Human and animal fascioliasis is emerging in many world regions, among which Andean countries constitute the largest regional hot spot and Peru the country presenting more human endemic areas. A survey was undertaken on the lymnaeid snails inhabiting the hyperendemic area of Cajamarca, where human prevalences are the highest known among the areas presenting a "valley transmission pattern", to establish which species are present, genetically characterise their populations by comparison with other human endemic areas, and discuss which ones have transmission capacity and their potential implications with human and animal infection.cox1 were sequenced by the dideoxy chain-termination method.Therefore, ribosomal DNA ITS-2 and ITS-1, and mitochondrial DNA 16S and Galba/Fossaria group: Galba truncatula, Lymnaea neotropica and L. schirazensis. Only one combined haplotype for each species was found. The ITS-1, 16S and cox1 haplotypes of G. truncatula are new. No new haplotypes were found in the other two species. This scenario changes previous knowledge, in which only L. viator (= L. viatrix) was mentioned. Galba truncatula appears to be the most abundant, with high population densities and evident anthropophyly including usual presence in human neighbourhood. Infection by Fasciola hepatica larval stages were molecularly confirmed in two populations of this species. The nearness between G. truncatula populations presenting liver fluke infection and both human settings and schools for children, together with the absence of populations of other lymnaeid species in the locality, suggest a direct relationship with human infection.Results indicate the presence of three, morphologically similar, small lymnaeid species belonging to the G. truncatula mainly involved in transmission to humans, L neotropica typically responsible for livestock infection, and L. schirazensis unable for transmission. Although several phenotypic characteristics may be helpful for a preliminary specimen classification, a definitive classification can only be obtained by marker sequencing. Aditionally, L. schirazensis increases the confusion, owing to its ability to mix with other Galba/Fossaria species and distort fascioliasis data such as transmission capacity and infection susceptibility. Second, a problem for epidemiological analysis, surveillance and control by methods as mathematical modelling and Remote Sensing - Geographical Information Systems. In Cajamarca, low resolution mapping may be insufficient, as already verified in Andean areas where different lymnaeid species overlap.The geographical overlap of three lymnaeid species poses problems for epidemiological studies and control action. First, a problem in classifying lymnaeid specimens in both field and laboratory activities, given their transmission capacity differences:  Fasciola hepatica distributed almost throughout and F. gigantica in large regions of Africa and Asia , H2 [EMBL: AJ243017] and H3  [EMBL: AJ272051] [L. cubensis H1 [EMBL: AM412223], H2 [EMBL: FN182200], H3 [EMBL: FN182201] and H4 [GenBank: JF514088]   [L. schirazensis H1 [GenBank: JF272601] and H2 [GenBank: JF272602] [-rDNA ITS-2: J272051] ,51; L. cF514088] ; L. viatM412224]  and H2 [M412224] ; L. neotM412225]  and H2 [F272602] .G. truncatula HA [EMBL: AJ243018], HB [AJ296270], HC  [EMBL: AJ272052] and HD [GenBank: JF514090] [L. cubensis HA from the type locality of Cuba [EMBL: AM412226], HB [EMBL: FN182202] and HC [EMBL: FN182203]   [L. schirazensis HA [GenBank: JF272603] and HB [GenBank: JF272604] [-rDNA ITS-1: F514090] ,41,50,51M412227] ; L. neotF272604] .G. truncatula 16S-A from Spain [EMBL: HE610431] (new sequence obtained for comparison purposes); L. cubensis 16S-A from the type locality of Cuba [EMBL: FN182204] (new sequence obtained for comparison purposes); L. viator 16S-A from the type locality of Rio Negro, Argentina, and also Chile [EMBL: HE610434] (new sequence obtained for comparison purposes); L. schirazensis 16S-A [GenBank: JF272605] and 16S-B [GenBank: JF272606] [Galba/Fossaria group such as Fossaria bulimoides [GenBank: AF485657] and F. obrussa [GenBank: AF485658] [-mtDNA 16S rRNA gene: F272606] ; and othcox1 gene: G. truncatula cox1a [EMBL: AM494011) [cox1b [GenBank: JF461487] [G. truncatula [GenBank: EU818799] [L. cubensis cox1a from the type locality of Cuba [EMBL: AM494009] [cox1b [GenBank: FN182205]  , cox1b [EMBL: FN356741], cox1c [GenBank: JF461485] and cox1d [GenBank: JF461486] [L. schirazensis cox1a [GenBank: JF272607], cox1b [GenBank: JF272608], cox1c [GenBank: JF272609] and cox1d [GenBank: JF272610]  , cox1c [F461487]  and G. tU818799] ; L. cubeM494009]  and cox1M494010] , cox1b [M494010] ; L. neotF461486] ,52; L. sF272610] .The rDNA ITS-1 sequence of the trematode larval stages was compared with the following sequences from GenBank-EMBL:F. hepatica from Spain, France, Poland, Ireland, Iran, Japan, Korea, Vietnam, Australia, Egypt, Bolivia, Peru, Uruguay, Argentina, Chile, Venezuela, Ecuador and Mexico  [-rDNA ITS-1: F612469] .Populations of lymnaeid species found are noted in Table cox1 nucleotide sequence data reported in this study are available in the GenBank™, EMBL and DDBJ databases under the accession numbers noted in Table Nuclear rDNA ITS-2 and ITS-1 and mtDNA 16S and L. viatrix or Lymnaea sp., proved to be G. truncatula by ribosomal and mitochondrial DNA markers , Yanamarca and Chaquicocha, preliminarily classified as rs Table .G. truncatula available in EMBL , this sequence proved to be identical to the previously described ITS-2 haplotype 1 (H1) for G. truncatula [EMBL: AJ243017].All the specimens showed identical ITS-2 sequence, of 401 bp and a 59.10% GC content. When compared with the three ITS-2 haplotypes of G. truncatula available in EMBL  and GenBank (HD) and proved to be different. It has consequently been ascribed to the new haplotype G.tru HE [EMBL: HE610430].Similarly, all specimens studied showed identical ITS-1 sequence, of 504 bp and a 57.74% GC content. This sequence was compared with the four ITS-1 haplotypes of G. truncatula present in Cajamarca, Peru (G.tru CH1E) and the Northern Bolivian Altiplano (G.tru CH3C) shows interesting information about the nucleotide differences shown by this lymnaeid vector in the two human fascioliasis endemic areas with the highest prevalences known , the Peruvian sequence proved to be identical to the previously described ITS-2 haplotype 1 (H1) for L. neotropica of its type locality [EMBL: AM412225]  and proved to be identical. This haplotype was previously reported in the type locality of Rio Lurin, Lima, Peru and also in Argentina [EMBL: AM412228].All specimens presented the same ITS-1 sequence of 533 bp and a 56.66 GC content. This sequence was compared with the ITS-1 haplotype of Only one halotype was detected in the specimens studied from this population. This partial sequence was 425 bp-long, presented a biased AT content of 69.48%, and was described as L.neo-16S-A provisional haplotype, as it proved to be identical to the one found in the type locality of this lymnaeid species [EMBL: HE610433].cox1 nucleotide sequence, of 672 bp and with a biased AT content of 69.5%. This sequence was compared with the four cox1 haplotypes of L. neotropica known so far and proved to be identical to the previously described haplotype L.neo-cox1a from the type locality of this species [EMBL: AM494008]. Nucleotide and amino acid differences between the four described haplotypes for L. neotropica are listed in Figure All of the specimens sequenced showed identical  EMBL: AM94008. NuL. viatrix, proved to be L. schirazensis after ribosomal and mitochondrial DNA marker sequencing , preliminarily identified as ng Table .L. schirazensis available in GenBank, it proved to be identical to the previously described L.schi-H1 [GenBank: JF272601].All the specimens presented the same ITS-2 sequence, of 436 bp and a 53.90% GC content. When compared to the two ITS-2 haplotypes of L. schirazensis available in GenBank  and proved to be the same as the previously described L.schi-HB [GenBank: JF272604].Similarly, all the lymnaeid individuals showed identical ITS-1 sequences, of 533 bp long and a 59.91% GC content. This haplotype was compared with two ITS-1 haplotypes of Only one halotype was detected in the specimens studied from this population. This partial sequence was 425 bp-long, presented a biased AT content of 69.48%, and corresponded to the provisional haplotype L.schi-16S-A [GenBank: JF272605].Galba/Fossaria species, including L. schirazensis plus G. truncatula, L. neotropica, L. viator, L. cubensis, F. bulimoides and F. obrussa are listed in Figure L. neotropica and L. cubensis, differing in only one polymorphic site in position 130.The nucleotide differences between the several haplotypes described for different cox1 nucleotide sequence, of 672 bp and with a biased AT content of 69.1%. This sequence was compared with the four cox1 haplotypes of L. schirazensis known so far and proved to be identical to the previously described haplotype L.schi-cox1d [GenBank: JF272610].All of the specimens sequenced showed identical Four DNA sequence alignments, one for each marker used, where made to show the total nucleotide differences between the respective haplotypes of the three lymnaeid species present in the fascioliasis hyperendemic area of Cajamarca.When comparing the ITS-2 sequences, a 487-bp-long alignment was obtained, in which a total of 172 variable positions (35.3%) including mutations and indels appeared Figure A. In thecox1 sequences, a total of 96 variable positions (14.2%) including only mutations were found in the 672-bp-long alignment. Most of these mutations were however silent, as indeed only two variable positions (0.8%) appeared in the 224-aa-long alignment of the corresponding partial protein sequences , with very few gaps  found in this species in Cajamarca pose a problem when attempting to elucidate the direct geographical source. In South America, the presence of G. truncatula has already been molecularly verified in the Northern Altiplano of both Bolivia [G. truncatula published so far [The aforementioned monomorphic genetic characteristics suggest an introduction of each one of the three lymnaeid species from only one source. In the case of  evident ,51, the  Bolivia , Argenti Bolivia ,77, Chil Bolivia  and Vene Bolivia . In Colod so far  appears d so far .Lymnaea neotropica, originally described from Lima and surroundings [oundings , appearsoundings  up to Veoundings . The comoundings .L. schirazensis present in Cajamarca appears to be a mix. It shares the ITS-2 haplotype L.schi-H1 with Spain, the Dominican Republic and Venezuela, and is different from the one in Rio Lurin, close to Lima. Its ITS-1 haplotype L. schir-HB has also been found in Mexico and Ecuador and in Peru it is present in both Cajamarca and Rio Lurin. The ribosomal 16S gene of the mtDNA does unfortunately not furnish any biogeographical information, as the haplotype L.schir-16S-HA found in Cajamarca is the same as everywhere. And finally, the mtDNA cox1 haplotype L.schir-cox1d appears to be unique [L. schirazensis with livestock and humans should have most probably occurred from northern Peru, probably by livestock transported between Quito and Lima through Cajamarca along the old rural Inca routes followed by the Spanish conquerors [The combined rDNA and mtDNA haplotype of e unique . Althouge unique , the intnquerors .G. truncatula, is responsible for such a human hyperendemic situation in the Northern Altiplano [Fascioliasis in Cajamarca has been the focus of several studies on both livestock ,37,61,81ltiplano , whereasGalba truncatula is considered the best F. hepatica transmitter known, with such a parasite/vector interaction as to support that this lymnaeid may be considered the original vector of this fasciolid [G. truncatula is known to give rise to very high human infection rates, both prevalences and intensities, at the very high altitude of the Northern Altiplano of both Bolivia [F. hepatica and G. truncatula as an adaptation response to the extreme conditions of the very high altitude [G. truncatula may behave in the 2600–3100 m high altitudes of Cajamarca similarly to in the 3800–4100 m altitudes of the Northern Altiplano, namely:Among the three lymnaeid species present in Cajamarca, asciolid . Moreove Bolivia ,18,19 analtitude . Several a) the results of the field surveys carried out showing that populations of this species are the most abundant in the endemic area; b) the typical anthropophylic characteristics of this lymnaeid appeared evident when proving to be the usual snail species in the neighbourhood of human communities such as Encañada, Santa Rosa de Chaquil, Tauripampa and Yanamarca Figure , close t c) the high population densities of thois species found in the high altitude localities studied Table ; d) the finding of infected specimens in two populations of this lymnaeid by means of the DNA sequencing methods Table , despiteG. truncatula populations from two different environments such as Cajamarca and the Northern Bolivian Altiplano should be emphasized  variety B elongata  has beerding to ,67,86. Trding to , as in Vrding to .L. schirazensis, experimental infection assays of different geographical strains of this snail species from different continents with F. hepatica and F. gigantica have proved that fasciolid larval stages are not able to fully develop within this lymnaeid, which does therefore not participate in disease transmission [L. schirazensis has an outstandingly broad distribution from lowlands to highlands, including from below sea level (−23 m) in Iran up to very high altitude areas in Ecuador (3158 m). Throughout this wide altitudinal range, it has always been confused with G. truncatula in the Old World and with G. truncatula and other similar Galba/Fossaria vector species in the Americas. This hitherto overlooked species has been masking not only the geographical distribution of fascioliasis, but also fasciolid population specificity/susceptibility analyses. It shall be considered that L. schirazensis has been shown molecularly to be able to cohabit with other Galba/Fossaria group species in such proximity that a natural population of specimens of an apparently unique lymnaeid species may in fact involve specimens from two different species unnoticeably mixed. Such mixed populations have already been described in the case of G. truncatula and L. schirazensis[With regard to smission . A largeirazensis.G. truncatula and L. neotropica, and a third non-transmitting lymnaeid, L. schirazensis, inhabit the fascioliasis hyperendemic area of Cajamarca. This new scenario pronouncedly changes the situation described so far, in which disease transmission was mentioned to be related to only one lymnaeid species, L. viator (= L. viatrix). All suggests that this has been the consequence of misclassifications due to the inaccuracy of traditional malacological methods for species differentiation among the Galba/Fossaria group of lymnaeids to which the three aforementioned, morphologically similar, small snail species belong [Sequences of the rDNA and mtDNA markers indicate that two lymnaeid vectors, s belong ,50.G. truncatula and L. schirazensis in Baños del Inca district, and of G. truncatula and L. neotropica in Cajabamba district, indicate that these three lymnaeid species geographically overlap inside the hyperendemic area. This fact poses two problems of increasing difficulty for epidemiological studies and control action.The nearby detection of populations of cox1. This problem becomes extremely important given the transmission capacity differences of these three lymnaeid species: G. truncatula as the vector mainly involved in disease transmission to humans, L neotropica as a typical responsible for livestock infection, and L. schirazensis as a lymnaeid unable to transmit fascioliasis. Moreover, from the ecological point of view, these three species may be found to show similar amphibious characteristics. Additionally, L. schirazensis pronouncedly increases the confusion problem, owing to its ability to mix its specimens and populations with those of other Galba/Fossaria species and distort fascioliasis data such as transmission capacity and infection susceptibility. Such L. schirazensis mixing has already been described with G. truncatula[First, there is a problem in classifying lymnaeid specimens in both field and laboratory activities in Cajamarca. Indeed, although several phenotypic characteristics may a priori be helpful for a preliminary specimen classification Table , a definruncatula.G. truncatula, is responsible for disease transmission. However, the same methods did not appear to be sufficiently accurate when analysing fascioliasis in the central regions of Chile where human fascioliasis is endemic and livestock prevalences are the highest in the country [Second, the complexity of the overlap of two different lymnaeid vector species poses a serious problem for the development of methods which have shown to be useful for epidemiological analysis, surveillance and control of human fascioliasis in Andean high altitude endemic areas, such as mathematical modelling through the application of different climatic indices  , and Rem country . Recent  country . The samThese conclusions should be considered within future control activities in the province of Cajamarca. In this hyperendemic area, a pilot intervention to assess human treatment strategies was successfully performed in 2007 and 2008, human fascioliasis treatment activities have since then been yearly implemented, thanks to the availability of Egaten® , provided by WHO through the Ministry of Health in Lima and the Dirección Regional de Salud of Cajamarca, and a large initiative for animal control to diminish risk infection has already been approved.The authors declare that they have no competing interests.MDB contributed to the design of the study, participated in field collections, analysed the sequences, and helped to draft the manuscript. PA carried out the DNA sequencing processes. MK performed the phenotypic characterisation. PO participated in laboratory procedures and facilitated logistics for both experimental and field work in the endemic areas of Cajamarca. CN participated in lymnaeid collections in the endemic areas of Cajabamba. SMC designed and supervised the study, participated in field collections, performed the epidemiological analyses, and wrote the manuscript. All authors read and approved the final manuscript.


Periodontal bacteria occur in both planktonic and biofilm forms. While poor oral hygiene leads to accumulation of bacteria, reducing these microbes is the first step toward good oral hygiene. This is usually achieved through the use of mouthwash solutions. However, the exact antibacterial activity of mouthwash solution, especially when bacteria form biofilms, is yet to be determined. In this study, we evaluated the antibacterial activity of common mouthwash solutions against standard bacteria in their planktonic and biofilm states.Standard bacterial strains were cultured, and biofilm were formrd. Thereafter, using standard method for determination of minimum inhibitory concentrations (MIC) values of various mouthwash solutions were determined.Results show that common mouthwash solutions have variable antibacterial activity depending on their major active components. Only mouthwash solutions containing chlorohexidine gluconate or cetylpyridinum chloride exhibited activity against majority, but not all tested bacterial strains in their biofilm state. Additionally, bacteria are generally less susceptible to all mouthwash solutions in their biofilm as compared to planktonic state.While mouthwash solutions have variable antibacterial activity, bacteria in their biofilm state pose a challenge to dental hygiene/care where bacteria become not susceptible to majority of available mouthwash solutions. When unicellular organisms come together forming a community and attached to a solid surface then encased in an exo-polysaccharide matrix, bacterial biofilms are formed . These bMore than three hundred known species of bacteria are considered as oral flora . They inMouthwash solutions are aimed at ensuring sufficient oral hygiene. Its ease of use in addition to the significant ability to reduce dental plaque formation, made mouthwashes a reasonable method to limit gingivitis and periodontitis , 11-13. Mouthwash solutions were obtained from the local market. Escherichia coli (ATCC 25922), Enterococcus faecalis (ATCC 19433), Staphylococcus aureus (ATCC 29213), Methicillin Resistans Staphylococcus Aureus MRSA (ATCC 43300), Pseudomonas aeruginosa (ATCC 27853), Staphylococcus epidermidis (ATCC 12228), Klebsiella pneumoniae (ATCC 13883), Streptococcus pyogenes (ATCC 19615), Proteus mirabilis (ATCC 12453), and Acinitobacter baumanni (ATCC 19606). All strains were stored in trypticase-soy broth with 20% glycerol  at -70 °C in a deep freezer until ready for susceptibility testing. They were thawed and passed 3 times to assure purity and viability before each experiment.The following bacterial strains were purchased and used in the study: Biofilm cells were performed as described by  under stS. aureus and P. aeruginosa planktonic and biofilm cells were tested against selected antibiotics, MIC were determined by using broth macrodilution method according to NCCLS [The MIC values of both to NCCLS . BrieflyP. aeruginosa and K. pneumoniae. Mouthwashes B, C, D, and E, have activity against all strains used in the study. In addition, mouthwash F exhibits high activity  against all strains except K. pneumoniae. However, mouthwashes H, G, and I show activity against some strains, but no activity against others. Although mouthwash B is active against all strains, its MIC values are larger than those for mouthwashes C, D, and E for almost all strains. Moreover, mouthwash B shows lower activity  except for K. pneumoniae when compared with mouthwash F. This indicates that mouthwash B exhibits less activity than C, D, E, and F. Mouthwash I has some activity against all bacterial strains except S. epidermidis, S. pyogenes. Moreover, it has low activity  against MRSA, K. pneumoniae, and A. baumannii. Mouthwash H exhibits little activity  against planktonic bacterial strains.The MIC values of tested mouthwash solutions against bacterial planktonic strains are shown in Mouthwash solutions usually encompass antimicrobial activity that ensures their work in eliminating harmful periodontal bacteria, which aids in preventing future dental carries, gingivitis and periodontitis , 11-13. Bacterial biofilms formation is associated with poor dental hygiene that provides better environment for biofilms formation, thus, rendering oral bacteria less susceptible to mouthwash or similar antiseptic formulation , 15, 17.Results of this study show that mouthwash solution possesses variable antibacterial activity depending on their chemical composition. For example, mouthwash solutions containing the antiseptics chlorhexidine are effective on most oral bacterial strains. This correlates with previous studies and is related to chlorohexidine’s mode of action as it works on different sites of the bacteria , 22. AddP. aeruginosa and K. pneumoniae. In fact, cetylpyridinum chloride is a quaternary ammonium compound known for its use as a cationic surface active agent that has antibacterial activity [S. aureus. This is in agreement with previously published work on cetylpyridinium chloride products [On the other hand, mouthwash solutions containing other ingredients such as cetylpyridinium chloride, sodium fluoride, hexetidine, povidone-iodine, eucalyptol, menthol, methylsalicylate, and thymol showed activity against some bacterial strains, but not others. For example, cetylpyridinium chloride (0.05%) mouthwash was shown to possess antimicrobial activity against all tested bacterial strains except activity , 25, 26.products .S. epidermidis, S. pyogenes. Moreover, It has low activity  against MRSA, K. pneumoniae, and A. baumannii. This mouthwash contains active ingredients of eucalyptol, menthol, methylsalicylate, and thymol. Eucalyptol, which is present in the essential oil of Eucalyptus, has been reported to posses some antibacterial activity against S. aureus and E.coli [The eucalyptol mouthwash has some activity against all bacterial strains except d E.coli . Howeverd E.coli . In addid E.coli .P. mirabilis. Upon comparison to mouthwashes that contains chlorhexidine as the main active ingredient, hexetidine 0.1% mouthwash exhibited less antibacterial activity against almost all strains. This is in agreement with work reported by Aznita et al [The hexitidine 0.1% mouthwash has high activity against all strains except ta et al .Results of the current study show that the MIC values of tested products against biofilm strains are greater compared with the planktonic strains. This is in agreement with what is known about biofilm resistance to anti microbial agents , 5. SeveCollectively, it is been shown that bacteria in their biofilm state is less susceptible to mouth wash solutions. Only mouthwash solutions containing chlorohexidine gluconate or cetylpyridinum chloride exhibited activity against majority, but not all tested bacterial strains in their biofilm state.


Pseuduvaria exhibits a diversity of floral sex expression. Most species are structurally andromonoecious (or possibly androdioecious), although the hermaphroditic flowers have been inferred to be functionally pistillate, with sterile staminodes. Pseuduvaria presents an ideal model for investigating the evolution of floral sex in early-divergent angiosperms, although detailed empirical studies are currently lacking. The phenology and pollination ecology of the Australian endemic species Pseuduvaria mulgraveana are studied in detail, including evaluations of floral scent chemistry, pollen viability, and floral visitors. Results showed that the flowers are pollinated by small diurnal nitidulid beetles and are protogynous. Pollen from both hermaphroditic and staminate flowers are shown to be equally viable. The structurally hermaphroditic flowers are nevertheless functionally pistillate as anther dehiscence is delayed until after petal abscission and hence after the departure of pollinators. This mechanism to achieve functional unisexuality of flowers has not previously been reported in angiosperms. It is known that protogyny is widespread amongst early-divergent angiosperms, including the Annonaceae, and is effective in preventing autogamy. Delayed anther dehiscence represents a further elaboration of this, and is effective in preventing geitonogamy since very few sexually mature flowers occur simultaneously in an individual. We highlight the necessity for field-based empirical interpretations of functional floral sex expression prior to evaluations of evolutionary processes.Unlike most genera in the early-divergent angiosperm family Annonaceae,  Anonidium Engl. & Diels Annona L. species previously recognized as Raimondia Saff. Diclinanona Diels Ephedranthus S. Moore Greenwayodendron Verdc. Klarobelia Chatrou Polyceratocarpus Engl. & Diels Pseudephedranthus Aristeg. Pseudomalmea Chatrou Pseuduvaria Miq. Stelechocarpus (Blume) Hook. f. & Thomson Uvariopsis Engl. The Annonaceae is a species-rich, early-divergent angiosperm family consisting of 112 genera and c. 2,440 species Pseuduvaria  Y.C.F. Su & R.M.K. Saunders, for example, is c. 20 µm in diameter , whereas fertile pollen is c. 25 µm The present study focuses on the genus Pseuduvaria, in contrast, is presumed to be fly-pollinated based on field observations of three species: an unidentified Australian species P. glabrescens (Jessup) Y.C.F. Su & R.M.K. Saunders based on published photographs and geographical location; P. froggattii (F. Muell.) Jessup P. megalopus (K. Schum.) Y.C.F. Su & Mols P. hylandii Jessup and P. villosa Jessup were also probably pollinated by flies P. froggattiiP. megalopusP. rugosa (Blume) Merr. and P. trimera (Craib) Y.C.F. Su & R.M.K. Saunders) possess light yellow petals, and lack prominent inner petal glands and dark-colored petal pigmentation Most Annonaceae species are beetle-pollinated, and this has been inferred as the plesiomorphic pollination mechanism in the family Pseuduvaria mulgraveana Jessup, a narrow endemic from northeastern Queensland, Australia Our research describes the floral phenology and pollination biology of Pseuduvaria mulgraveana is a species of small tree  which bears inflorescences with at most one or two flowers (ca. 15 mm long) Pseuduvaria species . Pseuduvaria mulgraveana flowers have previously been regarded as unisexual, with structurally hermaphroditic flowers inferred as functionally pistillate with non-viable pollen, although empirical studies are lacking P. mulgraveana were conducted in the Goldsborough Valley region  of the Wooroonooran National Park in northeastern Queensland, Australia . This area is mainly composed of lowland rainforest, c. 40 m asl, dominated by Apocynaceae, Araliaceae, Euphorbiaceae, Lauraceae, Moraceae, Myrtaceae, and Sapindaceae Field observations of P. mulgraveana  was selected for study within Goldsborough Valley, and 19 flowering individuals labelled with plastic tags for identification in phenological and experimental studies. Voucher specimens from this population are held in BRI herbarium .A population of P. mulgraveana was conducted over a one-month period (October–November 2010). Phenological changes in the flowers were monitored by tagging a total of 30 flower buds from 19 individual trees. All flowers encountered were used for the phenological observation irrespective of their positions on the plants. Observations on floral morphology and sexual functioning were taken at 1-h intervals from initial petal opening until the end of anthesis. The pistillate phase was recognized by the presence of stigmatic exudate  was used to record the temperature inside the floral chambers of 10 flowers from 10 different individuals during different phenological stages, with measurements taken at 1-h intervals. Ambient temperature immediately outside the floral chamber was recorded simultaneously.Volatile floral scent compounds were sampled using a purified SPME (solid-phase micro-extraction) fiber with a 65-µm divinylbenzene/polydimethylsiloxane coating, mounted on a manual sampling device . Flowers at different developmental stages  were collected and placed in polypropylene bags n-alkane standards to confirm compound identity The fibers were subsequently transferred to the School of Biological Sciences, The University of Hong Kong, for gas chromatography-mass spectrometry (GCMS) analysis using an Agilent 6890N gas chromatograph  coupled to an Agilent 5973 mass selective detector with a 30 m×0.255 mm i.d. DB-WAX capillary column and a 0.25 µm film . Helium was used as a carrier gas, with an injection temperature of 250°C for 1 min to allow vaporization of volatile compounds. The oven was maintained at 50°C for the first 5 min and then raised by 5°C/min to 230°C, and then at 230°C for 20 min. Electron ionization mass spectrometry was used with an acquisition range of 30–650 m/z. The volatile compounds were identified by comparing their mass spectra with the NIST 02 MS library bundle . Compounds with estimated identity likelihoods below 80 percent were considered as unknown 3BO3 and 50% Ca(NO3)2 by volume n.Pollen grains were obtained from both staminate flowers (staminate phase) and hermaphroditic flowers . In vitro pollen germination tests were used to evaluate the optimal sucrose concentration for pollen germination and also to assess pollen viability. Artificial sucrose solutions of different concentrations  were mixed with 50% HP. mulgraveana  were initiated as soon as flowers became sexually mature. Observations were made at 2-h intervals in order to monitor arrival/departure times of floral visitors and their activities during each visit. The following criteria were specifically adopted to determine whether the floral visitors qualify as effective pollinators: (1) the presence of P. mulgraveana pollen on the body of the floral visitor, assessed using a light microscope; (2) the coincidence of floral visits with the receptive periods of the flower; (3) relative visitation rates; and (4) evidence of inter-floral movement of the floral visitors.Observations of floral visitors to Samples of floral visitors (including 52 beetles from 11 flowers) were immobilized in Eppendorf tubes containing absorbent paper soaked with chloroform. After checking for the presence of pollen grain attachment, the insects were stored in 70% industrial methylated spirits for expert identification .Pseuduvaria mulgraveana, reflecting differences in sexual expression .The highest level of pollen germination was found in 30% sucrose solution . StaminaP. mulgraveana: a small nitidulid beetle, Aethina australis Kirejtshuk , and generally stayed inside the chambers throughout the sexually functional phases until the floral chamber abscised. In hermaphroditic flowers, no visitors were observed during the petal abscission phase or staminate phase. The beetles are active on the adaxial surface of the chamber, brushing against the stigmas and stamens and potentially pollinating the flowers or increasing their pollen load. Copulation of the beetles was observed both inside and outside the floral chamber  is inferred as an effective pollinator of Pseuduvaria mulgraveana based on the following criteria: the beetles were observed to be abundant on P. mulgraveana flowers, with their arrival and departure times coincident with the sexually functional phases of the flowers; and pollen of P. mulgraveana was observed on their bodies, including those collected from pistillate-phase hermaphroditic flowers, providing unequivocal evidence of inter-floral movement of beetles. Aethina Erichson species have previously been identified as pollinators of other Annonaceae species, including Uvaria semecarpifolia Hook. f. & Thomson, pollinated by Aethina (Circopes) subquadrata Motschulsky The beetle Pseuduvaria species have indicated fly pollination P. mulgraveana may act as a pollinator cannot be precluded; this would certainly be consistent with morphological characteristics of the flowers that are indicative of fly pollination, including the localized areas of dark red-purple pigmentation on the petals, and the presence of nectary glands. The observation of beetle pollination in flowers with adaptations indicative of myophily is also observed in Mitrephora (Blume) Hook. f. & Thomson, which shares a similar floral morphology to Pseuduvaria, with inner petals forming a small, partially enclosed mitriform chamber Mitrephora is likely to be pollinated by flies M. heyneana (Hook. f. & Thomson) Thwaites, is pollinated by small nitidulid beetles Previous studies of the pollination biology of P. mulgreaveana detectable by human olfaction contrasts with reports for other Pseuduvaria species such as P. froggattii, which apparently emits a floral scent reminiscent of ‘old dishwater and vomit’ Pseuduvaria species (probably P. glabrescens), with a ‘slightly perfumed’ fragrance P. mulgraveana, GCMS analysis of odors from sexually mature flowers reveal a mixture of volatile compounds previously implicated as insect attractants and/or pheromones. Of the eight volatiles identified from pistillate-phase hermaphroditic flowers (but absent from immature flowers), (Z)-3,7-dimethyl-1,3,6-octatriene and thymol methylether have been reported to be attractants and/or pheromones for Coleoptera and other orders of insects Anaxagorea brevipes Benth., Anaxagorea dolichocarpa Sprague & Sandwith, Annona neoinsignis H. Rainer, Desmos chinensis Lour., Duguetia asterotricha (Diels) R.E. Fr., Uvaria cordata  Alston, Uvaria semecarpifolia Hook. f. & Thomson, Xylopia aromatica (Lam.) Mart., and Xylopia benthamii R.E. Fr. The absence of a noticeable floral scent in Pseuduvaria was previously interpreted as an adaptation to attract flies P. mulgraveana clearly functions as a nutritive reward to A. australis, as the beetles were observed consuming it. Beetle- and fly-pollinated flowers often share similar traits, possibly explaining the attraction of both beetles and flies to the same flowers. Differentiating between fly-pollinated and beetle-pollinated Annonaceae species is often difficult, a problem also encountered in other fly- and beetle-pollinated families such as the Araceae Pseuduvaria, especially for species in early-divergent clades with pale-colored petals and lacking nectaries, such as P. galeata J. Sinclair, P. rugosa and P. trimeraAlthough nectar production in Annona L., Cymbopetalum Benth. and Duguetia A. St.-Hil. P. mulgraveana petals were observed to be eaten by A. australis , with no evidence of inter- and intra-individual flowering synchrony. The stamens in hermaphroditic flowers were shown to be fertile, despite differences in shape and size from those in staminate flowers. The possible selective advantage of andromonoecy in P. mulgraveana may be explained by considering the phenological timing of sexual maturation. The hermaphroditic flowers of P. mulgraveana are unusual in showing abscission of the corolla prior to anther dehiscence. This delayed anther dehiscence  is likely to have a profound impact on the functioning of the flower, since the floral chamber is the source of many of the attractants and rewards offered to the pollinators, including visual and olfactory cues, nutritive rewards , and possibly also as a sheltering and/or tryst site. Abscission of the corolla inevitably results in departure of the pollinators prior to pollen release, and hence precludes transfer of pollen from hermaphroditic flowers. The hermaphroditic flowers are therefore functionally pistillate, and the structural andromonoecy of P. mulgraveana populations actually functions as true monoecy. This mechanism to achieve functional unisexuality of flowers has not been reported previously in the Annonaceae (or apparently amongst angiosperms as a whole), and has implications for inferences regarding the evolution of unisexual flowers. Although structurally hermaphroditic  flowers co-occur with staminate flowers in P. mulgraveana, this functional monoecy is likely to effectively promote xenogamy: individual trees typically bear relatively few sexually mature flowers simultaneously, and the opportunities for intra-individual pollen transfer are therefore reduced, limiting the possibility of geitonogamy.Empirical data reveal that Pseuduvaria: P. trimera, for example, has been observed to bear staminate and structurally hermaphroditic flowers on separate branches within the same individual . Field-based observations are therefore essential for determining floral sex expression in such taxa.Difficulties are encountered in determinations of floral sex expression based solely on the examination of herbarium specimens. Staminate and hermaphroditic flowers may not always be present in the same branch in andromonoecious species, for example, leading to the erroneous interpretation of androdioecy. This problem is known to occur in AnaxagoreaFutabanthus asamigawensis Takahashi et al., which is hypothesized to have a phylogenetic position at the crown of the Annonaceae clade Pseuduvaria  Bakh. f., P. mindorensis Y.C.F. Su & R.M.K. Saunders and P. parvipetala Y.C.F. Su & R.M.K. Saunders P. fragrans Y.C.F. Su et al. and P. phuyensis  Y.C.F. Su & R.M.K. Saunders, which are monoecious or dioecious, only bear solitary or paired inflorescences Pseuduvaria froggattii (clade V), which produces large clusters of inflorescences, is possibly dioecious P. froggattii was observed by the first author in Atherton, Queensland, to produce only hermaphroditic flowers, however, suggesting that androdioecy cannot be satisfactorily ruled out .Most IV sensu  bear larP. mulgraveana and dioecy/androdioecy in P. froggattii clearly show that these two (or three) conditions coexist in the genus. The evolutionary relationship between andromonoecy and androdioecy is obscure. Lloyd proposed that dioecy may be derived from monoecy through the ‘paradioecy pathway’ Pseuduvaria could be a potential candidate genus for such investigations.Empirical data demonstrating the existence of andromonoecy in P. mulgraveana in its natural range in northeastern Queensland, Australia. Empirical studies and field surveys reveal that it is andromonoecious, with hermaphroditic and staminate flowers co-occurring within individuals. Both floral morphs were shown to produce viable pollen grains, contradicting the prevailing hypothesis that the stamens of structurally hermaphroditic flowers are sterile. A novel mechanism to promote xenogamy is reported. The structurally hermaphroditic flowers of P. mulgraveana exhibit delayed anther dehisce, with pollen only released after petal abscission once the pollinators have departed. Although structurally hermaphroditic, these flowers are therefore functionally pistillate, and effectively promote xenogamy by precluding autogamy and limiting geitonogamy. In contrast with previous studies of other species in the genus, P. mulgraveana was shown to be beetle-pollinated, with petal tissue and nectar provided as nutritive rewards to the pollinators.This study documents the floral phenology and pollination biology of Table S1Pseuduvaria mulgraveana. Phenological phases: II  =  immature; III  =  pistillate phase; IV  =  petal abscission phase; V  =  staminate phase.Putative chemical composition of floral volatiles emitted by hermaphroditic and staminate flowers in (DOC)Click here for additional data file.


Leishmania siamensis, is an emerging disease in Thailand. Although reported cases have been increasing, epidemiological information of the disease including host and vector aspects is not clearly known. This study was a preliminary survey of the potential vector of L. siamensis in an affected area of leishmaniasis, Trang Province, southern Thailand.Leishmaniasis, caused by hsp70) was used to identify L. siamensis DNA in sandflies.The collection of sandflies was performed around the area where a case of leishmaniasis was reported using CDC light traps. Species of sandfly were identified based on morphological characteristics according to Lewis’s key. PCR amplification and sequencing of the heat shock protein 70 gene (Sergentomyia (Neophlebotomus) gemmea, S. (Neophlebotomus) iyengari, S. (Parrotomyia) barraudi and Phlebotomus (Anaphlebotomus) stantoni. Among these species, S. (Neophlebotomus) gemmea was the most predominant species in all areas. DNA of L. siamensis was identified in S. (Neophlebotomus) gemmea. Nucleotide sequences of PCR products using DNA extracted from S. (Neophlebotomus) gemmea showed 99.8% identity to L. siamensis.A total of 146 male and female sandflies were collected in the affected areas. Of 71 female sandflies, four species were identified, i.e., S. (Neophlebotomus) gemmea might be a potential vector of L. siamensis in an affected area, Trang Province, southern Thailand. However further studies are needed to prove whether these sandflies can be natural vectors of leishmaniasis. Leishmania siamensis, firstly described from southern Thailand in 2008, is now considered an emerging disease in Thailand  x 1000), with the data representing a single sandfly species, collected over a time period and geographic area.Sergentomyia (Neophlebotomus) gemmea (49.3%) was the most predominant species, followed by S. (Neophlebotomus) iyengari (42.3%), S. (Parrotomyia) barraudi (4.2%) and Phlebotomus (Anaphlebotomus) stantoni (4.2%) (Table S. (Neophlebotomus) gemmea was found at all mentioned habitats, mostly at the cow corral and the thatched roof hut. None were trapped indoors.A total of 146 captured sandflies were collected with a female:male ratio of 1:1.1. Of 71 female sandflies, four species were identified of which hsp70 amplification showed identical amplicon sizes at 1,422 bp using extracted DNA of trapped S. (Neophlebotomus) gemmea, which showed 99.8% identity to the hsp70 of L. siamensis (GenBank accession number JX852709). GenBank accession numbers of the representative nucleotide sequences of L. siamensis detected in S. (Neophlebotomus) gemmea were assigned as JX852708. Positive samples were from two pools of S. (Neophlebotomus) gemmea females; the first pool was trapped at the cow corral near the house  and the other was collected at the store house . Unfortunately, we could not amplify the ITS1 from these samples. Determining the 18s rRNA of sandflies showed the consensus among nucleotide sequences from these L. siamensis- positive S. (Neophlebotomus) gemmea samples. One of these sequences was submitted to GenBank under accession number KF112045. The minimum infection rate (MIR) was 57.1 since there were two L. siamensis-positive pools and a total of 35 collected S. (Neophlebotomus) gemmea.All sequencing results from the cloning of positive S. (Neophlebotomus) gemmea as a potential vector of leishmaniasis caused by L. siamensis in Thailand. PCR amplification and sequence analysis of the hsp70 gene demonstrated L. siamensis DNA in S. (Neophlebotomus) gemmea captured in the affected area where a case of VL-HIV co-infection was reported. Unfortunately, the infection rate of S. (Neophlebotomus) gemmea could not be determined since we used the DNA of pooled samples. To date, no report has identified the possible role of S. (Neophlebotomus) gemmea as vector of any Leishmania species from any other geographical areas. In fact, Sergentomyia species were rarely considered as vector of medically important Leishmania since they prefer animal blood. Only a few studies could detect Leishmania DNA in Sergentomyia. A study in India identified L. donovani DNA in S. babu[L. major DNA in S. sintoni in Iran [L. major DNA was also detected in S. (Spelaeomyia) darlingi collected from zoonotic cutaneous leishmaniasis affected areas in Mali [L. major was also isolated from S. garnhami and successfully cultured in NNN medium [Sergentomyia sandflies are zoophilic, they might have a role in the transmission of zoonotic leishmaniasis. Leishmaniasis caused by L. siamensis might be also zoonotic since phylogenetic analysis showed that L. siamensis was closely related to L. enrietti, a Leishmania species infecting Brazilian guinea pigs [L. siamensis DNA was identified in animals such as rats and dogs .In the present study, we reported n S. babu, while P in Iran . Recentl in Mali . MoreoveN medium . Since Snea pigs . In addiS. (Neophlebotomus) gemmea was predominant in southern Thailand, the affected area of leishmaniasis caused by L. siamensis[L. siamensis by PCR in any samples of the previous survey [S. (Neophlebotomus) gemmea were found outdoors where the cow corral was the most common habitat. Habitat preference could be related to their blood meal feeding. In this study, we identified S. (Neophlebotomus) gemmea based on morphological characteristics [L. siamensis-positive S. (Neophlebotomus) gemmea was amplified and sequenced. The nucleotide sequence was then submitted to GenBank. Unfortunately no other 18s rRNA nucleotide sequence of S. (Neophlebotomus) gemmea is available to be compared in GenBank. Since the chromatogram of nucleotide sequences showed a completed consensus, we postulated that each pool contained only one species.The distribution of sandfly species in the present study was similar to our previous report showing that siamensis. Unfortus survey . Of the s survey , the diss survey ,14. All eristics ,16. The S. (Neophlebotomus) gemmea might be a vector of L. siamensis, the causative agent of leishmaniasis in Thailand. Further studies with larger samples are urgently needed to determine the potential sandfly vectors, their infection rate and behaviors such as host-preference behavior, blood meal feeding and breeding habitat in all affected areas of leishmaniasis. In addition, the natural reservoir host should be identified. The awareness of the disease by public health personnel and community health workers as well as having an effective vector control program are important keys to prevent the disease from spreading.Our study showed that The authors declare that they have no competing interests.KK, PT, MM and SL designed the study protocol. UN, SK and PJ collected and determined species of sandflies. SS, AH and CC performed PCR tests. KK, SS, MM and SL drafted and revised the manuscript. All authors have given approval to the final version of the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/13/333/prepub


We present a case of vertebral osteomyelitis presenting as chest pain. The patient initially underwent a CT chest angiogram to rule out a pulmonary embolism, which incidentally showed a soft tissue vertebral mass at T3-T4 disk space. Subsequent thoracic vertebral MRI was consistent with osteomyelitis with cord compression. Tissue culture from a CT-guided biopsy grew MRSA. The patient was successfully treated with Vancomycin. This is a rare presentation of vertebral osteomyelitis which poses an interesting diagnostic challenge. A 69-year-old African American male presented to the emergency department with a 4-day history of chest pain. He described the chest pain as band-like pressure across the chest, 10/10 in severity, constant, radiating to the back, worsened with coughing, and without relief by nitroglycerin. His past medical history included hypertension, chronic hepatitis C, prostate cancer s/p prostatectomy, a penile prosthesis removed the prior year due to infection, and several cystoscopies within the past year. Physical exam revealed a normotensive, afebrile patient with tenderness to palpation of the thoracic spine at T4 level. There were no focal neurologic deficits, and peripheral pulses were symmetric. Labs showed an elevated ESR without a leukocytosis. ECG showed normal sinus rhythm without any significant abnormalities. A CT angiogram of the chest ruled out a pulmonary embolism but showed a large soft tissue mass centered on the T3-T4 disk space. Subsequently, an MRI of the thoracic spine demonstrated osteomyelitis . Broad-sStaphylococcus aureus and Streptococcus species [Vertebral osteomyelitis can pose a diagnostic challenge when unusual symptoms predominate on presentation. Its incidence has been estimated to be approximately 2.4 cases per 100,000 population [ species . Back pa species , 6. Vert species –9. A lit species . From th species , 15. Ant species , 17. Yoo species . The diagnosis of vertebral osteomyelitis is challenging, considering the rarity of disease, high prevalence of back pain in the general population, and variability of presenting symptoms. Diagnosis is based on clinical, laboratory, and radiographic data, and a high index of suspicion is required for its prompt recognition and improvement in disease outcome.


Lactococcus lactis subsp. cremoris represents a cornerstone in the use of Gram-positive bacterial expression systems for biotechnological purposes. However, proteins that are subjected to such over-expression in L. lactis may suffer from improper folding, inclusion body formation and/or protein degradation, thereby significantly reducing the yield of soluble target protein. Although such drawbacks are not specific to L. lactis, no molecular tools have been developed to prevent or circumvent these recurrent problems of protein expression in L. lactis.The development of the Nisin Inducible Controlled Expression (NICE) system in the food-grade bacterium E. coli, two nisin-inducible expression vectors were constructed to over-produce various proteins in L. lactis as thioredoxin fusion proteins. In this study, we demonstrate that our novel L. lactis fusion partner expression vectors allow high-level expression of soluble heterologous proteins Tuc2009 ORF40, Bbr_0140 and Tuc2009 BppU/BppL that were previously insoluble or not expressed using existing L. lactis expression vectors. Over-expressed proteins were subsequently purified by Ni-TED affinity chromatography. Intact heterologous proteins were detected by immunoblotting analyses. We also show that the thioredoxin moiety of the purified fusion protein was specifically and efficiently cleaved off by enterokinase treatment.Mimicking thioredoxin gene fusion systems available for L. lactis. It was shown to prevent protein insolubility and degradation, allowing sufficient production of soluble proteins for further structural and functional characterization.This study is the first description of a thioredoxin gene fusion expression system, purposely developed to circumvent problems associated with protein over-expression in  L. lactis subsp. cremoris in conjunction with the Nisin Inducible Controlled Expression (NICE) system [L. lactis [L. lactis for heterologous protein overexpression and subsequently implemented in several other Gram-positive bacteria [L. lactis subsp. cremoris NZ9000 is employed as expression host, as its chromosome contains the signal transduction genes nisR and nisK involved in the nisin-induced transcriptional control of the PnisA promoter [nisA can be expressed in a controlled manner upon addition of nisin to the bacterial culture [L. lactis, as over-expressed proteins may be subject to poor expression, stability and/or solubility. Such drawbacks are intrinsically associated with the prokaryotic cell machinery limitations and therefore are inherent to all bacterial expression systems, representing a significant bottleneck in high level production of soluble proteins.The food-grade bacterium ) system -3 has be) system , metabol) system , or memb) system . The NIC. lactis ,3,7. It bacteria ,3,7-10. E. coli, a 'microbial cell factory' of choice for producing heterologous proteins [Schistosoma japonicum glutathione-S-transferase (GST) [E. coli maltose binding proteins (MBP) [Staphylococcus protein A [E. coli N-utilization substance (NusA) [E. coli thioredoxin (TrxA) [E. coli. Nevertheless, the adaptation of these existing fusion partner systems to other expression hosts is sparse, even though significant progress has been made to develop new molecular tools and methods in alternative prokaryotic and eukaryotic expression systems [L. lactis is currently lacking such a solubility-enhancing expression system to improve its spectrum of biotechnological applications, as L. lactis featured a number of benefits over other expression bacterial hosts, e.g. being a food-grade expression host, and the absence of endotoxins, extracellular proteinases and spores.In proteins ,12, the proteins . This tese (GST) , E. colins (MBP) , Staphylrotein A , E. colie (NusA)  and E. cn (TrxA) ,19. Alonn (TrxA) ,20,21. T systems ,23. The L. lactis. However, initial expression studies of individual protein subunits of Tuc2009 phage revealed such proteins often suffer from degradation, poor expression or result in insoluble protein aggregates, also called inclusion bodies (data not shown). The development of a fusion-based gene expression system in L. lactis could provide a novel strategy to express soluble proteins and avoid the use of laborious and spurious renaturation procedures. Among the numerous fusion partners employed, LaVallie et al. described the construction of an E. coli thioredoxin (TrxA) gene fusion system [E. coli thioredoxin fusion proteins were soluble, correctly folded and biologically active [E. coli thioredoxin thus appears to represent a good candidate for an L. lactis fusion-based gene expression system: small size of the fusion partner (11.67 kDa), ability to accumulate in a soluble form at high levels in the cytoplasm, steric accessibility of N- and C-termini of TrxA for protein fusions [i.e. immunoprecipitation or affinity chromatography [As part of our study on the structure-function analysis of lactococcal phage-host recognition and penetration, we attempted to over-express a number of proteins encoded by the lactococcal phage Tuc2009 in n system . In mosty active . The E.  fusions  and effitography ,24.L. lactis thioredoxin-fusion gene expression vectors harbouring the nisin-controlled expression (NICE) system. We evaluated the efficiency of the newly-constructed fusion gene expression system, by producing individual proteins or protein complexes that initially could not be expressed or were not soluble in L. lactis. Our data indicate that the L. lactis thioredoxin-fusion vectors represent a very valuable addition to the L. lactis genetic toolbox, in particular for the over-production of soluble proteins.In the present study, we report on the construction of two new L. lactis subsp. cremoris NZ9000 [L. lactis transformants were selected on GM17 agar plates supplemented with 5 μg/ml chloramphenicol (Sigma). The supernatant from overnight cultures of the nisin-producing strain L. lactis NZ9700 was filter-sterilized and used in this study as a source of the inducer nisin [Bacterial strains described in this study are listed in Table s NZ9000  and NZ97s NZ9000 ,6,7 weres NZ9000 ; Oxoid, B. breve UCC2003 was extracted as previously described [L. lactis NZ9000 as described by Holo et al. [Oligonucleotide primers were purchased from Eurofins MWG GmbH (Germany) and are listed in Table escribed ,27. Plaso et al. .E. coli trxA gene was amplified from the Gateway® plasmid pETG-20a and flanked with a poly-histidine tag and an enterokinase cleavage site by PCR using primers pTX48-F and pTX48-R  at maximum amplitude . Insoluble and soluble fractions were separated by centrifugation at 14, 000 × g for 10 min at 4°C and stored at -20°C for further analysis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as previously described [® Histidine-tagged Protein Purification kit . Protein content was measured using the Bio-Rad Protein Assay (Germany), based on the Bradford protein quantification method.Protein samples were prepared as described by Bahey-El-Din et al. . Bacteriescribed . Proteinescribed . Mouse pescribed . MonocloThe fusion protein TrxA-Bbr_0140 was expressed and His-tagged purified as described above. Purified enterokinase from calf intestine  was used to cleave TrxA-Bbr_0140 according to manufacturer's instructions. TrxA-Bbr_0140 was dialyzed against 50 mM Tris buffer, pH 8.0, since phosphate buffer is known to significantly reduce enterokinase activity as indicated in the user's manual. Typically, 25 μg TrxA-Bbr_0140 was incubated with 0.6 μg enterokinase for 16 h at 20°C or 37°C (enterokinase: TrxA-Bbr_0140 ratio = 1:42). We tested the effect of SDS on enterokinase cleavage efficiency as recommended in the manufacturer's manual, by supplementing the reaction mixture with 0.1% (w/v) SDS. An equal volume of 2X SDS-PAGE loading buffer was added and the samples were boiled at 95°C for 5 min to inactivate enterokinase. The cleaved protein products were analyzed by SDS-PAGE.L. lactis thioredoxin-fusion expression vectors, called pTX8048 and pTX8049  residues act as flexible joints within the fusion protein connecting the thioredoxin C-terminus to the recombinant protein N-terminus. It facilitates access to the enterokinase cleavage site (DDDDK), to facilitate release of the mature protein [E. coli fusion expression systems [E. coli thioredoxin gene trxA followed by a multiple cloning site to insert a gene of interest in an in-frame manner  with no known function and encoded by the lactococcal phage Tuc2009. We initially attempted to express the C-terminal His-tagged Tuc2009 ORF40 using the vector pNZ8048 in which ORF40 was cloned. The size of Tuc2009 ORF40 does not allow its detection by SDS-PAGE as the corresponding band would have been masked by the large amount of lyzozyme (14 kDa) required to lyse L. lactis NZ9000 . In Tuc2009 and also the closely related lactococcal phage TP901-1, the baseplate complex is associated with the initiation complex and the baseplate component BppU has been reported to be particularly vulnerable to degradation [L. lactis Trx-fusion expression system is also suitable for the production and purification of intact heteromultimeric protein complexes.Recombinant proteins may be improperly folded, preventing interactions with their protein partner(s). Such problems hamper further characterization of large hetero(multi)meric protein complexes, as certain protein-protein interactions may be prevented. The baseplate (Bpp) of Tuc2009 is a large multimeric protein complex involved in host recognition -43. It cradation . It was L. lactis. Bbr_0140 specifies a 200 amino-acid protein (23.5 kDa) encoded by the Bifidobacterium breve UCC2003 genome [L. lactis is also suitable to produce heterologous proteins from a completely unrelated bacterial origin.In an effort to demonstrate the versatility of our thioredoxin system, we also attempted to over-produce a protein encoded by a bacterium that is unrelated to 3 genome . We initotein 23. kDa encootein 23. kDa encootein 23. kDa encoe.g. up to 300 mg of lysostaphin per liter of culture on a industrial scale [L. lactis.The presence of a cleavage site is an important feature of this Trx-fusion expression system, as it allows the cleavage and release of the thioredoxin moiety from its fused protein of interest. Our vector pTX8048 possesses a peptide linker containing an enterokinase cleavage site (DDDDK) that connects the thioredoxin to the C-terminus of the fused protein. To test whether we could remove the Trx-moiety, purified thioredoxin-Bbr_0140 fusion protein was incubated with calf intestine enterokinase as described in the Methods section. As shown in Figure L. lactis that exhibit poor or no expression, or produce insoluble proteins using conventional expression vectors. In this study, we have described the construction of an L. lactis Trx-fusion expression system and demonstrated its applicability by over-producing and purifying various proteins or complexes as soluble thioredoxin fusions. The benefits of the original E. coli thioredoxin fusion expression system have previously been demonstrated [L. lactis, when combined with the NICE system. This expression and purification tool offers a wide spectrum of applications in L. lactis and also other Gram-positive bacteria that can accommodate the NICE system, such as L. plantarum [et al. [L. lactis using the thioredoxin fusion gene expression system allow further structural and biochemical analysis, such as X-ray crystallography analysis, antibody production, protein-protein interaction assays, and enzymatic assays.The thioredoxin gene fusion system represents an attractive system to over-produce and purify proteins in nstrated ,19, and lantarum . AlthougThe authors declare that they have no competing interests.FPD performed all experiments described in this study, designed the different expression vectors and drafted the manuscript, except MOCM who performed cloning and protein production of Bbr_0140 proteins. CC participated in its design and its coordination. FDP and DVS conceived of the study, and participated in its design and coordination and wrote the manuscript. All authors read, edited and approved the final manuscript.


Stratesaurus taylori gen et sp. nov.; Avalonnectes arturi gen. et sp. nov) and the most basal plesiosauroid, Eoplesiosaurus antiquior gen. et sp. nov. The initial radiation of plesiosaurs was characterised by high, but short-lived, diversity of an archaic clade, Rhomaleosauridae. Representatives of this initial radiation were replaced by derived, neoplesiosaurian plesiosaurs at small-medium body sizes during a more gradual accumulation of morphological disparity. This gradualistic modality suggests that adaptive radiations within tetrapod subclades are not always characterised by the initially high levels of disparity observed in the Paleozoic origins of major metazoan body plans, or in the origin of tetrapods. High rhomaleosaurid diversity immediately following the Triassic-Jurassic boundary supports the gradual model of Late Triassic extinctions, mostly predating the boundary itself. Increase in both maximum and minimum body length early in plesiosaurian history suggests a driven evolutionary trend. However, Maximum-likelihood models suggest only passive expansion into higher body size categories.Invasion of the open ocean by tetrapods represents a major evolutionary transition that occurred independently in cetaceans, mosasauroids, chelonioids (sea turtles), ichthyosaurs and plesiosaurs. Plesiosaurian reptiles invaded pelagic ocean environments immediately following the Late Triassic extinctions. This diversification is recorded by three intensively-sampled European fossil faunas, spanning 20 million years (Ma). These provide an unparalleled opportunity to document changes in key macroevolutionary parameters associated with secondary adaptation to pelagic life in tetrapods. A comprehensive assessment focuses on the oldest fauna, from the Blue Lias Formation of Street, and nearby localities, in Somerset, UK (Earliest Jurassic: 200 Ma), identifying three new species representing two small-bodied rhomaleosaurids ( The origin of Plesiosauria 200 million years ago (Ma) was a landmark event in tetrapod evolution. Spanning 135 Ma, plesiosaurians represent one of only three long-lived radiations of secondarily marine, non-mammalian tetrapods . Plesiosaurians possess an unusual body plan not seen in other marine vertebrates Despite its significance for understanding the macroevolutionary dynamics of adaptive radiations, the fossil record of the oldest plesiosaurs has been relatively understudied. This contrasts with early records of other pelagic tetrapod clades, the ichthyosaurs We present the results of a comprehensive review of the little-studied earliest Jurassic plesiosaurian fauna from Street, Somerset, UK , and a new phylogenetic dataset focussed on Lower Jurassic taxa. These are used as tools to study the earliest stage of plesiosaurian evolution.th century planorbis beds, which occur below the first occurrence of the ammonite Psiloceras planorbis. Thus, they probably fall within the earliest Hettangian P. tilmanni Chronozone, immediately following the Triassic–Jurassic boundary Eurycleidus arcuatus  Rhomaleosaurus’ megacephalus  Thalassiodracon hawkinsii  et al. Plesiosaurus’ cliduchus Seeley, 1865a The Blue Lias Formation at Street, Somerset, UK was extensively quarried for building stone in the 19Systematic Palaeontology). In total 16 plesiosaurian specimens from Street are taxonomically determinate . Other localities of similar age at ‘Street-on-the-Fosse’ (Pylle), Somerset and Barrow-on-Soar, Leicestershire have yielded additional specimens of ‘Rhomaleosaurus’ megacephalusT. hawkinsii is also known from Walton, Somerset Our taxonomic revision of the plesiosaurian fauna from Street indicates the presence of six species, including two new taxa identified here for the first time  can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix “http://zoobank.org/”. The LSID for this publication is: urn:lsid:zoobank.org:pub:5D0B1E45-4D2D-43F7-9480-F85C9832F815.The electronic version of this document does not represent a published work according to the International Code of Zoological Nomenclature (ICZN), and hence the nomenclatural acts contained in the electronic version are not available under that Code from the electronic edition. Therefore, a separate edition of this document was produced by a method that assures numerous identical and durable copies, and those copies were simultaneously obtainable (from the publication date noted on the first page of this article) for the purpose of providing a public and permanent scientific record, in accordance with Article 8.1 of the Code. The separate print-only edition is available on request from PLoS by sending a request to PLoS ONE, 1160 Battery Street, Suite 100, San Francisco, CA 94111, USA along with a check for $10 (to cover printing and postage) payable to “Public Library of Science”. In addition, this published work and the nomenclatural acts it contains have been registered in ZooBank <Pistosauria Baur 1887–1890 Plesiosauria de Blainville, 1835 Rhomaleosauridae Nopcsa, 1928 Stratesaurus n. gen.urn:lsid:zoobank.org:act:2C627698-239B-4436-81E5-E6FA5173F618Type and only species. Stratesaurus tayloriDiagnosis. As for the type and only species.Stratesaurus taylori n. gen. et sp.urn:lsid:zoobank.org:act:E2BA4E44-3E28-4987-928F-DF559BDCA98FPlesiosaurus sp. de la Beche and Conybeare; Conybeare 18221822 Thalassiodracon hawkinsi Owen; Storrs and Taylor 19961996 Thalassiodracon hawkinsi Owen; O'Keefe 20012001 Plesiosaurian faunal composition at Street).OUMNH  J.10337, a skull and partial postcranial skeleton including anterior cervical and pectoral vertebrae, a partial hindlimb and ilium  from Strsauros, Greek meaning lizard. Specific epithet after Michael A. Taylor, who carried out acid preparation of OUMNH J.10337 during an earlier study of Street plesiosaurians Genus name from ‘Strate’, the name for Street recorded in the Doomesday Book and S. taylori . AGT 11 is difficult to distinguish from Avalonnectes arturi (below), for which only the postorbital skull is known. However, in AGT 11, GSM 26035, and probably OUMNH J.10337, the premaxilla terminates posteriorly at approximately orbital midlength, whereas it extends further posteriorly in A. arturi. Furthermore, in AGT 11 and OUMNH J.10337 the squamosals contact each other dorsal to the parietals, unlike in A. arturi, in which this contact is more posterior. AGT 11 is otherwise indistinguishable from OUMNH J.10337 and GSM 26035 and is thus referred to S. taylori.AGT  11, a skull, and GSM 26035 , pl. 19,Small-bodied basal plesiosaurian (skull of holotype 180 mm long) with five premaxillary and 16 maxillary alveoli, lacking rostral constriction. Possesses two autapomorphies: prominent posterior processes on posterolateral surfaces of anterior cervical prezygapophyses; pectoral centra proportionally short (length:anterior height ratio = 0.7).Rhomaleosaurus’ megacephalusT. hawkinsii, which has four premaxillary teeth R. thorntoni: Natural History Museum, London, UK [NHMUK] R4853). Contrastingly, in pliosaurids and plesiosauroids the length of the posteroventral process is reduced The skull is crushed dorsoventrally, but has been prepared using acid and is almost free of matrix . It showE. antiquior n. sp. (below), but unlike the other taxa present at Street. The cervical centra have double-headed rib facets and concave anterior and posterior surfaces that are approximately 1.2 times as broad mediolaterally as high dorsoventrally , E. arcuatus (NHMUK 2047*), T. hawkinsiiAvalonnectes arturi and Eoplesiosaurus antiquior (see below). The condition cannot be determined in ‘Plesiosaurus’ cliduchus, for which only posterior cervical vertebrae are preserved. However, S. taylori has proportionally short pectoral centra .Eighteen anterior-middle cervical vertebrae are preserved, including most of the atlas-axis complex. The neurocentral sutures are convex, as in entrally . A smallentrally . This hapophyses . These al centra , unlike S. taylori is approximately straight in lateral view and has a broadly expanded dorsal end that is rotated approximately 20 degrees relative to the proximal end . A complete description of all material referred to S. taylori is beyond the scope of the present study but will form the basis of future work.The ilium of imal end . A rugosimal end . This isAvalonnectes n. gen.urn:lsid:zoobank.org:act:027222CA-DA5A-4F63-B59F-63A47567CD93Avalonnectes arturiAs for the type and only species.Avalonnectes arturi n. gen. et sp.urn:lsid:zoobank.org:act:27A8C8C1-5F02-461E-9778-2DB9E2645061NHMUK 14550, the posterior portion of the skull, and a partial postcranial skeleton .nectes, Greek meaning ‘swimmer’. Species epithet after Arthur Cruickshank (1932–2011), who with M. A. Taylor initiated the restudy of British Lower Jurassic plesiosaurians in the 1990s, and also as a reference to the legendary King Arthur of British folklore.Genus name from Avalon, an island from the legend of King Arthur, often identified with Glastonbury, near Street, and Plesiosaurian faunal composition at Street).AGT uncatalogued, a partial postcranial skeleton from Street, Somerset, UK  with 18–19 dorsal vertebrae, a low, autapomorphic number.S. taylori , it is unlikely that more than one or two additional cervical vertebrae were originally present. Thus, A. arturi has an estimated cervical count of 26–28, fewer than in T. hawkinsii . The cervical rib facets have two articular surfaces separated by a narrow horizontal groove. The neurocentral sutures are V-shaped in lateral aspect (E. arcuatus (NHMUK 2047*) and ‘P.’ cliduchus (CAMSM J.35180). This is unlike the condition in S. taylori and Eoplesiosaurus antiquior (see below), which have rounded neurocentral sutures. It is also unlike the condition in T. hawkinsii, in which the neurocentral suture contacts the rib facet . The complete dorsal series is not known in other plesiosaurians represented at Street. The apices of the dorsal neural spines of A. arturi alternate in morphology between being transversely thin and sheet-like, and transversely thicker  during the present study. A qualitatively different alternating morphology was reported in leptocleidid plesiosaurians Twenty-three cervical vertebrae are preserved . These dl aspect , as in E centrum , as in E thicker . This was victor , pl. 10,A. arturi possesses four sacral vertebrae, with short, robust ribs that converge laterally (E. arcuatus (NHMUK R1317) and T. hawkinsii (GSM 26035). It lacks the ventral projection at the distal end of the scapular blade of ‘P.’ cliduchus .Somerset County Museum, Taunton, UK [TTNCM] 8348, a postcranial skeleton  from WatEo, Greek meaning ‘dawn’, and Plesiosaurus, referring to a proportionally long neck, which is also present in Plesiosaurus. The species epithet is Latin meaning ‘more ancient’, in reference to the old geologic age of the specimen and Duria antiquior , a watercolour depiction of Lower Jurassic fauna, including plesiosaurs and ichthyosaurs, by Henry De la Beche.Genus name from Proportionally long-necked plesiosaurian (ratio of neck:trunk length>1.2) with at least 38 cervical vertebrae and one autapomorphy: small, conical lateral projections on the bases of the anterior cervical prezygapophyses.A. arturi , ‘R.’ megacaphalus  or T. hawkinsii , S. taylori and T. hawkinsii, only the anteriormost 12 preserved cervical neural spines curve posterodorsally. More posterior neural spines are inclined straight posterodorsally ; posteriormost dorsal rib facets split between centrum and neural arch  (146.1); medial surface of the iliac blade anteroposteriorly concave ; and a prominent flange extends anteriorly from the proximal half of the radius . This diagnosis focuses on unambiguous postcranial synapomorphies because cranial material of basal microcleidids is unknown.Plesiosauroids possessing: widely separated posterior cervical rib facets  brachypterygius (nov. comb), M. (Occitanosaurus) tournemiensis (nov. comb.), Seeleyosaurus guilelmiimperatoris, Eretmosaurus rugosus and Westphaliasaurus simonsensii.Großmman . This forms the nucleus of a new, formally-defined plesiosaurian family Microcleididae.Großmman , p. 556 Microcleidus Watson, 1909 Microcleidus homalospondylus  Microcleidus (Hydrorion) brachypterygius  comb. nov. Microcleidus (Occitanosaurus) tournemirensis ; jugal short, terminates around posterior orbital margin (33.1); cervical centra longer anteroposteriorly than high dorsoventrally (116.1); longitudinal ridge on lateral surface of cervical centrum (118.1); anteroposterior constriction at base of dorsal neural spines ; anterior process of coracoid long and transversely narrow .Microcleidid plesiosauroids with (selected unambiguous synapomorphies): jugal excluded from orbit margin by maxilla-postorbital contact . Pairwise dissimilarity (below: Character disparity) between all three species is very low; 7–17% of comparable characters have different scores . Most of these values are not substantially higher than those between Rhomaleosaurus  and Hauffiosaurus species (3–11%), and all are substantially lower than mean pairwise dissimilarity between all Toarcian plesiosaurians (0.37). On this basis, all three taxa are referred to a single genus, Microcleidus Watson, 1909 Three highly similar Toarcian long-necked microcleidids are recovered in a well-supported clade herein  We constructed a new phylogenetic data matrix based on a thorough review of all previously published characters and direct observation of most Lower Jurassic, European plesiosaurians. This resulted in analysis of 207 characters  of 0.4343, retention index (RI) of 0.5983 and rescaled consistency index (RC) of 0.2744. The strict consensus is well-resolved. Although basal pistosaurians form a polytomy, this results from the uncertain phylogenetic position of the skull of iosauria . A surprEoplesiosaurus) and pliosaurids , but most represent rhomaleosaurids . Branch support for nodes within Rhomaleosauridae is low, but Pliosauridae, Microcleididae, and nodes within both families, are generally well-supported . This is part of a temporal pattern in the distribution of branch support. Older nodes are generally less well-supported, probably reflecting poor knowledge of Triassic pistosaurians: 12 nodes within Plesiosauria occur prior to the Triassic–Jurassic boundary (i.e. probably within the Triassic) and have an (mean) average decay index of 1.33; six younger nodes occur within or prior to the Sinemurian and have an average decay index of 1.67; and seven younger nodes occur prior to or within the Toarcian and have an average decay index of 2.86. Most nodes within Rhomaleosauridae split in the Triassic, whereas most pliosaurid and plesiosauroid nodes split in the Jurassic.Specimens from Street and other earliest Jurassic localities include basal plesiosauroids  and Sthenarosaurus dawkinsi (Toarcian) were not included in our phylogeny but were added to the phylogenetic diversity estimate. This resulted in phylogenetic diversity estimates of 13 in the lower Hettangian; 12 in the Sinemurian and 10 (genera) or 16 (species) in the lower Toarcian. We prefer the genus-level estimate because German and British representatives of Hauffiosaurus (other than H. longirostris) and Microcleidus are not contemporaneous and may represent chronospecies that did not exist contemporaneously ‘Phylogenetic diversity estimates’ were calculated for the lower Hettangian, Sinemurian and Toarcian , countinet al.  in Ginkgo  using a negative eigenvalue correction (Cailliez method). Two disparity metrics were calculated for three narrow Lower Jurassic stratigraphic intervals : the mean pairwise dissimilarity . Minimum body size in the Triassic–Jurassic boundary fauna, as indicated by the proxy trunk length  is comparable to that among Triassic pistosaurians . Maximum body size also increases through time. The largest individual of ‘Rhomaleosaurus’ megacephalus from the earliest Hettangian fauna has a trunk 1820 mm long compared to 1500 mm for the Triassic pistosaurian BobosaurusRhomaleosaurus thorntoni (or 2460 mm for R. cramptoni). Sinemurian representatives of the large-bodied rhomleosaurid lineage are not currently known. Increase in the maximum and minimum values through time is suggestive of a driven trend of size increase Trunk length was used as a proxy for overall body size , definedet al. http://www.graemetlloyd.com/pubdata/functions_2.r (accessed 23 December 2011). Evolutionary models were fitted using the ‘fitContinuous’ function of the Geiger package Thalassiodracon hawkinsii. The results indicate overwhelming support for the BM+trend model when untransformed trunk length is analysed , and BM+trend, to our phylogenetic tree with temporal branch lengths estimated using stratigraphic age. This analysis was conducted in R version 2.10.1 analysed . Howeveranalysed . Thus, tNeck length excluding the skull was measured for the same individuals measured for trunk length (above). The ratio of neck length:trunk length was used as a proxy for overall body proportions . Other sR.’ megacephalus – 1.25 in Eoplesiosaurus [although this represents a slight underestimate because a small number of anterior cervical vertebrae are not preserved]). Sinemurian (0.71 in Archaeonectrus – 1.57 in Attenborosaurus) and Toarcian (0.57 in Meyerasaurus – 1.90 in Microcleidus tournemirensis) show small decreases in the minimum ratio and larger increases in the maximum ratio, demonstrating an increase in the range of plesiosaurian body plans though the Lower Jurassic.Body proportions, as indicated by the proxy neck length/trunk length, occupy a narrow range in the earliest Hettangian fauna (0.72 in ‘PlotosaurusPlotosaurus) or limb-driven locomotion . Most of these clades also show evidence of viviparity Archelon, the largest marine turtle, provides evidence that oviparity, and the necessity of terrestrial locomotion constrains maximum body size in pelagic tetrapods. Archelon attained a maximum length of approximately four metres  macromerus) and Kronosaurus; >10 metres Balaenoptera musculus; commonly 25–27 metres, but up to 33.58 metres During the Mesozoic, at least 12 tetrapod lineages independently became adapted to marine life e.g. . Of thesr metres : “3–5 m”Earliest Jurassic plesiosaurians possess their full suite of adaptations for specialised marine locomotion: reduction of intralimb flexibility, hyperphalangy, interlocking phalanges, and the appearance of supernumary ossiciations in the limbs resulting in the formation of a flipper; increased thoracic rigidity by modification of the limb girdles to form large ventral plates, and enlargement of the gastral basket; and shortening of the tail Rhomaleosaurus thorntoni, has a trunk almost twice as long as ‘Rhomaleosaurus’ megacephalus from the lower Jurassic fauna  macromerus has a humeral length of 840 mm and femoral length of 960 mm R. thorntoni (NHMUK R4853). Minimum body size also increased through the Lower Jurassic, though less prominently  are abundant in the earliest Jurassic fauna, but rarer in the Toarcian  The range of plesiosaurian body plans also increased through the Lower Jurassic . Althougn length , 5D. TheMaresaurus, Bajocian Borealonectes, Callovian The first appearance of plesiosaurians in the earliest Jurassic is characterised by a high diversity of rhomaleosaurids (five taxa). Despite the absence of derived plesiosaurians in older deposits, rhomaleosaurids from the earliest Jurassic are widely spread across morphospace , and repDespite their high diversity , the earThe observation of high taxic diversity in earliest Jurassic plesiosaurians, especially among rhomaleosaurids, suggests that an extinction event precisely at, or shortly before (∼100–150 kyrs et al. Thorne Appendix S1Measurements, age, provenance data and lists of specimens examined for taxa included in the phylogenetic analysis.[XLS]Click here for additional data file.Appendix S2Character list for the phylogenetic analysis.[DOC]Click here for additional data file.Appendix S3Phylogenetic data matrix.[PDF]Click here for additional data file.