lsid:zoobank.org:act:A6204B1E-D9BE-4543-9473-E5A33FBD5C29; Text S6Scrupocellaria sinuosa Canu & Bassler, 1927  the inner part of the opesia. Lateral avicularia present, with a slightly serrated rostrum and straight tip; lateral avicularium sometimes replaced by an avicularium. Frontal avicularia often present, small, monomorphic. Vibracular chamber almost triangular, with a rhizoidal foramen; setal groove straight, obliquely directed and occupying half of the length of the vibracular chamber; the setal groove is placed distally to the rhizoidal pore; 1 axillary vibraculum with lateral setal groove. Ooecium with some ectooecial pores.Licornia species.The generic name refers to the similarities of this genus with some et al.viz. presence of ooecia with ectooecial pores and single axillary vibraculum, to reassign some species of Scrupocellaria to two separate genera, Licornia and Cradoscrupocellaria. The genus Cradoscrupocellaria is quite distinct from Licornia and Paralicornia n. gen. in having a vibracular chamber with a transversal setal groove; Cradoscrupocellaria seems to be morphologically related to Aspiscellaria, but the two are distinct in the shape of the frontal scutum , the presence of dimorphic frontal avicularia (characteristic of some Cradoscrupocellaria) and the presence of branched distal spines (characteristic of Aspiscellaria). The genus Licornia resembles Paralicornia n. gen. in the direction of the setal groove of the vibraculum, but the two genera are distinct in the position of the joints in the outer zooids at the bifurcation , the presence of shorter opesia in Paralicornia n. gen. than in Licornia, the presence of bifurcated distal spines in Licornia, and the length of setal groove, longer in Licornia species than those of Paralicornia. The gigantic trifoliate lateral avicularia found in the three known species of Paralicornia n. gen. seem to be absent in Licornia.Vieira Scrupocellaria are transferred here to Paralicornia n. gen.: viz. Pa. hamata  n. comb. Pa. limatula  n. comb. Pa. obtecta  n. comb. Pa. pusilla  n. comb. Pa. sinuosa  n. comb. Pa. spatulatoidea  n. comb. Pa. unguiculata  n. comb. Paralicornia unguiculata has distinct dimorphic lateral avicularia with ligulate mandibles.Seven species previously assigned to Paralicornia will also need to be described  Pa. sinuosaspecies inquirenda, i.e. a species with doubtful identity needing further assessment.The examination of specimens deposited at the NHMUK, AMNH and USNM revealed that about a dozen new species of ibed see . Scrupocurn:lsid:zoobank.org:act:1E033805-798F-4680-B3F6-BDF303C967E8; Text S7Scrupocellaria californica Trask, 1857  + cellaria, used for some bryozoan genera.The generic name is composed from Pomocellaria n. gen. is erected to include four Eastern Pacific species: Po. californica  n. comb. Scrupocellaria brevisetis Hincks, 1882 Po. inarmata  n. comb. Po. talonis  n. comb. Po. varians  n. comb. Pomocellaria n. gen. is distinguishable from the other two genera with a trapezoidal vibracular chamber—i.e. Cradoscrupocellaria and Aspiscellaria n. gen.—in the position of the scutum, arising from the distal third of the inner edge of the opesia rather than the median part of the inner edge of the opesia; and in having an ooecium with a single frontal fenestra rather than one with many pseudopores. The dimorphic lateral avicularium and distal spines are absent in Pomocellaria inarmata.urn:lsid:zoobank.org:act:03E75D59-E2D3-45E5-83F0-412CFC7ABCE2; Text S8Scrupocellaria maderensis Busk, 1860  n. comb. Sb. gilbertensis  n. comb. Sb. maderensis  n. comb. Sb. ornithorhynchus  n. comb. The presence of a well-chitinized operculum placed in an obliquely truncate distal area, a toothed rim on the distal edge of ovicelled zooids, and the presence of two axillary vibracula led us to include four species in a new genus, Scrupocaberea maderensis has been reported to be widespread in tropical and subtropical waters worldwide e.g.Sb. maderensis by Harmer viz. Scrupocaberea dongolensis and Sb. gilbertensis; these species are distinct from Sb. maderensis (Sb. dongolensis and larger in Sb. gilbertensis than Sb. maderensis), shape of frontal avicularia, surface of the cryptocyst (granulose in Sb. gilbertensis), and size of the autozooids . Zooids of Sb. ornithorhynchus also have a well-chitinized operculum; this species differs from Sb. maderensis in having a vibracular chamber with a shorter setal groove and by the shape of scutum which is asymetrically developed in distal edge in Sb. ornithorhynchus rather than truncate as those of Sb. maderensis.derensis  by virtuScrupocellaria not included in this study probably belong to Licornia due to their having porous ooecia and similarly shaped abfrontal vibracula; they are here reassigned to Licornia: Licornia mexicana  n. comb. Licornia pugnax  n. comb. Licornia spinigera  n. comb. Licornia wasinensis  n. comb. L. spinigera the joints pass across proximal end of the opesia of outer zooids at the bifurcation, but pass more distally  in L. mexicana and L. pugnax. Licornia wasinensis is characterized by the absence of a scutum and by dimorphic lateral avicularia with forked mandibles; this forked mandible Paralicornia species.Four Recent species of Scrupocellaria elegantissima David & Pouyet, 1986 Notoplites Harmer, 1923 due to the presence of basal avicularia and joints passing across zooids FJ and GK at bifurcation Notoplites elegantissima  n. comb.We have examined the type specimens of Scrupocellaria micheli Marcus, 1955 Scrupocellaria profundis Osburn, 1950 Scrupocellaria uniseriata Liu, 1984 Scrupocellaria micheli is distinct in the irregular branching pattern of the colony and the presence of large aquiline lateral avicularia Scrupocellaria micheli and other Paralicornia n. gen. and Licornia species due to the presence of vibracula with a straight, obliquely directed setal groove occupying half of the length of the vibracular chamber, and an ooecium with some ectooecial pores; this species is tentatively assigned to Licornia, thus Licornia micheli  n. comb. Scrupocellaria profundis Osburn, 1950 is a deep water species  in the shape of its zooids, which are twisted at the axis of the maternal internode, and the position of the radicles chamber, lateral rather than proximally placed in the vibracular chamber and two axial vibracula with longitudinal straight setal groove . Bathycellaria n. gen. (urn:lsid:zoobank.org:act:362C7211-AE4D-4430-A385-B768B2E06FE8)  is erected to accommodate Osburn's species Bathycellaria profundis  n. comb. Scrupocellaria uniseriata Liu, 1984 has unique uniserial colonies Sinocellaria n. gen. (urn:lsid:zoobank.org:act:93013294-FD7C-40D4-8361-370CF74B631D)  is erected to accommodate Liu's (1984) species (type species by monotypy), thus Sinocellaria uniseriata  n. comb.Three Recent species  are unassigned to any genus of Candidae, Scrupocellaria. Scrupocellaria clausa Canu & Bassler, 1920 Notoplites, but this name is preoccupied by Notoplites clausus  Notoplites americanus n. name (urn:lsid:zoobank.org:act:C032937B-14AF-415D-86B4-E4270574EDFE) is proposed as a replacement name for Scrupocellaria clausa Canu & Bassler, 1920. Three species described from the Oligocene (Vicksburgian) Alabama, USA, are reassigned to Canda: Canda rathbuni  n. comb. Canda triangulata  n. comb. Canda williardi  n. comb. Scrupocellaria cookei Canu & Bassler, 1920 Scrupocellaria milneri Canu & Bassler, 1920 Scrupocellaria raigadensis Badve & Sonar, 1997  n. comb., Licornia milneri  n. comb., Licornia raigadensis  n. comb., and Licornia resseri  n. comb. Scrupocellaria marosticana Bizzarini & Braga, 2001 Scrupocellaria watersi Bizzarini & Braga, 1999 Scrupocellaria watersi Kluge, 1914 Aquiloniella, thus Aquiloniella marosticana  n. comb. The characteristics observed in the type specimens of Scrupocellaria dubia Canu & Bassler, 1920 Scrupocellaria vaughani Canu & Bassler, 1920 viz. Scrupocellaria elliptica Reuss, 1869 Scrupocellaria brendolensis Waters, 1891 Scrupocellaria crenulata MacGillivray, 1895 Scrupocellaria elliptica  Scrupocellaria gracilis Reuss, 1869 Scrupocellaria montecchiensis Waters, 1891 Scrupocellaria prolifera  Scrupocellaria rostrata Malecki, 1980 Scrupocellaria s. str. or other genera.About 19 fossil species have been assigned to the genus Table S1Character matrix used in phylogenetic analysis.(XLSX)Click here for additional data file.Text S1List of specimens examined and included in the phylogenetic analysis.(DOCX)Click here for additional data file.Text S2List of character state optimisations for semi-strict consensus tree . No List(DOCX)Click here for additional data file.Text S3Scrupocellaria species.List of type material of (DOCX)Click here for additional data file.Text S4Aquiloniella species.List of type material of (DOCX)Click here for additional data file.Text S5Aspiscellaria n. gen.List of type material of (DOCX)Click here for additional data file.Text S6Paralicornia n. gen.List of type material of (DOCX)Click here for additional data file.Text S7Pomocellaria n. gen.List of type material of (DOCX)Click here for additional data file.Text S8Scrupocaberea n. gen.List of type material of (DOCX)Click here for additional data file.


Mycoplasma hyopneumoniae is the causative agent of porcine enzootic pneumonia (EP), a mild, chronic pneumonia of swine. Despite presenting with low direct mortality, EP is responsible for major economic losses in the pig industry. To identify the virulence-associated determinants of M. hyopneumoniae, we determined the whole genome sequence of M. hyopneumoniae strain 168 and its attenuated high-passage strain 168-L and carried out comparative genomic analyses.M. hyopneumoniae strain 168 and its attenuated strain and made a preliminary survey of coding sequences (CDSs) that may be related to virulence. The 168-L genome has a highly similar gene content and order to that of 168, but is 4,483 bp smaller because there are 60 insertions and 43 deletions in 168-L. Besides these indels, 227 single nucleotide variations (SNVs) were identified. We further investigated the variants that affected CDSs, and compared them to reported virulence determinants. Notably, almost all of the reported virulence determinants are included in these variants affected CDSs. In addition to variations previously described in mycoplasma adhesins , cell envelope proteins (P95), cell surface antigens (P36), secreted proteins and chaperone protein (DnaK), mutations in genes related to metabolism and growth may also contribute to the attenuated virulence in 168-L. Furthermore, many mutations were located in the previously described repeat motif, which may be of primary importance for virulence.We performed the first comprehensive analysis of M. hyopneumoniae by comparative genomic analysis of virulent strain 168 and its attenuated high-passage strain 168-L. Our findings provide a preliminary survey of CDSs that may be related to virulence. While these include reported virulence-related genes, other novel virulence determinants were also detected. This new information will form the foundation of future investigations into the pathogenesis of M. hyopneumoniae and facilitate the design of new vaccines.We studied the virulence attenuation mechanism of  Mycoplasma hyopneumoniae causes porcine enzootic pneumonia, which is a mild, chronic pneumonia of swine m repeat region [n[(P/S)Q]m repeat region. Polyserine chains often function as a spacer region in proteins involved in complex carbohydrate degradation [The P146 adhesin-like protein of adhesion . In addiae cells . Comparet region . Interesradation , while sradation ,37. SuchM. hyopneumoniae[M. hyopneumoniae lysates [P159 is a proteolytically processed surface adhesin of neumoniae. Three p lysates , with eaM. hyopneumoniae[P216 (MHP168L_503/MHP168_503) is a proteolytically processed cilium and heparin binding protein of neumoniae. This suneumoniae. The 168neumoniae,34. CollM. conjunctivae. LppT is the second gene in a two gene operon with LppS, which was reported to be an adhesin in M. conjunctivae[M. hyopneumoniae, LppT encoded a protein of 954 aa with a calculated molecular mass 108 kDa. The gene product encoded by LppT is also a membrane protein, with a signal sequence of 34 aa at the amino-terminal end and a transmembrane structure. Notably, one of the amino acid substitutions in 168-L occurs near the C-terminus of LppT, resulting in a L814F replacement.The MHP168L_424 gene and its gene product LppT were analyzed in detail because they showed approximately 22% identity to the LppT protein from M. hyopneumoniae. In 168-L, MHP168L_103 (P95) is truncated by a nonsense mutation compared to 168, resulting in an E965* termination near the C-terminus of P95. Significant alteration in this outer membrane protein could conceivably cause a truncation in coding region, and in turn alter the function of P95 outer membrane protein.Cell envelope proteins and secreted proteins are involved in virulence, host cell interaction, and immune responses ,38,39. OM. hyopneumoniae contains an abbreviated membrane protein secretory system [secA (MHP168L_088), secY (MHP168L_128), secD (MHP168L_259), prsA (MHP168L_664), dnaK (MHP168L_069), trigger factor (MHP168L_154), and lepA (MHP168L_076). It has recently been demonstrated that some pathogenic bacteria use a type IV secretion system, composed of subunits related to the conjugation machinery, to deliver effector molecules to host cells [DnaK.y system . The patst cells , and thast cells . We founM. hyopneumoniae revealed several immunodominant proteins, including the P36 cytosolic protein [DnaK[M. hyopneumoniae[Studies on the antigenic properties of  protein ,42, the  protein -46, the  protein , the chaein [DnaK, the pyrein [DnaK, and theneumoniae.M. hyopneumoniae[Mycoplasmas commonly found in pigs demonstrated that the P36 proteins carry highly conserved species-specific antigenic determinants for M. hyopneumoniae[Mycoplasmas, including M. flocculare, M. hyorhinis, and Acholeplasma laidlawii[The cytosolic P36 protein is a lactate dehydrogenase  that indneumoniae. Comparaneumoniae. Hyperimlaidlawii. NotablyM. hyopneumoniae that is specifically recognized during disease [M. hyopneumoniae[P65 is an immunodominant surface lipoprotein of  disease . Analysi disease . The monneumoniae. In 168-Mycoplasmas are dependent on the exogenous supply of many nutrients, it has been predicted that they may need many transport systems [sgaA (MHP168L_422), sgaB (MHP168L_421), sgaT (MHP168L_563), mtlF (MHP168L_739), mtlA (MHP168L_561), nagE (MHP168L_582), and licA (MHP168L_041) [M. hyopneumoniae 168-L , cell envelope proteins (P95), cell surface antigens (P36), secreted proteins, chaperone protein (DnaK), and genes directly related to metabolism and in vivo growth may contribute to loss of virulence in 168-L. We then proceeded to characterize the alterations in gene functions caused by mutations at the protein level, and compared those mutations with previously described repeat motifs that may be of primary importance for virulence [in vivo studies are required to confirm and assess the relative importance of these suspected novel virulence determinants in the attenuation of virulence. The comparative genomic analysis presented here will not only provide insights into the basis of attenuation of virulence in 168-L, but may also provide targets for mutagenesis in the pursuit of development of a more efficacious vaccine.We successfully used a combination of sequencing genomics and comparative genomics strategies to provide a comprehensive analysis of virulent and attenuated irulence . InteresM. hyopneumoniae strain 168 and 168-L were selected for sequencing. Both of the strains were grown in KM2 cell-free medium at 37°C. The culture was harvested from 100 mL KM2 cell-free medium by centrifugation at 1,200×g for 30 min, and then total genomic DNA was extracted from mycoplasma cultures using a TIANamp Bacteria DNA Kit  according to the manufacturer’s instructions.Clonal isolates of Genomic libraries containing 8 kb inserts were constructed according to the manufacturer’s protocols. Whole-genome sequencing of strain 168-L was performed by combining GS FLX and Solexa paired-end sequencing technologies. A total of 242,507 reads (67.4% paired ends) were produced with the GS FLX system, giving 44.5-fold coverage of the genome. Eighty-eight percent  of reads were assembled into one large scaffold using Newbler . A total of 1,971,358 reads were generated with an Illumina Solexa genome analyzer IIx  and were mapped to the scaffold with the Burrows-Wheeler Alignment (BWA) tool . Gaps weM. hyopneumoniae strain 232 and strain J rRNA genes. Artemis (release 12) [http://www.genome.jp/kegg/pathway.html). Pseudogenes were detected by BLASTN analysis, comparing the genome sequences of 168-L with those of 232 and J, and then the annotation was revised manually.Open reading frames containing more than 30 amino acid residues were predicted using Glimmer 3.0  with modease 12)  was usedease 12) , GenBankease 12) , Swiss-Pease 12) , and COGease 12)  databaseNucleotide comparisons and single nucleotide polymorphism (SNP) analysis for strains 168 and 168-L were performed using the Artemis Comparison Tool (ACT)  and MauvM. hyopneumoniae strains 168 and 168-L genome sequences have been deposited in GenBank under accession numbers CP002274.1 and CP003131, respectively.EP: Porcine Enzootic Pneumonia; SNVs: Single Nucleotide Variations; MPS: Mycoplasmal Pneumonia Of Swine; COG: Clusters Of Orthologous Groups; EC: Enzyme Classification; IS: Insertion Sequence; ICE: Integrative Conjugative Element; SSB: Single Strand Binding Protein; PTS: Phosphotransferase; BWA: Burrows-Wheeler Alignment; tRNA: Transfer RNA; rRNA: ribosomal RNA; KEGG: Kyoto Encyclopedia of Genes and Genomes; ACT: Artemis Comparison Tool.The authors declare that they have no competing interests.Conceived and designed the experiments: WL SX LF HC. Performed the experiments: WL ML SL SG ZF. Analyzed the data: WL ZZ GS RL BL. Contributed reagents/materials/analysis tools: WL GS RL BL. Wrote the paper: WL LF SX. All authors read and approved the final manuscript.Nucleotide differences identified between genomes of 168 and 168-L.Click here for fileInsertions detected in 168-L compared to 168.Click here for fileDeletions detected in 168-L compared to 168.Click here for fileSNVs detected in 168-L compared to 168.Click here for fileAnnotated CDS of the elements ICEH168 and ICEH168-L.Click here for fileComplete list of "168-L-specific" genetic variations in intergenic.Click here for fileMutations affecting genes with ABC transporter family signatures.Click here for file


Lawsonia inermis L. and Mimosa pudica L. were proven for antibacterial properties against 15 Gram-positive and Gram-negative human pathogenic bacteria. Among the three types of extracts tested, ethanol extract was found to possess maximum antibacterial activity. The diameter of the zone of inhibition of bacterial growth showed that Gram-negative bacteria are more sensitive than Gram-positive bacteria to plant extracts. Between the two plants species studied, Lawsonia inermis extract showed more antibacterial activity compared to Mimosa pudica extract.Organic extracts  of two medicinal plants  Medicinal plants have played a significant role in ancient traditional systems of medication in many countries. They are rich source of bioactive compounds and thus serve as important raw materials for drug production2. Now-a-Lawsonia inermis L. and Mimosa pudica L. belong to the family of Lythraceae and Fabaceae, respectively. Both plant species are well-known for their medicinal properties and commonly distributed in Bangladesh. L. inermis is traditionally used for the treatment of headaches, migraine, albinism, skin abrasions and ulcers, burns, smallpox, leprosy boils, wounds, some mycotic infections. It was also used for the treatment of scalp and hair infections and ailments[M. pudica has been used traditionally in the treatment of various ailments including alopecia, diarrhoea, dysentery, insomnia, tumour and various urogenital infections[ ailments. It has  ailments, antican ailments and anti ailments8. M. pudnfections. It has nfections, antibacnfections12 and annfections. The purL. inermis L. and M. pudica L. were collected from Rajshahi University Flora, Bangladesh and identified at the Department of Botany, Rajshahi University, Rajshahi-6205. These specimens are preserved in the herbarium, Department of Botany, Rajshahi University, Rajshahi-6205, Bangladesh (Lawsonia inermis L.- Voucher Specimen No. 36 and Mimosa pudica L.- Voucher Specimen No. 52).The young leaf and stem (twig) of Staphylococcus aureus (BMLRU1002), Bacillus cereus (BMLRU1004), Staphylococcus haemolytica (BMLRU1006), Bacillus subtilis (BMLRU1008), Bacillus megaterium (BMLRU1010), Sarcina lutea (BMLRU1012) and Gram-negative Escherichia coli-B (BMLRU1001), Klebsiella sp. (BMLRU1003), Klebsiella pneumoniae (BMLRU1005), Pseudomonas aeruginosa (BMLRU1007), Salmonella typhi (BMLRU1009), Shigella dysenteriae (BMLRU1011), Shigella shinga (BMLRU1013), Shigella sonnei (BMLRU1015) and Pseudomonas sp. (BMLRU1017) species were tested. The strains were collected from the International Center for Diarrhoeal Disease Research of Bangladesh (ICDDRB), Mahakhali, Dhaka, Bangladesh.Following Gram-positive Lawsonia and Mimosa were cut into small pieces, oven dried at 40°. Well pulverized 5 g powder of the plant sample was continuously shaken with 20 ml each of ethanol, petroleum ether and chloroform on a water bath shaker for 10 h. The extracts were filtered through the Whatman No.1 filter paper, and the solvents were evaporated using a rotary evaporator to obtain brownish dark sticky residues. The evaporated extracts were dissolved in the respective solvents (10 mg/ml).Cleaned fresh twig samples of 2 in pressures at the temperature of 121° for 20 min to ensure sterilization.Bacterial strains were grown on Luria-Bertani (LB) medium which was prepared using the following compositions: 10 g of yeast extract supplemented with 10 g of bacto peptone and 5 g of NaCl dissolved in 1 liter of water. The pH L. inermis and M. pudica against the chosen 15 bacterial strains. Discs of 6 mm in diameter[L. inermis and M. pudica was determined by measuring zone of inhibition in millimeter scale against individual studied bacteria. Each assay was carried out in triplicate.Disc diffusion method was foll diameter were soa diameter. After i8cfu/ml), separately. Control test tubes contained only test organisms with distilled water instead of plant extract. The test tubes were incubated at 37° for 24 h. The least concentration of the plant extracts with no visible growth was taken as the MIC.Two-folds serial dilution method was follL. inermis, the zone of inhibition was ranging from 7.20 mm (E. coli) to 17.25 mm (S. dysenteriae). Lowest (156.25 μg/ml) and highest (2500 μg/ml) MIC was observed for S. dysenteriae and E. coli, respectively. In comparing with positive control it is shown that ethanol extract of L. inermis has good antibacterial properties. In petroleum ether extract highest and lowest zone of inhibition was 15.03 mm and 7.40 mm against S. dysenteriae and S. lutea, respectively and their corresponding MIC was 2500 μg/ml and 156.25 μg/ml. Here no zone of inhibition was observed for B. cereus and E. coli. In chloroform extract out of fifteen cases only in five cases, zones of inhibition were observed. This data is in close agreement with that published by other researchers[5L. inermis was found most effective for antibacterial activity especially on Gram negative bacteria.The results of the antibacterial screening are represented in Tables earchers517. CompaM. pudica, the highest and lowest zone of inhibition was 13.40 mm  and 9.35 mm  against P. aeruginosa and B. cereus, respectively. This data is in close agreement with Balakrishnan et al[B. subtilis with MIC value 156.25 μg/ml. Similar observations were made with L. inermis, where ethanol extract was found to exhibit most effective antibacterial activity against Gram-negative organisms. Negative control exhibited no zone of inhibition as expected whereas, positive control (disc containing antibiotics) exhibited zones of inhibition against all strains studied. Between the two plant species L. inermis showed more antibacterial activity than M. pudica.In ethanol extract of mm MIC, 3.5 μg/ml L. inermis and M. pudica have been determined for their antibacterial properties for controlling some Gram-positive and Gram-negative bacteria. Among the three extracts, ethanol extract was more effective than petroleum ether and chloroform extracts against Gram-negative bacteria than Gram-positive bacteria. Parekh and Chanda[Terminalia chebula, Mangifera indica and Eucalyptus citriodora showed maximum antibacterial activity. Khan et al.[Achyranthus aspera was much affective in Gram-negative bacteria than Gram-positive one. These observations may be attributed to the nature of biological active components whose activity can be increased in the presence of ethanol. Several types of alkaloids, glycosides, steroids and proteins have been reported to have the antibacterial activity[The screening of plant extracts, has led to the discovery of many clinically useful drugs that now play major roles in the treatment of human diseases. In the nd Chanda also repan et al. also rep activity.L. inermis and M. pudica. The antibacterial activity of the plant may be attributed to the various phytochemical constituents present in the crude extract. The work carried out was a basic approach to find out antibacterial activity residing in medicinal plants. This study indicated that medicinal plants could be a potential source of newer antibacterial agents. Further works on the types of phytoconstituents and purification of individual groups of bioactive compounds can reveal the exact potential of the plant to inhibit several pathogenic microbes and effective applications to control pathogenic bacteria causing severe illness in humans.In conclusion, the results of the study showed the antibacterial activity of twig extracts of


Duckweed can thrive on anthropogenic wastewater and produce tremendous biomass production. Due to its relatively high starch and low lignin percentage, duckweed is a good candidate for bioethanol fermentation. Previous studies have observed that water devoid of nutrients is good for starch accumulation, but its molecular mechanism remains unrevealed.Landoltia punctata). L. punctata was transferred from nutrient-rich solution to distilled water and sampled at different time points. Physiological measurements demonstrated that the activity of ADP-glucose pyrophosphorylase, the key enzyme of starch synthesis, as well as the starch percentage in duckweed, increased continuously under nutrient starvation. Samples collected at 0 h, 2 h and 24 h time points respectively were used for comparative gene expression analysis using RNA-Seq. A comprehensive transcriptome, comprising of 74,797 contigs, was constructed by a de novo assembly of the RNA-Seq reads. Gene expression profiling results showed that the expression of some transcripts encoding key enzymes involved in starch biosynthesis was up-regulated, while the expression of transcripts encoding enzymes involved in starch consumption were down-regulated, the expression of some photosynthesis-related transcripts were down-regulated during the first 24 h, and the expression of some transporter transcripts were up-regulated within the first 2 h. Very interestingly, most transcripts encoding key enzymes involved in flavonoid biosynthesis were highly expressed regardless of starvation, while transcripts encoding laccase, the last rate-limiting enzyme of lignifications, exhibited very low expression abundance in all three samples.This study globally analyzed the response to nutrient starvation in order to investigate the starch accumulation in duckweed (L. punctata under nutrient starvation, which indicates that nutrient starvation down-regulated the global metabolic status, redirects metabolic flux of fixed CO2 into starch synthesis branch resulting in starch accumulation in L. punctata.Our study provides a comprehensive expression profiling of  Liquid biofuels, such as bioethanol, converted from biomass are considered as a promising alternative for traditional fossil fuels. Biofuels development can reduce greenhouse gas emission and meet the world's rapidly growing demand for energy. Currently, bioethanol is mainly produced from feedstocks with relatively high starch or sugar percentage, such as corn, sugarcane, sweet potato and cassava -3. HowevLemnacecae family), a small flowering plant that has a global adaptability across a broad range of climates  was conducted by a locally installed blast program to investigate functional annotation of each contig. Of the 74,797 contigs, 51,968 (69.5%) had significant BLASTX hits and matched 25,581 unique protein accessions  of NCBI [http://main.g2.bx.psu.edu/]  for RNA sequencing.Total RNA were extracted by using OMEGATo get a comprehensive transcriptome and investigate the transcriptomic response to nutrient starvation, fronds harvested at 0 h, 2 h and 24 h were used for RNA-Seq study. More than 20 μg qualified total RNA extracted from each sample were used for RNA sequencing by Hiseq 2000. The poly (A)+ RNAs were purified using poly-T oligo-attached magnetic beads and eluted with Tris-HCl under heating condition. mRNAs was mixed with fragmentation media and then fragmented. Fragmented mRNAs were copied into first strand cDNA using reverse transcriptase and random primers. This is followed by second strand cDNA synthesis using DNA Polymerase I and RNaseH. The ends of these dscDNAs were repaired by adding a single ‘A’ base and then Illumina adapters ligated to the repaired ends. 200 bp cDNAs fragment were purified from a gel and used for further templates enrichment by PCR with two primers that anneal to the ends of the adapters to construct a fragmented cDNAs library. Quality control analysis was performed by Agilent 2100 Bioanalyzer.The validated 200 bp fragments cDNAs library constructed above was submitted to Illumina Hiseq 2000 to perform transcriptome sequencing. Then the Illumina sequencing-by-synthesis, image analysis and base-calling procedures were used to obtain PE read sequence information and base-calling quality values.http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc/] and PE reads were de novo assembled by using Trinity (v2012-06-08)  [http://pgrc.ipk-gatersleben.de/misa/misa.html].Before de novo assembly, the GC percentage was calculated basing on the PE reads. After sequence assembly, the GC percentage and codon usage bias of the transcriptome were analyzed using EMBOSS  on the Gsu.edu/] ,56. We ahttp://www.ncbi.nlm.nih.gov/] by using local blast program. For BLASTX search, the threshold was set to E-value <103. The blast results were imported into the Blast2GO [http://wego.genomics.org.cn/cgi-bin/wego/index.pl] [http://www.genome.jp/kegg/]. KEGG and GO function cluster was conducted by common perl scripts.All of the contigs produced by Trinity (v2012-06-08)  were useBlast2GO ,88 and pBlast2GO  was achindex.pl] . Enzyme To investigate the express level of each contig in different samples, all PE reads for each sample were aligned back to the final assembly by using perl scripts in Trinity (v2012-06-08) package  under deBased on the expression levels, the effect and bias introduced by library size and/or RNA composition were eliminated by edgeR  , then siTo verify the reliability of the NGS-based expression levels, 50 transcripts were chose randomly from the annotated transcripts set for primer design (Additional file DW: Dry weight; NGS: Next-generation sequencing; PE: Paired-end; NR: Non-redundant protein database; ORF: Open reading frame; FPKM: Fragments Per Kilobase of transcripts per Million mapped fragments; DET: Differentially expressed transcript; log2FC: log2 fold-change; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; EC: Enzyme codes.The authors declare that they have no competing interests.XT carried out the data analysis, drafted and revised the manuscript. YF participated in the design of the study and revised the manuscript. YX carried out the biochemical assays, drafted and revised the manuscript. Y-lJ participated in the design of the study and revised the manuscript. X-rM revised the manuscript. YZ revised the manuscript. K-zH participated in the design of the study. HZ conceived the study and revised the manuscript. H-yW participated in the design of the study and revised the manuscript. All authors read and approved the final manuscript.Sequence annotations of L. punctata transcripts and the gene expression profiling of three samples.Click here for fileCodon usage table of L. punctata coding sequences.Click here for fileStatistics of cDNA-derived SSR markers in L. punctata.Click here for fileDifferentially expressed transcripts identified among three L. punctata samples.Click here for fileGO classification of L. punctata transcriptome and differentially expressed transcripts indentified among three samples. A: GO classification of the L. punctata transcriptome; B,C,D: GO classification of the differentially expressed transcripts for 0 h vs 2 h, 0 h vs 24 h, 2 h vs 24 h.Click here for fileKEGG classification of L. punctata transcriptome and differentially expressed transcripts indentified among three samples.Click here for fileComparison of expression patterns between RNA-Seq expression and qRT-PCR.Click here for file


Francisella had been genome-sequenced. Previously published Francisella genome sequences were largely restricted to the zoonotic agent F. tularensis. Only limited data were available for other members of the Francisella genus, including F. philomiragia, an opportunistic pathogen of humans, F. noatunensis, a serious pathogen of farmed fish, and other less well described endosymbiotic species.Prior to this study, relatively few strains of Francisella species, including some for which the phylogenetic positions were previously uncertain. The genus Francisella could be divided into two main genetic clades: one included F. tularensis, F. novicida, F. hispaniensis and Wolbachia persica, and another included F. philomiragia and F. noatunensis.We determined the phylogenetic relationships of all known Francisella species were found to have significant recombination frequencies. However, the fish pathogen F. noatunensis subsp. noatunensis was an exception due to it exhibiting a highly clonal population structure similar to the human pathogen F. tularensis.Some Francisella can be divided into two main genetic clades occupying both terrestrial and marine habitats. However, our analyses suggest that the ancestral Francisella species originated in a marine habitat. The observed genome to genome variation in gene content and IS elements of different species supports the view that similar evolutionary paths of host adaptation developed independently in F. tularensis  and F. noatunensis subsp. noatunensis (infecting fish).The genus  Francisella tularensis. Due to its infectious nature, ease of dissemination and associated high fatality rate , F. tularensis has been included in military biological weapons programs and is regarded as one of the top six agents which have a high potential for misuse in bioterrorism [Tularemia is a zoonotic disease caused by the highly infectious, virulent, Gram-negative bacterium rrorism .Francisella has recently expanded substantially and the boundaries between taxonomic units within the genus is currently under debate [F. tularensis subspecies: subsp. tularensis  and subsp. holarctica  [F. tularensis type A infections, for which mortality rates up to 30% have been reported [F. tularensis type B causes a milder disease, for which fatalities rarely occur. A third accepted subspecies, F. tularensis subsp. mediasiatica is found in central Asia. This subspecies has been found to display level of virulence comparable to strains of subspecies holarctica and very rarely cause human infections [F. novicida  and F. philomiragia  [The known diversity of the genus  debate ,3. In masphere) . Nearly eported . In contections ,7. Disea water) -10.Francisella lineages also exist that do not appear to be associated with human disease. Strains closely related to F. philomiragia, which have recently been designated as Francisella noatunensis, have been identified as etiological agents of Francisellosis in fish [F. noatunensis is further divided into two subspecies, subsp. noatunensis[orientalis[Francisella from soil and water [F. tularensis (Francisella-like endosymbionts) [Wolbachia persica[alpha-proteobacteria [Candidatus F. noatunensis subsp. endociliophora’ [Francisella cantonensis[Francisella genus has become increasingly apparent. Members of Francisella appear to be associated with a multitude of different ecological niches, ranging from specialised endosymbionts and pathogens with different host spectrums, to generalists believed capable of a free-living existence.A number of in fish -15. F. natunensis and subsrientalis,18. Reced water -23, as wbionts) . One isoa persica, incorreacteria . Furtherntonensis. Thus, iFrancisella strains other than those belonging to F. tularensis. Analysis of gene content and genetic relationships may help to improve our understanding of the biology and evolution of these bacteria, including that of the human pathogen F. tularensis. Sequencing multiple strains of environmental Francisella may also reveal important sequence differences between environmental species and F. tularensis. This would allow the design of increasingly specific F. tularensis detection assays for use in anti-bioterrorism applications and epidemiological studies, particularly important since current methods frequently falsely detect F. tularensis in environmental samples [There are several reasons for sequencing samples .Francisella and includes genome sequences for 18 environmental Francisella strains, four F. tularensis and two distant but genetically related species Fangia hongkongensis[Piscirickettsia salmonis[. Here we describe the population structure of the genus Francisella, suggest that ancestral Francisella strains originated in marine habitats, show that present-day strains can be divided into two main genetic clades occupying continental and aquatic habitats and provide evidence that similar evolutionary paths of host adaptations have occurred independently in F. tularensis  and F. noatunensis subsp. noatunensis (infecting fish).The genome sequence data generated by the present study represents a considerable advancement of knowledge of the genus kongensis and Pisc salmonis. Here weFrancisella genus : ANI for F. philomiragia compared to F. noatunensis subsp. noatunensis is higher (95%) than F. philomiragia compared to F. noatunensis subsp. orientalis (92–93%) Additional file Analysis of the whole-genome phylogeny showed that strains of F. philomiragia  shared 1359 genes , which grouped at three different locations in the genome. No unique genes were found in F. novicida genomes. F. hispaniensis, F. noatunensis and F. philomiragia exhibited seven, nine and ten unique genes, respectively. The complete clade 2 (F. noatunensis and F. philomiragia) genomes displayed 41 genes that were not present in clade 1. However, the clade 2 species F. noatunensis subsp. noatunensis and F. noatunensis subsp. orientalis shared 32 and 114 genes, respectively, which were not present in other genomes.An overview of the gene distribution in the genus F. hispaniensis contained a few copies of ISFtu2, ISFtu3, ISFtu4, ISFpi1 and IS4502, while W. persica contained very few copies of ISFtu2 and ISFtu3. The isolates in clade 2 exhibited a different pattern of IS elements. Whereas F. noatunensis subsp. orientalis was almost completely lacking IS elements , F. noatunensis subsp. noatunensis exhibited an expansion trend similar to that previously reported for F. tularensis[Notable differences in IS element content were identified between different species and clades. In clade 1, ularensis. The expFa. hongkongensis and P. salmonis, contained completely different sets of IS elements compared with Francisella. The genome of P. salmonis was enriched with ISPsa1 and ISPsa2 [Fa. hongkongensis. The IS element distributions of F. tularensis, F. novicida and F. philomiragia have been reported previously [The more distant relatives studied, i.e.  ISPsa2 , whereasviously ,38.Francisella core genome demonstrating evidence of recombination was 32%, although the presence of recombination was highly variable across the different Francisella lineages. For some species/subspecies, recombination was entirely absent , whereas recombination in F. philomiragia and F. novicida occurred frequently.Results of the recombination analyses are presented in Figure Francisella genomes have mainly been of the human pathogenic species F. tularensis. Consequently, conclusions about evolutionary processes within the genus Francisella have largely been based on F. tularensis genomes [To date, sequenced genomes ,38.F. tularensis genome sequences have been shown to give false-positive results. These have been attributed to the presence of the same sequence signatures as in other environmental Francisella lineages [Moreover, a number of identification assays developed on the basis of ineages .Francisella, with the exception of the recently published F. cantonensis[. Despite a generally high sequence similarity within the genus, we found that different genetic clades demonstrated different population structures (recombination frequencies). This suggests differences in lifestyle and host association.In the present study, we provide details of the genetic content of nearly all currently known members of the genus ntonensis. DespiteF. tularensis has been found to be a highly clonal species, differentiating it from environmental lineages characterised by moderate recombination rates. We also found evidence that the fish pathogen F. noatunensis subsp. noatunensis also possesses a highly clonal lineage. The low recombination frequency in F. noatunensis subsp. noatunensis may indicate that a close host association reduced contact with other Francisella species, and thereby, lowered opportunities for gene acquisition by recombination, as previously hypothesised for F. tularensis[F. novicida opposed to F. tularensis subspecies tularensis, holarctica and mediasiatica strains should be taken in consideration for the on-going discussion whether it should be a separate species or included as a subspecies in F. tularensis.ularensis. The datFrancisella genomes showed that recognised species could be divided into two main genetic clades  than other Francisella genomes. Moreover, in W. persica, regulatory genes were found to be depleted, while biosynthesis pathways supplying amino acids to their insect host were retained, and there was a low abundance of IS elements. Our results confirm that W. persica and other Francisella-like endosymbionts (FLE) [Francisella and that W. persica has undergone a significant reduction in its genome.In agreement with other studies ,40, our s (FLE) ,24,41,42F. novicida[F. hispaniensis clade [An important outcome of the present study was reclassification of the isolate 3523 from Australia. It was initially classified as an unusual  novicida, a class novicida. It was s clade . This reFrancisella was similar to that reported for the genus Bacillus  are organised in an operon and were predicted to be involved in exopolysaccharide synthesis. It has been suggested that an exopolysaccharide capsule plays a role in F. tularensis virulence [The seven specific genes in rulence .wbt locus of F. tularensis. Genes in this locus encode proteins involved in lipopolysaccharide O-antigen synthesis. The O-antigens of F. tularensis contain an insertion element that differentiates it from F. novicida[Shigella sonnei[Francisella is not straight-forward. Our data suggest that F. tularensis virulence cannot be easily explained by a simplistic model which just considers the presence or absence of specific “virulence genes” [The second gene cluster  is present within the  novicida. A similla sonnei. Finally genes” .Fa. hongkongensis and P. salmonis both contain many genes that are absent from the Francisella genus and are therefore likely to occupy distinctly different ecological niches.Francisella genus exhibit several similarities and differences in IS element content and composition. F. noatunensis subsp. noatunensis contains an expanded set of IS elements similar to those reported for F. tularensis. The expanded IS elements in F. tularensis are ISFtu1 and ISFtu2, whereas the expanded IS element in F. noatunensis is ISFpil. Interestingly, the two subspecies of F. noatunensis exhibited contrasting contents of IS elements. While F. noatunensis subsp. noatunensis contained the highest number of IS elements of all species in the genus, F. noatunensis subsp. orientalis contains very few IS elements. This may have arisen because of different evolutionary modes in these subspecies.The two principal clades of the Francisella genus is highly variable, ranging from undetectable in F. tularensis to moderate in F. novicida[Recombination is an important process for generating new genetic diversity within bacterial populations and is a known driver of evolution in many bacterial species . The rec novicida.F. noatunensis exhibited apparently low rates of recombination at levels similar to the human pathogen group of F. tularensis. Otherwise, both clade 1 and II displayed moderate recombination rates, largely due to frequent recombination within F. novicida (clade 1) and F. philomiragia (clade 2). According to our findings, 33% of the core gene set provided evidence of past recombination events, similar in magnitude to estimates for Campylobacter[Neisseria[Francisella[F. novicida and F. philomiragia have been recombined, but our data suggests that this is could be an underestimate due to a lack of genomic sequences available at that time.We found that the subspecies of ylobacter and NeisNeisseria, but higancisella. It was Yersina pestis[Bacillus anthracis[Mycobacterium tuberculosis[Francisella genus in the present work, such effects were minimised.Our findings are similar to reports for other bacterial genera that include highly specialized pathogens. Variable recombination rates have previously been reported for several pathogenic populations, . It shoerculosis,56. By eFrancisella genomes, and therefore provides an important resource for assessing evolutionary and functional relationships within the genus. We identified the core genome in the Francisella genus and single nucleotide polymorphisms within these genes were used to infer a robust phylogeny. A high degree of clonality confirmed for F. tularensis type A and B, was also identified in F. noatunensis subsp. noatunensis. Despite infecting different species, it is apparent that these lineages also share certain important evolutionary properties.This work significantly expands the number of sequenced Francisella strains.Next-generation sequencing enables rapid generation of high quality data for the fast assembly of novel genomes . Growing2 for 5 days (F. tularensis strains) or up to ~30 days (non-F. tularensis strains).All strains Table , except 9 CFU/ml and heat-killed at 95°C for 15 minutes. The bacteria were lysed using Buffer ATL , or similar non-denaturing lysis buffer. DNA was prepared using phenol extraction (PE), or extraction with Biorobot EZ1  according to the manufacturer’s protocol. Sterility was verified by lack of growth on Modified Thayer-Martin (McLeod) plates [A loopful of bacteria was suspended in 1 x TRIS-EDTA (TE) buffer to 10 plates  after inWolbachia persica) was propagated in cells, and bacteria were collected by differential centrifugation. Bacterial cell pellets were suspended in phosphate-buffered saline (PBS) and DNA was prepared as above. For FSC772 and FDC178, DNA was prepared using the Gentra Puregene Cell kit  and was re-suspended in a hydration solution supplied with the kit. DNA concentrations were measured using Nanodrop .The strain FSC845  was calculated by pairwise genome comparisons with the MUMmer and BLAST algorithms using JSpecies v1.2.1 . A thresFrancisella species and subspecies  in the included genomes. Pairwise BLAST alignments of both left and right flanking regions, and full length IS sequence elements, were constructed for all genomes. The results were evaluated by e-value (<10-3), identity (90%), bit score (>40) alignment length and proximity to contig ends. Inherently, de novo assembly of next-generation sequence data implies problems in resolving repeat structures, resulting in an increased risk of underestimating the IS element copy number.This study focuses on newly sequenced genomes of previously uncharacterized Francisella and two genetic relatives were downloaded from IMG/ER [To infer recombination events in the ex (Phi), neighboex (Phi) and maxiex (Phi). These mex (Phi). A subseFrancisella-like endosymbionts; MaxChi: Maximum chi-square; NSS: Neighbour similarity score; Phi: Pair wise homoplasy index.ANI: Average nucleotide identity; FLE: No competing interests for any of the authors exist.Francisella isolates that infect fish and knowledge of fish pathogenic Francisella, and contributed to completion of the manuscript. PLA and MF conceived the study. AS, KS, JA, PLI, AJ, PLA, MF drafted the manuscript. All authors read and approved the manuscript.AS assembled all genomes and performed analysis of genome and gene content. JA performed the 16 S rRNA alignment, analysis of over-represented COGs and recombination. CO performed the analysis of average nucleotide identity, IS elements and clade specific genes. SD and DJC provided The views and conclusions contained in this document are those of the authors and should not be interpreted as necessarily representing the official policies, either expressed or implied, of the U.S. Department of Homeland Security or the Swedish Civil Contingencies Agency.is a table of the average nucleotide identity.Click here for fileis a figure showing the 16 S rRNA phylogenetic tree.Click here for fileis a table summarising the recombination results.Click here for fileis a table of sequencing statistics for included isolates.Click here for fileis a table of the IS element results.Click here for fileis a table giving information of public genome.Click here for fileis the results from orthoMCL.Click here for file


The purpose of this study was to estimate hand hygiene opportunities (HHOs) in two types of hospitals -- large-teaching and small-community, and three different clinical areas -- medical-surgical intensive care, general medical wards, and emergency departments.Hand hygiene opportunity data were collected through direct observations using the World Health Organization’s (WHO) monitoring methodology. Estimates of HHOs were developed for 12-hour AM/PM shifts and 24-hour time frames.During 436.7 hours of observation, 6640 HHOs were identified. Estimates of HHOs ranged from 30 to 179 per patient day on inpatient wards and 1.84 to 5.03 per bed hour in emergency departments. Significant differences in HHOs were found between the two hospital types and between the three clinical areas.This study is the first to use WHO data collection methodology to estimate HHO in general medical wards and emergency departments. These data can be used as denominator estimates to calculate hand hygiene compliance rates when product utilization data are available.C. Steed Grant/Research support from DebWorldwide, J. Kelly Grant/Research support from DebWorldwide, D. Blackhurst Grant/Research support from DebWorldwide, S. Boeker Employee of DebWorldwide, P. Alper: None declared, E. Larson Grant/Research support from DebWorldwide.


Ascaris suum (large roundworm of pigs) is a parasitic nematode that causes substantial losses to the meat industry. This nematode is suitable for biochemical studies because, unlike C. elegans, homogeneous tissue samples can be obtained by dissection. It has large sperm, produced in great numbers that permit biochemical studies of sperm motility. Widespread study of A. suum would be facilitated by more comprehensive genome resources and, to this end, we have produced a gonad transcriptome of A. suum.A. suum gonad, respectively. 86% of the high-quality (HQ) reads were assembled into 9,955 contigs and 69,791 HQ reads remained as singletons. 2.4 million bp of unique sequences were obtained with a coverage that reached 16.1-fold. 4,877 contigs and 14,339 singletons were annotated according to the C. elegans protein and the Kyoto Encyclopedia of Genes and Genomes (KEGG) protein databases. Comparison of TES and VAS transcriptomes demonstrated that genes participating in DNA replication, RNA transcription and ubiquitin-proteasome pathways are expressed at significantly higher levels in TES tissues than in VAS tissues. Comparison of the A. suum TES transcriptome with the C. elegans microarray dataset identified 165 A. suum germline-enriched genes (83% are spermatogenesis-enriched). Many of these genes encode serine/threonine kinases and phosphatases (KPs) as well as tyrosine KPs. Immunoblot analysis further suggested a critical role of phosphorylation in both testis development and spermatogenesis. A total of 2,681 A. suum genes were identified to have associated RNAi phenotypes in C. elegans, the majority of which display embryonic lethality, slow growth, larval arrest or sterility.Two 454 pyrosequencing runs generated 572,982 and 588,651 reads for germline (TES) and somatic (VAS) tissues of the A. suum. By comparison with C. elegans datasets, we identified sets of genes associated with spermatogenesis and gonad development in A. suum. The newly identified genes encoding KPs may help determine signaling pathways that operate during spermatogenesis. A large portion of A. suum gonadal genes have related RNAi phenotypes in C. elegans and, thus, might be RNAi targets for parasite control.Using deep sequencing technology, this study has produced a gonad transcriptome of  Ascaris, also known as the "giant intestinal roundworms", contains the largest intestinal nematode species. Ascaris lumbricoides causes the commonest helminth infection of humans, whereas a closely related species, Ascaris suum, typically infects pigs and causes substantial financial losses to the meat industry. The A. suum female is capable of producing more than 200,000 eggs per day and these eggs can survive and remain infective after many years in soil [e.g., RNA interference) for parasite control.The genus  in soil . At presA. suum [Caenorhabditis elegans biology provides opportunities to elucidate functionally conserved mechanisms in nematode biology. Employment of NGS technologies has greatly accelerated the 959 Nematode Genomes project http://www.nematodes.org/nematodegenomes/index.php/Main_Page. Genome sequencing of A. suum somatic cells is ongoing http://www.sanger.ac.uk/resources/downloads/helminths/ascaris-suum.html, and a draft genome and transcriptome of A. suum is now available http://www.nematode.net/NN3_frontpage.cgi?navbar_selection=home&subnav_selection=asuum_ftp. To date, there have been 38 A. suum EST libraries with ~55,000 sequences available in the NEMBASE4 database http://www.nematodes.org/nembase4/ and these ESTs were obtained using conventional cDNA library sequencing technology.To better understand the molecular and biochemical basis of nematode development, nematode EST projects have generated more than 250,000 ESTs from 30 species, including A. suum ,5. This A. suum gonad, the organ for reproduction. A. suum males have a large gonad that can be readily isolated by dissection to provide large numbers of sperm that are suitable for biochemical and cell biological studies [A. suum gonad is composed of three distinct regions; the testis and seminal vesicle form germline tissue and the glandular vas deferens forms somatic tissue. Sperm are stored in the seminal vesicle. During copulation, the spherical, non-motile sperm are activated into bipolar, amoeboid spermatozoa by an unknown component secreted by the glandular vas deferens. The motility of amoeboid sperm is driven by the regulated assembly and disassembly of major sperm protein (MSP) cytoskeleton [e.g., MPOP, MFPs and PP2A) that participate in the dynamics of the MSP cytoskeleton have been identified [A. suum, there have been few studies focusing on sperm chromatin or on distinctions between germline and somatic tissues in A. suum. In addition, chromatin diminution in A. suum represents a fascinating exception to the general rule of the constancy of the genome. However, the complex mechanism of this phenomenon, involving DNA degradation and new telomere addition remain an enigma [A. suum.In the present study, we applied 454 pyrosequencing technology to unravel the transcriptome of the male  studies . The malskeleton ,8. The mentified -12. Despn enigma -18. One A. suum, we acquired the transcriptomes of germline and somatic tissues of A. suum gonad using the RNA-seq approach. Comparison of these two tissues showed that the nucleic acid metabolic and proteasome-ubiquitin pathways are more active in the germline than in the soma. Further comparison with C. elegans microarray data identified 165 conserved germline-enriched genes in A. suum. We also categorized the RNAi phenotypes for A. suum gonadal genes, taking advantage of the C. elegans RNAi phenotype database. Therefore, these A. suum transcriptome data provide a valuable platform for both fundamental biological studies  and for research concerning parasite control (e.g. use of RNAi).To facilitate diverse studies concerning reproductive biology in A. suum gonad was dissected into two parts: testis and seminal vesicle (TES) and glandular vas deferens (VAS), and both samples were subjected to total RNA extraction followed by cDNA synthesis. Second-strand cDNAs with trimmed poly(A) tails were used for high-throughput sequencing on a 454 GS FLX Titanium platform. We performed two runs that produced ~1.16 million raw reads constituting a total of ~0.4 billion base-pairs (bp). The majority of the reads were over 400 bp and the average length of the reads was ~356 bp. The size distribution of the raw reads from both samples is shown in Figure de novo assembly using Newbler (Version 2.3). 999,214 reads from either TES or VAS were assembled into 9,955 contigs. These contigs range from 100 bp to 6,649 bp and 97.6% of them were assembled from three or more reads. The size distribution of the contigs is shown in Figure http://159.226.118.206/miaolab/index.htm. The contigs longer than 200 bp have been deposited in the GenBank Transcriptome Shotgun Assembly (TSA) database under the accession numbers JO467643-JO475858. A summary of 454 sequencing and assembly is shown in Table The male A. suum testis EST library in NEMBASE4 has collected 2,868 ESTs. These ESTs correspond to 595 homologous genes in C. elegans . In contrast, our A. suum gonad transcriptome corresponds to 4,207 homologous genes in C. elegans and 3,686 novel gonadal genes were identified  protein databases using BLASTX . A total of 4,877 contigs (49%) and 14,339 singletons (20.5%) were annotated. A large portion of the 454 sequences have not been functionally defined. Some sequences can be annotated by increasing the E-value and others may represent A. suum specific genes. In summary, 9,822 unique sequences  were annotated in the TES dataset and 12,123 unique sequences  were annotated in the VAS dataset , whose activity is essential for gonad and body morphology in C. elegans [Tubulin genes  are the most abundant transcripts in the  elegans , are alsA. suum gonad metabolic pathways, we mapped the C. elegans homologues of A. suum genes to the KEGG pathways. A total of 5,426 unique A. suum sequences corresponding to 850 homologous genes in C. elegans were assigned to metabolic pathways  having significantly higher levels of expression in TES than in VAS for semi-quantitative RT-PCR analysis. These genes are involved in processes including, protein processing, ubiquitin-proteasome pathways, Wnt signaling, cell division and nucleic acid metabolism. The results  [C. elegans germline-enriched genes and 139 (32.8%) of the soma-enriched genes have homologues in A. suum TES and VAS tissues, respectively . Substantial numbers of serine/threonine kinases and phosphatases (KPs), as well as tyrosine KPs were identified, suggesting pivotal roles of phosphorylation during spermatogenesis. It should be noted that the genes encoding KPs are over-represented among the sperm-enriched genes in C. elegans [Using microarray technology, Reinke ormBase) . BLAST ad 139 32.% of the  elegans .When comparing the TES and VAS datasets, we noticed an enriched expression of genes encoding KPs in TES tissues. There are 242 contigs in the TES dataset encoding KPs that have levels of expression 10-fold higher than those in VAS; in contrast, there are only 17 contigs encoding KPs with VAS/TES expression ratios above 10  [A. suum gonad transcriptome  analysis showed most of them regulate reproduction, embryo development, growth, locomotion and development of anatomical structures, as shown in Figure Through a genome-wide RNAi screen, Kalis ormBase) . BLAST aC. elegans [A. suum larval development has been reported [C. elegans and A. suum to query the C. elegans RNAi phenotype database. RNAi phenotypes associated with 2,681 genes were identified  was first discovered in  elegans  and has reported . TherefoA. suum causes massive losses to the swine industry worldwide. The 959 Nematode Genomes Project has included this species for whole-genome sequencing and a draft genome and transcriptome of A. suum has just become available. In this study, we adopted 454 sequencing technology to determine the transcriptome of the A. suum gonad so as to facilitate further studies of this organism.Nematodes are one of the most diverse phyla and they make up approximately 80% of all individual animals on earth ,26. As tC. elegans exome (26 million bp). 86% of the high-quality reads were assembled into longer contigs, suggesting that these sequencing data had a high coverage. We annotated half of the contigs and 20% of the singletons; a large fraction of sequences have not been functionally assigned. The unannotated 454 ESTs may contain precursor non-coding RNAs  as well as the polyadenylated ncRNA classes; for example, over 13% and 26% of full-length cDNAs in mice and human, respectively, are proposed to be polyadenylated mRNA-like ncRNAs [A total of 0.4 billion bp were obtained by 454 sequencing, which were assembled into 25 million bp, which is equivalent to the e ncRNAs -29. The C. elegans [A. suum germline. MSP comprises 10-15% of the total proteins in nematode sperm [To investigate germline-soma distinctions, we compared the digital transcriptomes of TES and VAS, and identified numerous TES-specific pathways, including DNA replication and proteasome and ubiquitin-mediated proteolysis pathways. These pathways might be required to regulate germline proliferation and differentiation. The proteasome has been documented to regulate the balance between cell proliferation and meiotic entry in  elegans . The gende sperm  and sperde sperm ,8,32,33.A. suum spermatogenesis. Based on the C. elegans microarray data, 165 A. suum genes (83% are spermatogenesis-enriched) were identified as conserved genes controlling germline development. The most abundant proteins involved in A. suum spermatogenesis consist of sperm specific proteins, PDZ domain proteins, tyrosine kinases and phosphatases (KPs), and serine/threonine KPs. Identification of the genes encoding KPs in this analysis underpins the essential role of phosphorylation in the regulation of spermatogenesis. As the most common posttranslational modification, phosphorylation has been established to link to sperm function in a variety of species. In mammalians, the processes regulated by phosphorylation include capacitation, hyperactivated motility, zona pellucida binding, acrosome reaction and sperm-oocyte binding and fusion [C. elegans, the genes encoding KPs are over-represented among the sperm-enriched genes [A. suum. A phosphorylated membrane protein (named MPOP) recruits a soluble casein kinase 1 (named MPAK) to the inner leaflet of the plasma membrane to initiate sperm motility [A. suum. We provide evidence that a ~45 KD protein in sperm is associated with strong tyrosine phosphorylation (pY). Because tyrosine phosphorylation and dephosphorylation act as a molecular switch to regulate MSP assembly [A. suum spermatogenesis/spermiogenesis.We have a particular interest in d fusion -36. In Ced genes . Clues tmotility ; MPAK, imotility ,11. A pumotility . The newassembly , we propC. elegans genes that are homologous to genes represented in the A. suum gonad transcriptome were examined for associated RNAi phenotypes. Although the genes involved in spermatogenesis are possibly insensitive to RNAi [C. elegans database. This RNAi phenotypic categorization is in line with the functional classification of the gonad developmental genes, the majority of which control reproduction, embryo development and growth. Due to the growing concern of anthelminth resistance, RNAi provides a new means to combat parasitic nematodes. RNAi has been successfully used to knock down target genes in a few parasitic nematode species, including B. malayi [H. glycines [G. pallida [O. volvulus [T. colubriformis [A. suum [A. suum gonad transcriptome sequencing data will provide opportunities to use RNAi as a novel anti-parasite agent for parasite control.Lastly,  to RNAi , a varie. malayi , H. glycglycines , G. pall pallida , O. volvvolvulus , T. coluriformis  and nota[A. suum . Recentl[A. suum . TherefoA. suum males were collected from the intestines of infected hogs at Zhongrui Pork Processors  and were stored in worm buffer . A. suum gonads were dissected into two parts: (1) testis and seminal vesicle; (2) glandular vas deferens. Dissected samples were immediately frozen in liquid nitrogen prior to storage at -80°C.A. suum testis and vas deferens were prepared using TRIzol reagent  before removal of trace genomic DNA using DNAse I . Poly A+ RNA was purified using an Oligotex Direct mRNA Kit  followed by first and second-strand cDNA synthesis using the Universal RiboClone cDNA Synthesis System (Promega) with a modification as follows. We designed a poly(T) adaptor with a BsgI site flanking the poly(T) sequence (5'-CGTGTGCAGT(20)VN-3') for cDNA synthesis. The purified second-strand cDNAs were digested by BsgI and recovered with a QIAquick PCR Purification Kit (Qiagen). Both double-strand cDNA samples were subjected to 454 pyrosequencing using the GS FLX Titanium Kit.Total RNAs from C. elegans confirmed protein database (derived from Wormbase) as well as the KEGG protein databases for all organisms http://www.genome.jp/kegg/download/ by BLASTX; the BLAST cutoff E-value was set at < 1e-5. The A. suum gonad developmental genes were functionally assigned by GO Slimer http://amigo.geneontology.org/cgi-bin/amigo/slimmer?session_id. The A. suum genes were mapped onto metabolic pathways according to C. elegans pathways http://www.genome.jp/kegg/download/. Total read numbers of TES and VAS datasets were normalized to equal levels, and the relative gene abundance was defined by log10 of the normalized read number. Heat-maps were generated using R software (version 2.12.0).High-quality sequences (> 99.5% accuracy on single base reads) were filtered to remove short ESTs (< 50 bp) before assembly using Newbler (version 2.3). For assembly, the quality score threshold was set to 40. All unique sequences containing both contigs and singletons were compared against the A. suum testis and vas deferens were isolated before being reverse transcribed into cDNA using the SuperScript III First-Strand Synthesis System (Invitrogen). The cDNA products were diluted 10-fold for use as RT-PCR templates. PCR was performed using High Fidelity PCR SuperMix  under the following conditions: 94°C for 3 min; 30 cycles of 94°C for 30 s, 57°C for 30 s, 72°C for 1 min; 72°C for 8 min. The genes Act-1 and eIF-4A were used as controls.Total RNAs from A. suum TES and VAS tissues were disrupted in HKB buffer  in a homogenizer. An equal volume of lysis buffer  was added and samples were then placed on ice for 30 min before centrifugation for 30 min at 20,000 rpm. Supernatants were then collected for Western blot analysis. To prepare sperm extracts (S100), frozen sperm were thawed on ice for 1 hr and centrifuged for 10 min at 13,000 rpm. Supernatant was centrifuged at 100,000 × g for 1 hr at 4°C. Supernatant (S100) was further analyzed by Western blotting.Fresh SDS-PAGE and Western blotting were performed as previously described . WesternA. suum sample collection. XM prepared ds cDNA samples and performed RT-PCR. CL conducted library construction and 454 pyrosequencing. XM and YS extracted total proteins from TES and VAS tissues and performed Western blot analyses. XM and YZ performed data analyses. XM and LM wrote the manuscript. All authors have read and approved the final manuscript.XM and LM conceived and designed the experiments. SC helped to conceive and coordinate the experiments. FM participated in A. suum testis EST library in NEMBASE4 and for the A. suum gonad transcriptomeSummary of gene assignments for the . Sheet GenBank EST summarizes the A. suum testis EST library; Sheet Gonad 454 data summarizes the A. suum gonad transcriptome.Click here for fileA. suum germline (TES) and soma (VAS) transcriptomesAnnotation of .Click here for fileA. suum TES and VAS transcriptomesMetabolic pathway assignment of .Click here for fileTranscripts with relatively high expression levels in TES and VAS. Sheet TES summarizes transcripts having a 10-fold or higher level of expression in TES compared with VAS; Sheet VAS summarizes transcripts having a 10-fold or higher level of expression in VAS compared with TES.Click here for fileGene/Protein association network for genes highly expressed in TES. The functions of the highlighted gene clusters are as follows: gray, proteasome pathway; yellow, ubiquitin pathway; green, nucleotide metabolism; pink, ribosome members; blue, MSP family.Click here for fileRT-PCR verification of highly expressed genes in TES. Genes Act-1 and eIF-4A are used as controls. Gene codes are from C. elegans.Click here for fileA. suumPredicted germline and soma-enriched genes in . Datasets were acquired by comparison of the A. suum gonad transcriptome with published C. elegans microarray data.Click here for fileA. suum candidate genes conserved in nematode germline developmentSummary of the 165 .Click here for fileContigs encoding kinases and phosphatases that are highly expressed in TES and VAS tissues. Sheet TES summarizes the transcripts of KPs having a 10-fold or higher level of expression in TES compared with VAS; Sheet VAS summarizes the transcripts of KPs having a 10-fold or higher level of expression in VAS compared with TES.Click here for fileA. suumGenes predicted to control gonadogenesis in . Dataset was acquired by comparison of the A. suum gonad transcriptome with published C. elegans genes that regulate gonad development.Click here for fileA. suum gonadal genesRNAi phenotypic categorization of . Each A. suum sequence ID is in bracket. RNAi phenotype codes are from Wormbase.Click here for file