Alternanthera philoxeroides, and its native congener, A. sessilis, after inoculation with Rhizoctonia solani. Pathogenicity tests showed significantly slower disease progression in A. philoxeroides compared to A. sessilis. Expression analyses revealed jasmonic acid (JA) and ethylene (ET) expressions were differentially regulated between A. philoxeroides and A. sessilis, with the former having prominent antagonistic cross-talk between salicylic acid (SA) and JA, and the latter showing weak or no cross-talk during disease development. We also found that JA levels decreased and SA levels increased during disease development in A. philoxeroides. Variations in hormonal gene expression between the invasive and native species  were identified during R. solani pathogenesis. Thus, plant hormones and their cross-talk signaling may improve the resistance of invasive A. philoxeroides to pathogens, which has implications for other invasive species during the invasion process. Invasive plants are a huge burden on the environment, and modify local ecosystems by affecting the indigenous biodiversity. Invasive plants are generally less affected by pathogens, although the underlying molecular mechanisms responsible for their enhanced resistance are unknown. We investigated expression profiles of three defense hormones  and their associated genes in the invasive weed,  However, many of these species have become invasive and pose a serious threat to agriculture, biodiversity and ecosystem function . Followiissions) . Much ofissions) ,5. Due tissions) , the genInvasive plants may encounter novel abiotic and biotic stresses across the introduction–naturalization–invasion continuum. These stresses can affect their survival and reproduction, and can act as barriers to plant invasion. Successful invasive plant species possess various attributes , and have effective defenses against natural enemies, which allow them to overcome barriers to invasion ,8. HigheCirsium arvense, in response to nutrient deficiency and shading [C. arvense [Ambrosia artemisiifolia), an invasive species to Europe, candidate genes were identified using oligonucleotide microarrays under light and nutrient stress conditions that were thought to contribute to invasiveness [Helianthus annuus naturalized in the USA were tested for variations in gene expression compared to wild non-weedy species [The improved performance of invasive plants in a competitive environment to enemy attack may be due to genetic changes acquired during the invasion process ,28. The  shading . This st arvense . Similarsiveness . In addi species . This st species . HoweverR genes) [Generally, a multitude of plant defense pathways are activated in response to microbial pathogens . The firR genes)  are usedR genes) ,39. The R genes) , where sR genes)  for evolR genes)  and therR genes) ,13. Arabidopsis [Cirsium arvense. Interestingly, the plant hormone auxin and ABA-signaling was found to regulate paradormancy, allowing plants to resist weed control methods , thereby enhancing their invasiveness [The timely recognition of the invading pathogen and a rapid, effective induction of defense responses are required for resistance to disease in plants . Plant hbidopsis ,54, tobabidopsis  and tomabidopsis , but aresiveness . CurrentAlternanthera philoxeroides (Martius) Grisebach, is an amphibious stoloniferous perennial herb [A. philoxeroides growing in China have extremely low genetic diversity, which is attributed to the predominance of a single genotype (likely due to a single recent introduction) and extensive vegetative propagation by cuttings since being introduced [The alligator weed, ial herb . It is nial herb  and was ial herb . It is tial herb ,62 whereial herb . Populattroduced ,64.Alternanthera philoxeroides is highly vulnerable to insect herbivore attack. For example, more than 15 generalist insects were found to feed on A. philoxeroides in China [Agasicles hygrophila  from South America was introduced to the USA [A. philoxeroides. However, only a few disease incidences in A. philoxeroides have been reported. For example, species of Nimbya have been found to cause leaf and stem spot on A. philoxeroides in Australia [Nimbya alternantherae has been identified as a biocontrol agent in Brazil [Fusarium have been used as a biocontrol for alligator weed in China [Rhizoctonia solani has been shown to be pathogenic to A. philoxeroides, and has also been found to infect a related species, Alternanthera sessilis, in the USA [R. solani (ACCC 30374) is more virulent on A. philoxeroides compared to Fusarium oxysporum f.sp cubense (ACCC 36369) . Therefore, A. philoxeroides represents a good model to study pathogen resistance and defenses in an invasive plant species following invasion. in China . A speci the USA  and Chin the USA  to contrustralia . Nimbya n Brazil , while sin China . Rhizoct the USA  and Indi the USA . EarlierA. philoxeroides and its native congener A. sessilis to test for differences in gene expression between the species against a generalist necrotrophic fungus, Rhizoctonia solani. Although signaling of these defense hormones and their cross-talk in response to pathogens are well documented in Arabidopsis and other model plant species [A. philoxeroides. Specifically, we ask the following questions: (1) Is invasive A. philoxeroides less susceptible to the pathogen (R. solani) compared to its native congener A. sessilis? (2) Are there differences in gene expression between the native and invasive species after inoculation with R. solani? (3) Are there differences in resistance between infected and un-infected neighboring leaves? To address these questions, we performed in vitro and in planta leaf inoculations using R. solani. Six hormones and their responsive genes  were successfully isolated from both invasive A. philoxeroides and native A. sessilis for expression analysis using RT-qPCR in response to three treatments. The first included R. solani inoculation for susceptibility tests, the second comprised un-infected samples for systemic resistance tests, and the third included hormone pretreatments using SA, JA, and ET (prior to inoculation with R. solani).In this study, we aimed to isolate the defense hormones  and associated genes in invasive  species ,54,55,56R. solani was tested on invasive A. philoxiroides and native A. sessilis. Disease symptoms and necrotic lesions developed on leaves of both plant species  were quantified in both the local and systemic leaves of the two species against R. solani  was selected on the basis of core signaling component for detailed functional analysis in both A. philoxiroides and A. sessilis. Each sequence was assembled to a single long contig to predict protein coding genes along with mRNA and the amino acid translation , JAR1 (JA amido synthetase 1), and PR6 (Proteinase inhibitor), were tested in A. philoxiroides following inoculation with R. solani. We found the expressions of JAR1 and PR6 were reduced in A. philoxiroides, whereas LOX expression was inconsistent at each time after inoculation  levels had increased (R. solani in A. philoxiroides because JA was induced earlier (up to 6 hpi). During disease progression (after 48 hpi), R. solani may overcome JA-resistance signaling by suppressing the defensive responses in A. philoxiroides  and SA (PAL and PR3) transcripts had prominent antagonistic cross-talk in A. philoxiroides at both earlier (at 6 hpi) and later (>24 hpi) time intervals following R. solani inoculations   . These rEIN3, gene expression was induced in both treatments in A. philoxiroides  transcripts  during pathogenesis in A. philoxiroides, we quantified the endogenous contents of each hormone in R. solani infected plants at each time interval  using ELISA. We detected a 6.9-fold higher SA content  and JA (JAR1) engage in antagonistic cross-talk during pathogenesis. The ET-EIN3 expression was reduced at each time interval  there were interspecific differences in hormone gene expression  following inoculation by R. solani; and (3) there were differences in the hormone signaling and their cross-talk between infected local and the un-infected systemic leaves.Plants are exposed to numerous biotic and abiotic stresses in their environment. Invasive plants may have evolved to adapt to these stresses and to fluctuating environmental conditions via plasticity in growth and development ,28,70. SR. solani may affect host plant defense hormones, specifically by inducing the major defense hormone SA . Furthermore, our results indicate that the stronger antagonistic cross-talk between SA and JA in the invasive A. philoxiroides may delay disease progression. To our knowledge, this is the first study that demonstrates a necrotrophic pathogen influencing plant defense hormone pathways in an invasive clonal weed differentially compared to a native congener  [We found that there were interspecific differences in JA and ET-signaling, as well as in the degree of antagonistic cross-talk between SA and JA (which was stronger in the invasive species and weaker in the native species). This variation in the expression of defense hormone genes between species suggests congener . A previsferase) . Gene exsferase) ,30,31. A. philoxeroides  may influence the resistance response of each species to pathogens. The data from our study provide an appropriate baseline for investigating this line of inquiry in the future. The differences in defense hormone signaling between the invasive and native species observed in our study during pathogenesis may be the result of interspecific genetic variation. It would be interesting to study how differences in genetic variation between the invasive agation, ,64) and A. philoxeroides (using RT-qPCR). Our results showed that both JA-JAR1 and PR6 transcript expressions decreased during disease development following inoculation with R. solani AG4 HGI  allowing for the successful infection of an invasive species,  AG4 HGI a,b, indisistance . In the pression c. This wR. solani induced SA (PAL and PR3), which may antagonize JA during pathogenesis in invasive A. philoxeroides , a virulence factor that elicits SA to activate antagonism to JA, thereby enhancing the ability of the fungus to infect tomato [R. solani needs to be investigated further. A follow-up study by El Oirdi et al. [Alternaria solani) in tomato [SAR8.2k gene induced by SA may be involved in disease susceptibility caused by R. solani in tobacco [In our study, xeroides a,b. Earl tobacco . Like in tobacco ,80. For d tomato . The nect tomato . Our rest tomato , althougi et al.  showed Sn tomato . It has  tobacco ). PR genes are often associated with specific signaling pathways and their expression can be regulated by different plant hormones [PR3 showed a 10-fold higher expression to R. solani infection  [A. philoxeroides  represses the SA biosynthesis gene ICS/SID2, thereby reducing SA accumulation [Our results indicate that, as with SA,  factor)  enhancesxeroides c. Althouculation c,f. Prevbidopsis . In contmulation .Invasive plants may encounter multiple pathogens with different infection strategies in their non-native range. Hormonal cross-talk provides signals to adjacent leaves from local infected tissues to resist a forthcoming infection ,91,92. TPAL and JA-JAR1 showed antagonism in the neighboring leaves, whereas other gene transcriptions  were not suppressed  is one of the 100 worst invasive species in the world [A. philoxeroides, including areas in Southeast Asia, Southern Africa, and Southern Europe [he world . Climaten Europe . In Chinn Europe .Alternanthera sessilis (L.) DC. is native to China, and was used for comparison with A. philoxeroides in our study [A. philoxeroides and A. sessilis were randomly collected in August (summer) 2016 from naturally occurring sites in Fuzhou National Forest Park  used in this study was obtained from the Agriculture Culture Collection of China . This strain was further sequenced to identify the specific anastomosis group (AG4-HGI), which was confirmed by PCR amplification of AG common and subgroup specific primers [globally . The mos primers , and int primers . The funA. philoxiroides and A. sessilis. Although there is currently a lack of hormone specific sequence information for many invasive and native plant species (including Alternanthera species), the species from our study are in the Amaranthaceae family (which was used for sequence isolation). Twenty-one hormones and their responsive gene sequences  were retrieved from species in the Amaranthaceae  were successfully isolated from both nthaceae . Each genthaceae  to compaPAL and PR3 for SA; LOX, JAR1 and PR6 for JA; EIN3 for ET) were successfully isolated based on the PCR amplification of similar amplicon lengths obtained from both A. philoxiroides and A. sessilis . Genomic DNA was further precipitated by adding sodium acetate and ethanol and purified using the method of Dellaporta et al. . PCR wassessilis . FurtherA. philoxeroides and A. sessilis inoculated with 5-mm agar plugs (R. solani culture). Both detached and in planta leaf assays were performed using the method used by El Oirdi and Bouarab [Pathogenicity tests consisted of detached (in vitro) and in planta leaves from four-week old stem cuttings of  Bouarab . InoculaA. philoxeroides and A. sessilis were used for gene expression experiments by quantitative RT-qPCR. Inoculated leaf samples were harvested at different time intervals, including 0, 6, 12, 24, 48, 72, and 96 h post-inoculation (hpi). There were three biological replicates per time interval . The 0 hpi time interval refers to un-inoculated control samples. All inoculated plants were grown at 25 °C and 70% humidity with a 16 h photoperiod. Harvested leaf material at different time intervals was quickly frozen in liquid nitrogen for RNA extractions. For the systemic acquired resistance (SAR) tests, samples of un-inoculated neighboring leaves  were investigated. For SAR gene expression experiments, samples of three biological replicates were taken per time interval . That is, one plant per replicate for each species was investigated. The experiments of R. solani infection and SAR were repeated twice.In planta inoculations of R. solani was tested using RT-qPCR by spraying each hormone two days before inoculation with R. solani. Hormone pretreatments were performed on four-week old plants of A. philoxeroides and A. sessilis. There were three treatments in this experiment group for each species . Hormones SA , methyl jasmonate , and ethephon  were dissolved in water. Hormones were sprayed directly on plant leaves (in each treatment group) until drenched (surface run-off) once per day, for three consecutive days. All treatment plants were grown at 25 °C and 70% humidity with a 16 h photoperiod until they were harvested. Each of the hormone pretreated samples was harvested at different time intervals: 0, 6, 12, 24, 48, 72, and 96 hpi with R. solani. The hormone pre-treatment samples for SA and ET spray were unable to be collected after 72 and 96 h. This was because the ET-spray samples wilted early and could not be sampled . The control treatment (0 hpi) consisted of only water pretreated and un-inoculated. There were three biological replicates for each time interval  for each species. All harvested samples were quickly frozen in liquid nitrogen for RNA extractions. The experiment was repeated twice.The effect of each hormone  on resistance to β-tub (β-tubulin), EF1α , and Act (Actin), in R. solani infected samples based on Cycle threshold (Ct) difference and the coefficient of variance method [Actin had the lowest Ct difference in R. solani infected samples across different time intervals  and was used for normalizing the expression level of each target gene. Relative gene expression was calculated using the comparative 2CT−ΔΔ method [Total RNA was isolated from leaves of invasive and native plants in each treatment using the TaKaRa MiniBEST Plant RNA Extraction Kit according to the manufacturer’s instructions . First-strand cDNA was synthesized from 500 µg total RNA using PrimeScript RT Master Mix . The targets were amplified using primers that are listed in e method . PrimersT method . Three bR. solani infected A. philoxiroides and A. sessilis. Fungal inoculations were performed on four-week old plants (as described above). Samples collected at different time intervals  were subjected to hormone quantification by Lengton Bio. Tec. Co., Ltd.  using the ELISA method [The endogenous hormone contents  was determined for A method . The 0 hA. philoxiroides and A. sessilis. Leaves were removed at each time interval  and stained with lactophenol-trypan blue, followed by de-staining with saturated chloral hydrate using the method of Koch and Slusarenko [To visualize dead plant cells and to measure the cell death area of infected leaves, trypan blue staining was performed on the leaves of four-week old infected usarenko . Samplesusarenko . To furtusarenko . A. philoxeroides and A. sessilis (PAL (SA), JAR1 (JA), and EIN3 (ET) was selected for additional flanking sequence isolation for both A. philoxiroides and A. sessilis . Flanking (5′ and 3′) sequences were isolated from a known sequence region  that may affect the signaling pathways differentially between invasive and native species [R) genes, such as the non-expresser of PR genes 1 (NPR1) of hormone SA, which is a major transcriptional activator that activates antagonism to JA or ET during disease development [During the invasion process, an invasive species from a small founding population may face difficulties due to adverse abiotic and biotic stresses, impacting its survival and reproduction. Invasive species can undergo genetic changes to overcome these natural barriers. Our findings are consistent with previous studies, which identified that many stress induced genes are differentially expressed between invasive and native plant species ,30,31. O species . More spelopment .


During Q1 2019, the vancomycin-variable enterococci (VVE) ST1421-CT1134 vanA E. faecium became the most dominant vanA E. faecium clone and has spread to all five regions in Denmark. Among 174 E. faecium isolates with vanA, vanB or vanA/vanB genes in Q1 2019, 44% belonged to this type.We describe clonal shifts in  The aim is to highlight the importance of using molecular methods for detecting vancomycin-variable enterococci (VVE), and to alert other countries about this emerging nosocomial clone.We describe the clonal shift for E. faecium harboring the vanA gene complex, but being phenotypically vancomycin susceptible [in vitro and in vivo has been described [1546 (vanA transposon). Tn1546 was truncated in vanX by a 252 bp 3' deletion explaining the vancomycin susceptibility of Efm-V1511. In ST1421-CT1134 isolates resistant to vancomycin, resistance could be attributed to changes in ddl disrupting gene function sometimes accompanied by changes in vanS, increased pHVH-V1511 copy number or the existence of an additional vanA-containing plasmid encoding a functional vanX [Vancomycin-variable enterococci (VVE) are ceptible ,2. VVE cescribed ,3-5. ThiE. faecium and 30 E. faecalis) were tested for the presence of vancomycin resistance genes vanA and vanB by PCR from 2005 through 2014.We have previously described the surveillance of vancomycin-resistant enterococci (VRE) in clinical isolates in Denmark from 2005 to 2015 . In the van genes were extracted in silico. The isolates were further subtyped in SeqSphere + ) using the cgMLST scheme by de Been et al. [E. faecium.From 2015 through Q1 2019, all clinical VRE/VVE isolates  underwent whole-genome sequencing (WGS) as previously described . From thn et al.  for E. fE. faecium isolates from blood cultures for the presence of vanA/vanB genes by PCR was introduced in the DCMs in the Capital Region. During 2018, this was expanded to testing of all clinical E. faecium isolates. During 2018, molecular testing by PCR of E. faecium from all clinical samples was also implemented in one of the four DCMs in the Region of Southern Denmark. Furthermore, E. faecium isolates from blood cultures were tested by PCR for vanA/vanB genes in another DCM in the Region of Southern Denmark and in the DCM in the Central Denmark Region in 2018. In Q1 2019, diagnostic algorithms to detect VVE have expanded. Most of the DCMs across Denmark test at least all blood culture E. faecium isolates for the presence of vanA genes using PCR.VVE diagnostic algorithms have differed substantially over time and between the five Danish regions. In 2017, testing of phenotypically vancomycin-susceptible vanA E. faecium, 74 vanB E. faecium, 32 vanA/vanB E. faecium, 12 vanA E. faecalis, and 43 vanB E. faecalis from clinical samples were submitted to SSI . ST80 (22%), ST203 (65%) and ST1421 (9%) were most prevalent. Typing by cgMLST revealed 156 different complex types (CTs).The vanA,vanB and vanA/vanBE. faecium from 2015 to Q1 2019 are shown in vanA E. faecium, ST203-CT859 vanA E. faecium and ST1421-CT1134 vanA E. faecium  during 2015 to 2017, but decreased in 2018  (vanA E. faecium spread to the Faroe Islands during 2018 and 2019 (data not shown).In 2017, 3% of the  Denmark , Table 2pe (44%) . It was vanA E. faecium isolates. This study shows that predominating clones shifted over time and, importantly, the emergence of a vancomycin-variable clone, ST1421-CT1134 vanA E. faecium, that has spread to all the five Danish regions in 2019.During 2005 to Q1 2019, most of the Danish clinical VRE isolates have been E. faecium isolates belonged to 156 CTs, three types  have dominated during the last 4 years.Although the vanA E. faecium was highly prevalent in the Capital Region during 2012 to 2015 [vanA E. faecium constituting Group2_ST80 in the paper by Pinholt et al. [vanA E. faecium decreased during 2016 to 2018, and this clone was not detected during Q1 2019.ST80-CT14  to 2015 . The vant et al.  belongedvanA E. faecium emerged during 2015 through 2017 and nearly disappeared 2019. This clone has spread to Sweden, the Faroe Islands and Greenland [ST203-CT859 reenland ,7.vanA E. faecium have been under-reported in some regions at least during some periods. Thus, the rising incidence could partly be explained by increasing molecular testing of vancomycin susceptible isolates. However, a sharply increasing incidence has also been seen in DCM with extensive testing for VVE.Because of differences in diagnostic algorithms, there is a detection bias of VVE. It seems very likely that ST1421-CT1134 vanA E. faecium and ST203-CT859 vanA E. faecium are still unknown. vanA E. faecium isolates belonging to ST1421-CT1134 have also been reported from Australia, but these isolates have not been VVE [The origin of ST80-CT14 been VVE . Why thevanA E. faecium, in Denmark is of concern, especially since VVE diagnostic is challenging. Because of this, the clone is likely to be underdiagnosed, which facilitates further spread. Since cross-border spread has been described for VRE, countries with patients transferred from Denmark should be aware of the vancomycin-variable ST1421-CT1134 vanA E. faecium clone.The spread of the VVE clone, ST1421-CT1134


Here we have developed the generic and nine species-specific succinylation site classifiers through aggregating multiple complementary features. We optimized the consecutive features using the Wilcoxon-rank feature selection scheme. The final feature vectors were trained by a random forest (RF) classifier. With an integration of RF scores via logistic regression, the resulting predictor termed GPSuc achieved better performance than other existing generic and species-specific succinylation site predictors. To reveal the mechanism of succinylation and assist hypothesis-driven experimental design, our predictor serves as a valuable resource. To provide a promising performance in large-scale datasets, a web application was developed at http://kurata14.bio.kyutech.ac.jp/GPSuc/.Lysine succinylation is one of the dominant post-translational modification of the protein that contributes to many biological processes including cell cycle, growth and signal transduction pathways. Identification of succinylation sites is an important step for understanding the function of proteins. The complicated sequence patterns of protein succinylation revealed by proteomic studies highlight the necessity of developing effective species-specific  Lysine et al. proposed a predictor SucPred based on Support Vector Machine (SVM), in which four types of encoding methods were used [et al., adopts the pseudo amino acid composition encoding scheme to improve the prediction performance [et al. developed another SVM-based predictor SuccFind considering amino acid composition (AAC), an amino acid index (AAindex) physicochemical properties and k-space amino acid pair composition (CKSAAP) [et al. developed two predictors, iSuc-PseOpt [et al. developed a structure-based predictor SucStruct using a decision tree classifier [et. al. developed two predictors termed as SuccinSite and SuccinSite2.0 based on the amino acid frequency and properties with combined RF classifier scores [k-spaced amino acid pairs (pCKSAAP) and binary amino acid codes (BE) with a RF classifier. Dehzang et al.http://www.sciencedirect.com/science/article/pii/S0022519317302072 developed two predictors, PSSM-Suc and SSEvol-Suc, based on position-specific scoring matrix (PSSM) encoding and secondary structure information [et al. developed another predictor, termed Success, using evolutionary and structural properties of amino acids[To date, numerous of bioinformatics implementations have been established to predict succinylation substrates –27. Zhaoere used . The encformance . Xu et a(CKSAAP) . Jea et c-PseOpt  and pSucc-PseOpt , by usinassifier . Hasan er scores , 23. Theormation . Lopez eino acids. A specivia logistic regression (LR), the GPSuc outperformed other existing generic and species-specific succinylation site predictors. It provides valuable insights into the processes and functions of succinylation. Moreover, we systematically analyzed critically important features that influence the performance of classifiers. The GPSuc predictor was implemented as a web application at http://kurata14.bio.kyutech.ac.jp/GPSuc/.However, the overall performance of the above-mentioned existing predictors is still not satisfying and there is further room to improve the prediction performance. In the current study, we develop generic and 9 species-specific succinylation classifiers named Global Prediction of Generic and Species-specific Succinylation Sites (GPSuc) based on combining of five sequence encoding features: pCKSAAP, AAC, AAindex, BE, and PSSM features. We optimized the consecutive feature vectors and trained them by a random forest (RF) classifier. With an integration of RF scores One of the main challenges in predicting succinylation sites is to obtain the suitable dataset for model development. Since the training data should be derived from experiments, experimentally identified 10,000 succinylated proteins were collected from nine species. Then the redundant protein samples were removed by using CD-HIT with a 30% identity threshold cutoff [H. sapiens, M. musculus, M. tuberculosis, E. coli, T. gondii, S. cerevisiae, and S. lycopersicum were retrieved from the SuccinSite2.0 [T. capsulatus (150 succinylated proteins were set as training samples while 33 proteins randomly as test samples) and T. aestivum (53 succinylated proteins were set as training samples while 20 proteins randomly as test samples) [http://kurata14.bio.kyutech.ac.jp/GPSuc/.The generic and seven species-specific datasets of nSite2.0 . They wesamples) . It is nAn overview of the computational framework of the proposed GPSuc predictor is shown in To establish an accurate species-specific prediction model, the individual sequence fragment was encoded into a numeric feature vector. It is a critical step to represent the collective architecture of the classifier. Therefore, to obtain the local information around each succinylated lysine, a high-quality sequence encoding method was essential. As a substitute for retaining a general binary representation of corresponding amino acid sequences, five types of feature encodings were adopted: AAC, AAindex, BE, PSSM, and pCKSAAP schemes. Details in each feature encoding scheme was described as follows.AAC feature encoding is one of the most popular schemes and widely used in protein bioinformatics research , 31. It http://www.genome.jp/aaindex/AAindex/list_of_indices) and MIYS990104[In AAindex database (version 9.1), the primary physicochemical and biochemical properties of the amino acids were extracted . After sIYS990104 were traA 20-dimensional binary vector for each residue in the sliding window was generated by BE scheme . Through−4 and 3, respectively. Then, the feature vectors were extracted using sliding sequence fragments. To each sequence, the dimension of the PSSM vector was 820 (41×20). We considered 20 amino acids without counting any gap (-).The PSI-BLAST (version 2.2.26+) against the whole Swiss-Prot non-redundant database (December 2010) was used to generate PSSM matrix , which ik-space amino acid pairs, pCKSAAP feature vectors, were extracted from the generated PSSM profile for each sequence window[T times between r and r+k+1, the pCKSAAP feature scores were calculated and normalized using the following equation:ni and nj  represent 20 types of amino acid residues. The PSSM  denoted the amino acid pair of ni with the rth row position of the PSSM score in ni{k}nj. The PSSM  represents the amino acid pair nj at the (r+k+1)th row position of PSSM. Details in the pCKSAAP scheme are available in our previous study [k = 0,1,2,3 and 4).The compositions of ce window. If the us study . For eacBased on succinylated and non-succinylated samples, five types of features were generated. Among the generated features, there may be some redundant and uncorrelated information, which can affect the speed and accuracy of a predictor. Therefore, feature selection strategies are necessary to pick out informative features that can prevent overfitting, to improve the prediction performance and to understand inherent properties of succinylation sequences. We employed the WR test to select differentially expressed features.r scores/observations and a negative-group has s scores /observations with test statistics, W was defined as the sum of the ranks of the annotations for the positive-group (or negative-group). The following steps were conducted for the WR test.Assuming that a positive-group has r + s annotations with rank observations from the smallest to largest group, where r ranks are allocated to the positive-group and s ranks are allocated into the negative-group. Calculate W of the positive-group.Associate the Discover all the possible permutation of the ranks.p-value is calculated as follows.Each permutation of the rank is calculated and the To classify the models of lysine succinylation sites, a supervised statistical learning approach, RF was employed . RF is ok is the number of individual features with probability P, βn is the regression coefficient with prediction score Sn and α is the constant term. A generalized linear model of an R package software (http://www.R-project.org/) was considered to access the LR.For prediction of succinylated and non-succinylated sites, the outputs of distinct RF scores were combined using an LR method. The LR scheme was successfully used in protein ubiquitin site prediction . The finhttps://cran.r-project.org/web/packages/pROC/index.html). Using the threshold dependent parameters, four statistical indexes: accuracy (Ac), specificity (Sp), sensitivity (Sn), and Matthews correlation coefficient (MCC), were calculated, defined as follows:nTP represents the number of the observed positive residues predicted to be the positive sample, nTN the number of the observed negative residues predicted to be the negative sample, nFP the number of the observed positive residues predicted to be the negative, and nFN the number of the observed negative residues predicted to be the positive sample, respectively.To calculate the prediction performance of each model of GPSuc, the threshold-independent and threshold-dependent indices were measured. The values of area under the curve (AUC) were calculated and the receiver operating characteristic (ROC) curve was depicted using threshold independent parameters by an R-package  were enriched and depleted in H. sapiens, M. musculus, E. coli and S. cerevisiae. In M. tuberculosis, S. lycopersicum and T. aestivum the neutral residues  were depleted.First, given that distinct distribution patterns of the sequence surrounding the succinylation sites in the 9 species datasets, a two-sample graph software was used  to classH. sapiens, H. capsulatum, M. musculus, S. cerevisiae, S. lycopersicum and T. aestivum. Amino acid ‘R’ showed higher AAF scores in H. sapiens, E. coli, M. musculus, M. tuberculosis and T. aestivum than the other species. Here, a non-parametric Kruskal-Walis hypothesis test was accessed to identify whether two samples were significantly different. The p-values were filtered in the corresponding window positions of neighboring succinylated and non-succinylated sites and corrected by the Bonferroni test. For many amino acids surrounding succinylation sites in the nine species, statistical differences were observed between the succinylated and non-succinylated samples, with a p-value of less than 0.05  scores for each amino acid residue in the surrounding succinylated and non-succinylated sequence windows, as shown in han 0.05 . These rH. sapiens, H. capsulatum, M. musculus, M. tuberculosis, E. coli, T. gondii, S. cerevisiae, S. lycopersicum and T. aestivum, respectively . Initially, to inspect the performance for generic site prediction by ‘GPSuc’, the sequence windows were encoded as numerical feature vectors based on the five consecutive features of AAC, BE, AAindex, PSSM, and pCKSAAP. The calculated feature vectors often have redundant and uncorrelated information that impairs the prediction performance. Therefore, feature selection strategies are essential to reduce the dimensionality and optimize the collective contribution features. The feature vectors were optimized using the WR scheme in this study. The WR scheme reduced the dimensionality of the high dimensional pCKSAAP and AAindex features more than other methods. After several trials in the generic classifier, top 390 and 250 feature vectors were collected from the pCKSAAP and AAindex schemes, respectively. The collected feature vectors were transformed into a new ordered feature based on low to high WR values. The corresponding features were adopted from the other three feature vectors .via the LR method provided the highest AUC values of the generic classifier were 0.840 and 0.779 for the training and test datasets of the generic model, respectively . The proposed nine species-specific classifiers were trained and tested based on the consecutive five sequence features of AAC, BE, AAindex, PSSM, and pCKSAAP. To optimize the model features, a WR feature selection strategy was employed by applying a 10-fold CV test to the training dataset of each species. After several trails, the WR feature selection test was found effective in the pCKSAAP and AAindex schemes compared to other model features vectors. Therefore, the optimal feature vectors were transformed from the pCKSAAP and AAindex schemes for nine species. In Five final feature vectors for each species, including the two optimal feature vectors of pCKSAAP and AAindex, were trained by RF classifiers. The collected RF scores were combined by the LR method to construct the GPSuc. Then we plotted the ROC curves and calculated the AUC.via the LR method (GPSuc) provided a more powerful predictor than single encoding models. Use of pCKSAAP, AAindex and BE features performed a little higher than the other two features. Using the ROC curves, the performance on the training dataset by the combined model (GPSuc) reached AUC values of 0.882, 0.807, 0.826, 0.811, 0.732, 0.866, 0.926, 0.859 and 0.847 for H. sapiens, H. capsulatum, M. musculus, E. coli, M. tuberculosis, T. gondii, S. cerevisiae, S. lycopersicum, and T. aestivum, respectively  were plotted in a bar plot . The comectively . Finallyectively . The aboH. sapiens, M. musculus, M. tuberculosis, E. coli, T. gondii, S. cerevisiae, and S. lycopersicum [We compared the performance of the species-specific classifier of GPSuc with SuccinSite2.0, which represents the state-of-the-art predictor available, as shown in persicum . To makein vitro or in vivo species-specific succinylation site.We designed a generic and nine species-specific predictors to classify potential succinylation sites. The GPSuc predictor interpreted high prediction performance in both general and species-specific models. It greatly improved the prediction results compared to previous predictors. Our analysis shows the sequence patterns of succinylation sites are significantly different in the nine species, and the GPSuc combining multiple features using LR analysis improved the prediction performance. To identify the designated succinylation site, a user-friendly online server for GPSuc was established that is particularly beneficial for some hypothesis-driven experiments. GPSuc serves as a complementary and powerful predictor for identification S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Tablep-values were calculated using the Kruskal-Wallis test and corrected by the Bonferroni multiple comparison test. ‘*’ represents p values < 0.05.The (DOCX)Click here for additional data file.S4 Tablep-values were calculated using Chi-square and corrected by the Bonferroni test at each window position (~-5 to +5). Nine species of H. sapiens, H. capsulatum, M. musculus, E. coli, M. tuberculosis, S. cerevisiae, T. gondii, S. lycopersicum and T. aestivum are used.The (DOCX)Click here for additional data file.S5 Tablep-values were calculated using the Kruskal Walis test and corrected Bonferroni test. ‘*’ represents p values < 0.05.The (DOCX)Click here for additional data file.S6 TableThey were used for generic and species-specific models.(DOCX)Click here for additional data file.S7 Tablep values were calculated using the Kruskal Walis and corrected by Bonferroni test. ‘*’ represents p values < 0.05.The 12 types of AAindex are used to calculate the MPPs for each position of the flanking sequence located in the window positions of ~-20 to +20. The (DOCX)Click here for additional data file.S8 Table(DOCX)Click here for additional data file.S1 FigThe PSSM was used to calculate the MPV at each position of the flanking sequence located in the window positions of ~-20 to +20.(DOCX)Click here for additional data file.S2 FigNAKH920108 was used to calculate the MPP at each position of the flanking sequence located in the window positions of ~-20 to +20.(DOCX)Click here for additional data file.


Plants have been used since approximately 5000 BC not only for their medicinal properties, but also as food aroma and for their preservative activity . SeveralTheir health-promoting effects were largely demonstrated . MoreovePhytochemicals characterized by antioxidant activity remove free radicals and inhibit oxidative reactions by being oxidized themselves . Some anNatural antioxidants have been found to be promising strategies to counteract the undesirable effects of oxidative stress. Oxidative stress has been identified as a major causative factor in the development and progression of several diseases, including cardiovascular and neurodegenerative diseases, as well as cancer . In the Brassica oleracea (broccoli) on its phytochemical content with particular reference to carotenoids, phenols, chlorophyll, and radical-scavenging activity. For this purpose, broccoli was extracted by ethanol at 30 °C for 20 min with ultrasound assistance. The assumption of carotenoids and chlorophyll content were considerably affected by gastric digestion whereas phenols were significantly decreased after intestinal digestion. This study confirmed that carotenoids are instable at low pH values (gastric conditions).Scrob et al.  investigHowever, their bioaccessibility depends on the different types of carotenoids as well as on the food matrix in which these compounds are present, or how this food is cooked . RegardiCitrus limon cv Femminello comune is a widely consumed Italian IGP  product. Its juice was subjected to ultrafiltration (UF) process using a cellulose acetate membrane in order to evaluate if this process enhances its healthy properties [operties .Physicochemical parameters, and total phenols and flavonoids contents were evaluated. The most abundant flavonoids, such as rutin, hesperidin, eriocitrin, and neohesperidin, were quantified.The potential antioxidant and hypoglycemic activities were examined. UF juice obtained at 1.5 bar showed the highest antioxidant activity and exhibited promising α-amylase and α-glucosidase inhibitory activity. Prunus armeniaca and P. domestica leaves’ essential oils. All investigated samples were characterized by an interesting 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical scavenging activity. P. domestica essential oils resulted as more active than P. armeniaca in inhibiting lipid peroxidation. A different behavior was observed in cholinesterase inhibitory activity. In fact, P. armeniaca oils were more active than P. domestica against acetylcholinesterase (AChE). Terpenes represent another promising class of bioactive natural compounds. Bonesi et al.  studied Coffea anthonyi, C. arabica, C. canephora, C. charrieriana, C. liberica, C. liberica var. liberica, C. humilis, and C. mannii). 5-CQA resulted to be the most abundant isomer, and some species contained a very low amount of CQAs. Fluctuations were observed depending on the Coffea species and harvesting period. Data suggested the preferential selection of C. liberica leaves collected in July due to the high total content in CQAs. Coffee is one of the beverages extensively consumed in the world. Roasted coffee beans are the most used, while coffee leaves are mainly used as infusion for traditional medicinal purposes in several countries. They showed some antioxidant compounds. Rodríguez-Gómez et al.  developeN-docosahexaenoyl vanillylamine (DHVA) and N-eicosapentaenoyl vanillylamine (EPVA), were synthesized from their corresponding long chain polyunsaturated fatty acids, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) both important dietary components, and studied for their antioxidant and carbohydrateshydrolyzing enzymes’ (α-amylase and α-glucosidase) inhibitory activity together with capsaicin and the corresponding n-3 polyunsaturated fatty acids [Two capsaicin derivatives, namely ty acids . Both faN-acetylcysteine (NAC), commonly found in onion. This compound is a precursor of glutathione. Actually, NAC is used in food supplements and cosmetics. Moreover, there are several uses in preclinical and clinical phases. Although NAC is considered safe, results from clinical trials are sometimes controversial or incomplete. Šalamon et al.  criticalRubus idaeus (red raspberry) fruits are rich in antioxidants. One promising food industry application was described by Giuffrè et al. [è et al. . Red rasThe fruits stored in the nanoactive film A showed better performance regarding storage in the fridge or freezer.In conclusion, natural antioxidant use in food products can increase quality and add value. Therefore, novel environmentally friendly procedures of extraction, purification, identification and quantification of natural antioxidants from plants need to be developed to improve the extraction yields and market value of natural products.


Lepotrema Ozaki, 1932 from marine fishes in the Indo-West Pacific. Prior to the present study six species were recognised. Here we propose eight new species on the basis of combined morphological and molecular analysis: Lepotrema acanthochromidis n. sp. ex Acanthochromis polyacanthus from the Great Barrier Reef (GBR); Lepotrema hemitaurichthydis n. sp. ex Hemitaurichthys polylepis and H. thompsoni from Palau and French Polynesia; Lepotrema melichthydis n. sp. ex Melichthys vidua from Palau and the GBR; Lepotrema amansis n. sp. ex Amanses scopas from the GBR; Lepotrema cirripectis n. sp. ex Cirripectes filamentosus, C. chelomatus and C. stigmaticus from the GBR; Lepotrema justinei n. sp. ex Sufflamen fraenatum from New Caledonia; Lepotrema moretonense n. sp. ex Prionurus microlepidotus, P. maculatus and Selenotoca multifasciata from Moreton Bay; and Lepotrema amblyglyphidodonis n. sp. ex Amblyglyphidodon curacao and Amphipron akyndynos from the GBR. We also report new host records and provide novel molecular data for two known species: Lepotremaadlardi Bray, Cribb & Barker, 1993 and Lepotremamonile Bray & Cribb, 1998. Two new combinations are formed, Lepotrema cylindricum  n. comb. (for Preptetos cylindricus) and Lepotrema navodonis  n. comb. (for Lepocreadium navodoni). With the exception of a handful of ambiguous records, the evidence is compelling that the host-specificity of species in this genus is overwhelmingly oioxenous or stenoxenous. This renders the host distribution in three orders and ten families especially difficult to explain as many seemingly suitable hosts are not infected. Multi-loci molecular data  demonstrate that Lepotrema is a good generic concept, but limited variability in sequence data and differences in phylogenies produced for different gene regions make relationships within the genus difficult to define.We review species of the genus  Lepotrema Ozaki, 1932 is a case in point. It is encountered in a wide range of fish families in the orders Tetraodontiformes and Perciformes, with one record from a pleuronectiform. Most of the literature reports, including some from all three orders, are listed under the type-species, Lepotrema clavatum Ozaki, 1932, suggesting a very low level of specificity. Our molecular evidence presented here, based mainly on ITS2 rDNA and cox1 mtDNA sequences, together with sampling evidence, however, indicates that in general specificity is high. Most species appear to be oioxenic or stenoxenic, with no clear evidence that any individual species parasitizes multiple orders.Members of the digenean family Lepocreadiidae Odhner, 1905 are common parasites of fishes of the Indo-West Pacific region, particularly of coral reef fishes. Our systematic studies on this family in the waters around northern Australia and other sites in the region have hitherto been mostly reliant on comparative morphology  = 1.5”, “Nb bins = 20” and “distance = Jukes-Cantor”.ITS2 rDNA and Mobahincia Bray, Cribb & Cutmore, 2018 (MH157068), Multitestis Manter, 1931 (MH157071) and Neomultitestis Machida, 1982 (MH157072) were designated as functional outgroup taxa, following Bray et al.  (as Monacanthus c.), from the Japanese coast. The exact site of collection was not given, but this fish species is said to be distributed between Otaru and Nagasaki. A “genital sucker” is described “at the bottom of the chamber  lying directly inside of the end part of the metraterm”. This feature, along with the distinctly dorsal excretory pore, represent the main distinguishing features of the genus. Yamaguti (Thamnaconus modestus (Günther) (as Cantherhines unicornu) (Monacanthidae) and the cinnamon flounder Pseudorhombus cinnamoneus (Temminck & Schlegel)  from Japanese waters. Some measurements were given but no illustration. The morphological features of the genus were not discussed. Four years later, Yamaguti   , the brown-and-white butterflyfish Hemitaurichthys zoster (Bennett) (Chaetodontidae) and the Hawaiian dascyllus Dascyllus albisella Gill (Pomacentridae), all from Hawaii, but again without any illustration. Yamaguti   from off Hawaii with the metraterm provided with a “bulb of lamellar muscle fibers”, and a “dorsoterminal” excretory pore. Dyer et al. (Lepocreadium clavatum in the white-banded triggerfish Rhinecanthus aculeatus (Linnaeus)  off Okinawa, Japan, but again without any illustration. Bray et al.  and the banded parma Parma polylepis Günther, from off Heron Island on the southern Great Barrier Reef, providing illustrations of individuals from both fishes. The terminal part of the metraterm was decribed as a “large, circular, folded muscular pad (not a sphincter)” and the excretory pore as “mid-dorsal, about halfway between caecal ends and posterior extremity”. They also decribed Lepocreadium adlardi Bray, Cribb & Barker, 1993 from the Bengal sergeant Abudefduf bengalensis (Bloch) (Pomacentridae), with the distal extremity of the metraterm “clamped in prominent folded muscular pad” and the excretory pore “mid-dorsal, close to level of posterior extremity of caeca”.The genus by Ozaki  for L. cYamaguti  recognisYamaguti  re-exami. Hanson  followedritchard  reportedYamaguti  re-recorr et al.  reportedy et al.  reportedLepotrema based on the structure of the distal metraterm and the dorsal excretory pore. This action was then taken by Bray & Cribb  (Monacanthidae) (illustrated) and the halfmoon triggerfish Sufflamen chrysopterum (Bloch & Schneider)  (not illustrated) from off Heron Island and described Lepotrema canthescheniae Bray & Cribb, 1996 from the endemic large-scaled leatherjacket Cantheschenia grandisquamis Hutchins from off Heron Island. Bray & Cribb  from off Heron Island. This species is problematical in that the distal metraterm is surrounded only by a “distinct, but narrow, sphincter”. The excretory pore, however, is “dorsal, between ends of caeca”. Machida & Kuramochi (Lepotrema in reporting (but not illustrating) L. clavatum in T. modestus and the red-toothed triggerfish Odonus niger (Rüppell)  off Japan. Machida & Uchida  from off Japan. They gave some measurements but did not describe the metraterm or excretory pore or give an illustration. In his review of the family Bray  reported by Bray et al.  (s.l.), the Japanese sea nettle Chrysaora pacifica (Goette) and the ghost jellyfish Cyanea nozakii Kishinouye, from the Seto Inland Sea, Japan. They also reported metacercariae and juveniles of L. clavatum from juvenile Pacific rudderfish Psenopsis anomala (Temminck & Schlegel) (Centrolophidae) and Thamnaconus modestus. The first intermediate hosts are unknown.The only evidence of the life-cycle of o et al. , who desOverview of new findingsLepotrema in the possession  of a distinct folded muscular bulb on the distal metraterm and a postero-dorsal excretory pore. These were from 29 host/parasite/locality combinations. These forms are superficially highly similar to each other. The specimens were therefore assessed iteratively by morphology and analysis of ITS2 rDNA and cox1 mtDNA sequences for as many combinations for which suitable specimens were available. As discussed in greater detail below, we found that for host/locality combinations for which there were multiple specimens and multiple sequences, there was a strong tendency for genetic and morphological distinctions to be detectable. Using this rationale, we here characterise nine species on the basis of combined morphological and molecular data; of these seven are described as new. One further species is described as new on the basis of morphological data only. In addition, six existing species for which no molecular data are available are recognised. Finally, we summarise reports, old and new, of five host/parasite combinations which may well comprise further new species but for which the evidence is presently inadequate.In the present study we examined new specimens consistent with the concept of Molecular datacox1 mtDNA sequences for 15 host/locality combinations. Eleven genotypes/clades were present in both the ITS2 and cox1 datasets, several of which exhibited some low-level intra-genotypic variation. The complete ITS2 region ranged between 275–293 bp in length . The ITS2 alignment (including flanking 5.8S and 28S regions) comprised 11 genotypes (each represented by 1–6 replicates) and consisted of 466 bp. The level of distinction between ITS2 genotypes in the final dataset ranged from 1–9 bp  was 1,339 bp long. The level of differences between species ranged between 0–23 bp; two genotypes that had no bases different in the final dataset (those from P. wardi and those from Cirripectes spp.) differed by a single indel only. A phylogram representing analyses of the 28S dataset is shown in Fig. We generated 32 5.8S-ITS2-28S rDNA sequences and 31 partial cox1 sequences are consistent with those suggested by host distribution and morphology. The 11 main genotypes/clades are thus considered to represent 10 species. The disparity between 11 genotypes/clades and the recognition of 10 species relates to the samples from Hemitaurichthys polylepis from Palau and the Austral and Marquesas Archipelagos in French Polynesia. Of the three sources of evidence available, host clearly gave no basis for distinction between these forms. The ITS2 distinction (a single base) was unique; no other putative species showed any intraspecific variation in ITS2 sequence data. The cox1 distinction was at a level lower than between any combination of species but far greater than within any of the other species which exhibited intraspecific variation. However, no other species was sequenced over such a wide geographical range, so we are unable to interpret this distinction in context. There was no difference in the partial 28S sequence data for samples from Palau and the Austral Archipelago. The morphology of the forms from H. polylepis suggests subtle distinctions, but nothing that amounts to a reliable difference. In the face of these combined data, we propose a conservative approach, interpreting all specimens from H. polylepis as a single species that demonstrates geographical genetic variation.All data, molecular, morphological and biological , were considered iteratively. Overall, the distinctions suggested by ITS2 and Species ofLepotremaLepotrema clavatumOzaki, 1932Type-host: Stephanolepis cirrhifer (Temminck & Schlegel) (Tetraodontiformes: Monacanthidae), threadsail filefish.Type-locality: “Otaru southwards to Nagasaki”, Japan.Material studied: Voucher specimens collected by Ozaki, probably the type-series, 3 slides with 10 worms, three mature and measured. Worms flattened.Locality: Off Hiroshima, Hiroshima Prefecture, Japan.Voucher specimens: Meguro Parasitological Museum: Vouchers 30029, 30030.Site in host: Upper part of intestine.Records: 1. Ozaki  , Thamnaconus modestus (Günther) .Doubtful definitive hosts: Balistidae: Melichthys niger (Bloch) , Melichthys vidua (Richardson) , Odonus niger (Rüppell) (10), Rhinecanthus aculeatus (Linnaeus) (8); Chaetodontidae: Hemitaurichthys zoster (Bennett) (5); Paralichthyidae: Pseudorhombus cinnamoneus (Temminck & Schlegel) (2); Pomacanthidae: Genicanthus semifasciatus (Kamohara) (11); Pomacentridae: Dascyllus albisella Gill (5).Second intermediate hosts: Cnidaria, Scyphozoa: Aurelia aurita (Linnaeus) (s.l.) (12), Chrysaora pacifica (Goette) (12), Cyanea nozakii Kishinouye (12).Freshly ingested immatures: Centrolophidae: Psenopsis anomala (Temminck & Schlegel) (12); Monacanthidae: Thamnaconus modestus (12).Localities: Japan , Hawaii .Lepotrema, was known mainly from its original description. We reproduce here one new illustration , have been deleted from the list of hosts for this species. We base our ideas on the morphology of this worm on the original description  Bray & Cribb, 1996Lepocreadium adlardi Bray, Cribb & Barker, 1993Syn. Type-host: Abudefduf bengalensis (Bloch) (Perciformes: Pomacentridae), Bengal sergeant.Type-locality: Off Heron Island, Great Barrier Reef, Australia.Records: 1. Bray et al. .New materialHost: Abudefduf bengalensis.Localities: Off Heron Island , off Lizard Island , Queensland, Australia; Ningaloo Reef , Western Australia.Prevalence: Off Heron Island: in 20 of 43 fish examined; off Lizard Island: in 2 of 5 fish examined; Ningaloo Reef: in 3 of 13 fish examined.Voucher material: Off Heron Island ; off Lizard Island (QM G237460); Ningaloo Reef Representative DNA sequences: ITS2 rDNA, two identical replicates (one submitted to GenBank MH730000); cox1 mtDNA, two replicates (both submitted to GenBank MH730027–28); 28S rDNA, one sequence (submitted to GenBank MH730015).L. adlardi from the northern Great Barrier Reef (Lizard Island) and the Indian Ocean . The small samples sizes available  , the scissortail sergeant Abudefduf sexfasciatus (Lacépède) , the blackspot sergeant A. sordidus (Forsskål) , the Indo-Pacific sergeant A. vaigiensis (Quoy & Gaimard) (12 specimens from the GBR and Moreton Bay) or Whitley’s sergeant A. whitleyi Allen & Robertson . Forty-one other pomacentrid species have been investigated without the recovery of L. adlardi.This species is morphologically and genetically distinct, being narrow, with an even narrower, long forebody, a long prepharynx and a pre-vitelline distance similar to the forebody length Figs. –7. New mle Table  give no Lepotrema acanthochromidisn. sp.L. clavatum of Bray et al.  (Perciformes: Pomacentridae), spiny chromis.Type-locality: Off Heron Island , Great Barrier Reef, Australia.Other locality: Off Lizard Island , Great Barrier Reef, Australia.Type-material: Off Heron Island: holotype (QM GL 14769); paratypes ; off Lizard Island .Site in host: Intestine.Prevalence: Off Heron Island: in 21 of 65 fish examined; off Lizard Island: in 21 of 74 fish examined.Representative DNA sequences: ITS2 rDNA, two identical replicates (one submitted to GenBank MH729999); cox1 mtDNA, two identical replicates (both submitted to GenBank MH730025–26); 28S rDNA, one sequence (submitted to GenBank MH730014).ZooBank registration: To comply with the regulations set out in article 8.5 of the amended 2012 version of the International Code of Zoological Nomenclature .[Based on 26 whole-mounted specimens, 9 from off Heron Island, 17 from off Lizard Island; measurements in Table Testes 2, oval, entire, virtually tandem or slightly oblique, in mid-hindbody. External seminal vesicle usually obscured by eggs, but small when seen. Cirrus-sac claviform, mainly dorsal to ventral sucker. Internal seminal vesicle rounded to oval. Pars prostatica vesicular. Ejaculatory duct long, muscular. Genital atrium distinct. Genital pore sinistral, ventral to sinistral caecum at bifurcal level or just posterior.Ovary trilobate, immediately pre-testicular, close or adjacent to ventral sucker. Laurer’s canal opens at about level of anterior edge of anterior testis. Seminal receptacle dorsal or dorso-lateral to ovary. Mehlis’ gland dorsal to ovary or anterior part of anterior testis. Uterus intercaecal, mostly pre-testicular, passes ventrally to ovary, overlaps ventral sucker. Eggs tanned, operculate. Metraterm shorter than cirrus-sac, distal extremity with large folded muscular pad. Vitellarium follicular, reaching from posterior edge of pharynx to posterior extremity, fields may be confluent in forebody (as narrow band) and post-testicular region; lateral and ventral to caeca.Excretory pore dorsal, in anterior post-testicular region; vesicle reaches to testes, not traced further.Lepotrema clavatum is larger, with a relatively longer forebody, relatively larger oral sucker and pharynx, a longer cirrus-sac, a shorter post-testicular region, longer ventral sucker to bifurcal distance and ventral sucker to ovary distances, a slightly shorter pre-vitelline distance, a smaller sucker ratio and slightly longer caeca. Lepotrema incisum has deeply incised testes, a relatively larger pharynx, a longer pre-vitelline distance and cirrus-sac, a relatively shorter post-testicular region and smaller eggs. Lepotrema monile lacks a strong muscular pad around the metraterm, has a small sphincter and has a relatively shorter cirrus-sac.This species is characterised by molecular means Table  and distcox1 sequence data. 28S sequence data for the specimens from off Heron Island differed from samples from off Lizard Island (GenBank: FJ788483.1) by 2 bp; this is a level greater than between conspecific Lepotrema samples infecting Hemitaurichthys polylepis from Palau and French Polynesia, which had identical 28S data across these regions. In addition, specimens of L. acanthochromidis n. sp. from off Heron Island tend to have slightly longer eggs than those from off Lizard Island. However, given the host and generally similar morphology and the lack of ITS2 and cox1 data for samples from off Lizard Island, we take a conservative approach and recognise both sets of samples as the same species. This anomaly is worthy of further study.The two worms sequenced from off Heron Island have identical ITS2 and Acanthochromis polyacanthus, having been found in that species at least 40 times on the GBR but never in 1,228 individuals of 55 other pomacentrid species examined on the GBR. Acanthochromis polyacanthus can be found together with Abudefduf bengalensis (the host of L. adlardi) and Pomacentrus wardi  but there is no evidence of any sharing of the three Lepotrema species by these three pomacentrid species. Notably, according to Cribb et al.  pomacentrid host for a bivesiculid, Bivesicula unexpecta Cribb, Bray & Barker, 1994.This species has strongly oioxenous specificity for b et al. , A. polyLepotrema hemitaurichthydisn. sp.Type-host: Hemitaurichthys polylepis (Bleeker) (Perciformes: Chaetodontidae), pyramid butterflyfish.Other host: Hemitaurichthys thompsoni Fowler (Perciformes: Chaetodontidae), Thompson’s butterflyfish.Type-locality: Off Palau .Other localities: Ex H. polylepis: off Tubuai , off Rimatara , Austral Islands, French Polynesia; ex H. thompsoni: off Fatu Hiva, Marquesas, French Polynesia .Type-material: Holotype (QM G237483), paratypes: ex H. polylepis off Palau ; off Tubuai ; off Rimatara (QM G237494). Voucher: ex H. thompsoni (QM G237495).Site in host: Intestine.Prevalence: Ex H. polylepis: off Palau (in 13 of 15 fish examined); off Tubuai (in 2 of 5 fish examined); off Rimatara (in 1 fish examined). Ex H. thompsoni: off Fatu Hiva (in 2 of 3 fish examined).Representative DNA sequences: ITS2 rDNA, four replicates (two submitted to GenBank MH730006–07); cox1 mtDNA, four replicates ; 28S rDNA, two identical replicates (both submitted to GenBank MH730019–20).ZooBank registration: To comply with the regulations set out in article 8.5 of the amended 2012 version of the International Code of Zoological Nomenclature , a smaller pharynx, a longer intestinal bifurcation to ventral sucker distance, a larger cirrus-sac, a longer ventral sucker to ovary distance, relatively shorter post-testicular and post-caecal distances and possibly slightly larger eggs. Lepotrema acanthochromidis n. sp. is relatively narrower, with a smaller pharynx and possibly ventral sucker, tending to have a longer prepharynx and slightly longer post-testicular and post-caecal distances. Lepotrema incisum has deeply incised testes, relatively larger pharynx and pre-vitelline distance, a shorter cirrus-sac, a relatively shorter post-testicular region and smaller eggs. Lepotrema monile has a small sphincter rather than a strong muscular pad around the metraterm and a relatively shorter cirrus-sac.The specimen from ns Table  and distns Table . LepotreLepotrema have been found only in the two species Hemitaurichthys that we have examined. Multiple infections were detected in Hemitaurichthys polylepis off Palau (13 of 15) and in the Austral Archipelago of French Polynesia (3 of 6).In our investigations of Indo-Pacific fishes, we have examined over 1,600 individuals of 35 species of chaetodontids. Species of Lepotrema melichthydisn. sp.Lepotrema clavatum of Bray & Cribb (Syn.  & Cribb Type-host: Melichthys vidua (Richardson) , pinktail triggerfish.Type-locality: Off Palau .Other locality: Off Heron Island , Great Barrier Reef, Australia.Type-material: Off Palau: holotype (QM G237496); paratypes ; off Heron Island .Site in host: Intestine.Prevalence: Off Palau: in 1 fish examined; off Heron Island: in 1 fish examined.Representative DNA sequences: ITS2 rDNA, two identical replicates (one submitted to GenBank MH730008); cox1 mtDNA, two replicates (both submitted to GenBank MH730046–47); 28S rDNA, one sequence (submitted to GenBank MH730021).ZooBank registration: To comply with the regulations set out in article 8.5 of the amended 2012 version of the International Code of Zoological Nomenclature , a longer intestinal bifurcation to ventral sucker distance, a larger anterior testis, and relatively smaller pharynx and post-testicular and post-caecal distances. Lepotrema acanthochromidis n. sp. has a less distinctive pre-oral lobe, a relatively smaller pharynx, a longer prepharynx, a relatively longer oesophagus and pre-vitelline distance, a relatively shorter cirrus-sac and the gonads tend to be slightly larger. Lepotrema hemitaurichthydis n. sp. has a less distinct pre-oral lobe, a relatively longer oesophagus, a relatively shorter pre-vitelline distance, a longer ventral sucker to ovary distance, the gonads are slightly larger, the post-testicular distance is shorter and the caeca are longer. Lepotrema incisum has deeply incised testes. Lepotrema monile has a small sphincter rather than a strong muscular pad around metraterm.This species is characterised by molecular means and distinguished from similar congeners by the following morphological characteristics Table . MoleculThe Palau and Heron Island specimens differ slightly, with the Palau specimens having a relatively longer pre-oral lobe, shorter oesophagus, longer ventral sucker to ovary distance and smaller gonads. These differences are not of the magnitudes that distinguish this form from other species, so they are considered intraspecific variation here.M. vidua at each of Palau and Heron Island, both being infected with multiple specimens of L. melichthydis n. sp. Four individuals of M. vidua examined in French Polynesia were not infected. In addition, we have examined 344 specimens of 13 species of Balistidae in the Indo-West Pacific region without finding infections of this species. We thus infer that it is oioxenous to M. vidua, or potentially stenoxenous to Melichthys, which has just three recognised species.We have examined only a single specimen of L. clavatum from M. vidua from Hawaiian waters. Yamaguti  (Tetraodontiformes: Monacanthidae), broom filefish.Type-locality: Off Heron Island , Great Barrier Reef, Australia.Type-material: Holotype (QM G237503); paratypes .Site in host: Intestine.Prevalence: In 5 of 7 fish examined.Representative DNA sequences: ITS2 rDNA, four identical replicates (one submitted to GenBank MH730001); cox1 mtDNA, four replicates ; 28S rDNA, one sequence (submitted to GenBank MH730016).ZooBank registration: To comply with the regulations set out in article 8.5 of the amended 2012 version of the International Code of Zoological Nomenclature , relatively smaller suckers and pharynx, relatively larger testes and relatively shorter cirrus-sac and post-testicular and post-caecal distances. Lepotrema acanthochromidis n. sp. is usually distinctly larger, with a longer pre-oral lobe, relatively slightly smaller suckers and pharynx, a longer oesophagus, a relatively slightly shorter cirrus-sac and larger gonads. Lepotrema hemitaurichthydis n. sp. is usually distinctly larger and broader, with a tendency to have a longer oesophagus, a longer pre-vitelline distance, a relatively slightly shorter cirrus-sac, larger testes and longer caeca. Lepotrema incisum has deeply incised testes. Lepotrema melichthydis n. sp. is distinctly larger, with a distinctly longer pre-oral lobe, a relatively shorter prepharynx, relatively smaller suckers, a relatively shorter cirrus-sac and a relatively longer post-testicular distance. Lepotrema monile has a small sphincter rather than a strong muscular pad around metraterm.This species is characterised by molecular means Table  and distAmanses scopas on the Great Barrier Reef; five of these were infected with L. amansis n. sp. No infection of this or any other species of Lepotrema has been seen by us in 130 individuals of 28 other species of Monacanthidae examined in the region, apart from the records of L. canthescheniae in the southern Great Barrier Reef/ New South Wales endemic fish Cantheschenia grandisquamis.We have examined nine individuals of Lepotrema cirripectisn. sp.Type-host: Cirripectes filamentosus (Alleyne & Macleay) (Perciformes: Blenniidae), filamentous blenny.Other hosts: Cirripectes chelomatus Williams & Maugé, Lady Musgrave blenny; Cirripectes stigmaticus Strasburg & Schultz, red-streaked blenny (both Blenniidae).Type-locality: Off Lizard Island , Great Barrier Reef, Australia.Other locality: Off Heron Island , Great Barrier Reef, Australia.Type-material: Holotype (QM G237521); paratypes: ex C. filamentosus ; ex C. chelomatus ; ex C. stigmaticus (QM G237551).Site in host: Intestine.Prevalence: Ex C. filamentosus (in 7 of 8 fish examined); ex C. stigmaticus (in 2 of 2 fish examined); ex C. chelomatus (in 4 of 4 fish examined).Representative DNA sequences: ITS2 rDNA, six identical replicates (two submitted to GenBank MH730004–05); cox1 mtDNA, six replicates ; 28S rDNA, one sequence (submitted to GenBank MH730018).ZooBank registration: To comply with the regulations set out in article 8.5 of the amended 2012 version of the International Code of Zoological Nomenclature  and 8 from off Heron Island ; measurements in Table [Based on 40 whole-mounted specimens, 32 from off Lizard Island , a shorter pre-vitelline distance, a relatively longer cirrus-sac, and shorter ventral sucker to ovary, post-testicular and post-caecal distances. Lepotrema acanthochromidis n. sp. has a longer pre-oral lobe, a shorter ventral sucker to ovary distance and larger testes. Lepotrema adlardi is narrower, particularly in the forebody, the forebody is longer, the pre-oral lobe is distinct, it has longer prepharynx and pharynx, the intestinal bifurcation is more posterior, the extension of the vitellarium into forebody is much shorter and the ventral sucker to ovary distance is longer. Lepotrema amansis n. sp. has relatively larger suckers and pharynx, a longer cirrus-sac and shorter ventral sucker to ovary and post-testicular distances. Lepotrema hemitaurichthydis n. sp. is broader, with a longer forebody and pre-oral lobe, slightly larger suckers, a larger pharynx, a shorter ventral sucker to ovary distance, larger testes and a shorter post-testicular distance. Lepotrema incisum has deeply incised testes. Lepotrema melichthydis n. sp. has a distinct pre-oral lobe, a relatively shorter prepharynx and oesophagus, a larger pharynx, shorter pre-vitelline and ventral sucker to ovary distances and a relatively smaller ovary. Lepotrema monile has a small sphincter rather than a strong muscular pad around metraterm.This species is characterised by molecular means Table  and distLepotrema cirripectis n. sp. has been detected in five of 11 individuals of two species of Cirripectes examined at Lizard Island and in one species of Cirripectes examined at Heron Island. No specimens relating to Lepotrema have been found by us in 240 individuals of 28 species of other blenniid genera from the region, allowing the inference that this species is stenoxenous for the genus Cirripectes. Heron Island specimens are all smaller than the Lizard Island specimens, but with similar proportions.Lepotrema justinein. sp.Lepotrema cf. clavatum of Bray & Justine (Syn.  Justine Type-host: Sufflamen fraenatum (Latreille) , masked triggerfish.Type-locality: Interior Lagoon near Recif Toombo , New Caledonia.Other localities: Inside Lagoon, facing Recif Toombo , Interior Lagoon near Recif Toombo , New Caledonia.Type-material: Holotype (MNHN JNC2772Aa); paratypes .Site in host: Intestine.ZooBank registration: To comply with the regulations set out in article 8.5 of the amended 2012 version of the International Code of Zoological Nomenclature , a relatively smaller pharynx, a relatively longer oesophagus (cf. “type series”), a more anterior intestinal bifurcation and vitelline extent, a relatively smaller ventral sucker, a longer ventral sucker to ovary distance, a shorter post-testicular distance and longer caeca. Lepotrema acanthochromidis n. sp. has smaller suckers, a longer prepharynx, a smaller pharynx, a longer oesophagus, a more anterior intestinal bifurcation, the ovary is separated from the ventral sucker and the gonads are smaller. Lepotrema amansis n. sp. is distinctly narrower, with a shorter pre-oral lobe, a longer prepharynx, a relatively longer cirrus-sac, a longer ventral sucker to ovary distance, relatively distinctly smaller gonads and a shorter post-testicular distance. Lepotrema cirripectis n. sp. is narrower, with a shorter pre-oral lobe, a longer prepharynx, a shorter pharynx, longer intestinal bifurcation to ventral sucker and ventral sucker to ovary distances, smaller testes and longer caeca. Lepotrema hemitaurichthydis n. sp. has a shorter pre-oral lobe, smaller suckers, longer prepharynx and cirrus-sac, the ovary separated from the ventral sucker, the gonads are smaller, the post-testicular region is shorter, and the caeca are longer. Lepotrema incisum has deeply incised testes. Lepotrema melichthydis n. sp. has a slightly longer pre-oral lobe, slightly smaller suckers, a shorter pre-vitelline distance, a longer ventral sucker to ovary distance and relatively slightly smaller gonads. Lepotrema monile has a small sphincter rather than a strong muscular pad around metraterm.It is the only species with more or less symmetrical testes, and, probably as a result, it tends to be broader than the other species. Lepotrema justinei n. sp. has been found only in S. fraenatum from off New Caledonia, where three of 14 fish examined were infected. We have not seen this species in eight S. fraenatum examined from off Heron Island or in seven examined at Ningaloo Reef. In addition, examination of a further 167 individuals of S. bursa  and S. chrysopterum from the Indo-West Pacific region  have not been infected with this species. The single specimen reported as Lepotrema clavatum from S. chrysopterum by Bray & Cribb (1996) from off Heron Island is clearly distinct (see below as Lepotrema sp. 3).Lepotrema moretonensen. sp.Type-host: Prionurus microlepidotus Lacépède (Perciformes: Acanthuridae), sixplate sawtailOther hosts: Prionurus maculatus Ogilby (Acanthuridae), yellowspotted sawtail; Selenotoca multifasciata (Richardson) (Perciformes: Scatophagidae), spotbanded scat.Type-locality: Off Amity , North Stradbroke Island, Queensland, Australia.Other localities: P. maculatus: off Amity, North Stradbroke Island, Queensland; S. multifasciata, off Green Island, Moreton Bay , Australia.Type-material: Holotype (QM G237552); paratypes: ex P. microlepidotus ; ex P. maculatus ; ex S. multifasciata (QM G237562).Site in host: Intestine.Prevalence: Ex P. microlepidotus (in 8 of 8 fish examined); ex P. maculatus (in 2 of 2 fish examined); ex S. multifasciata (in 2 of 36 fish examined).Representative DNA sequences: ITS2 rDNA, six identical replicates (three submitted to GenBank MH730011–13); cox1 mtDNA, five replicates ; 28S rDNA, two identical replicates (one submitted to GenBank MH730023).ZooBank registration: To comply with the regulations set out in article 8.5 of the amended 2012 version of the International Code of Zoological Nomenclature ; measurements in Table Testes 2, oval, entire, virtually tandem or slightly oblique, in mid-hindbody. External seminal vesicle saccular, often obscured by eggs. Cirrus-sac claviform, sigmoid or flexed, mainly dorsal to ventral sucker but reaches distinctly into hindbody. Internal seminal vesicle oval. Pars prostatica vesicular. Ejaculatory duct long, thick-walled. Genital atrium distinct. Genital pore sinistral, ventral to sinistral caecum, distinctly anterior to ventral sucker, may be at bifurcal level.Ovary trilobate, immediately pre-testicular, separated from ventral sucker. Laurer’s canal opens dorsally to anterior part of anterior testis, not easily detected. Seminal receptacle dorsal or dorso-lateral to ovary. Mehlis’ gland dorsal to ovary. Uterus intercaecal, pre-testicular, passes ventrally to ovary. Eggs tanned, operculate. Metraterm shorter than cirrus-sac, distal extremity with large folded muscular pad. Vitellarium follicular, reaching from level of posterior part of pharynx, oesophagus or intestinal bifurcation to posterior extremity, confluent in forebody and post-testicular region; lateral and ventral, but not dorsal to caeca.Excretory pore dorsal, in anterior post-testicular region or at level of caecal ends; vesicle reaches to testes, not traced further.Lepotrema clavatum is larger with a slightly longer forebody, a shorter pre-oral lobe, longer prepharynx (cf. “type-series”) and oesophagus, a shorter pre-vitelline distance, a longer cirrus-sac, a relatively shorter ventral sucker to ovary distance, slightly larger testes, a shorter post-testicular distance and longer caeca. Lepotrema acanthochromidis n. sp. is very similar but the prepharynx tends to be longer, and there is a tendency for a slightly shorter pharynx and ventral sucker to ovary distance. Lepotrema adlardi is slightly narrower, with a distinctly narrower forebody, a smaller oral sucker, a longer prepharynx, a shorter intestinal bifurcation to ventral sucker distance, a much longer pre-vitelline distance and a slightly shorter cirrus-sac and ventral sucker to ovary distance. Lepotrema amansis n. sp. is smaller, but relatively slightly narrower, it has a less distinct pre-oral lobe, a slightly shorter pre-vitelline distance, a shorter ventral sucker to ovary distance and a smaller posterior testis. Lepotrema cirripectis n. sp. has a less distinct pre-oral lobe, slightly smaller suckers, a longer prepharynx, a smaller pharynx, the ventral sucker to ovary distance tends to be longer, the testes tend to be smaller as does the cirrus-sac but the post-testicular distance may be slightly larger. Lepotrema hemitaurichthydis n. sp. is very similar, but is wider, with possibly a slightly longer prepharynx, possibly a slightly longer oesophagus, a longer ventral sucker to ovary distance and slightly longer caeca. Lepotrema justinei n. sp. is broader, with a relatively slightly larger ventral sucker, a shorter pre-bifurcal distance, a distinct separation of ventral sucker and ovary, symmetrical testes and smaller gonads. Lepotrema melichthydis n. sp. has a more prominent pre-oral lobe, a shorter pre-vitelline distance and a slight tendency for the ventral sucker to ovary distance to be smaller.This species is characterised by molecular means Table  and distLepotrema moretonense n. sp. have been found in all seven P. microlepidotus and both P. maculatus examined from Moreton Bay. In addition, a single gravid adult and a single immature specimen (that was sequenced for the ITS2 and cox1 datasets) have been found in two of the 36 individuals of Selenotoca multifasciata (Scatophagidae) examined from Moreton Bay. We think that these data suggest that this species is effectively stenoxenous to the genus Prionurus. We have only examined a single uninfected individual of a Prionurus species elsewhere and so cannot comment on the geographical distribution of this species.Numerous specimens of Lepotrema amblyglyphidodonisn. sp.Lepocreadium sp. of Bray et al.  (Perciformes: Pomacentridae), staghorn damselfish.Other host: Amphipron akyndynos Allen, Barrier Reef anemonefish (Pomacentridae).Type-locality: Off Heron Island , Great Barrier Reef, Australia.Type-material: Holotype (QM G237563); paratypes: ex A. curacao (QM GL 14775–6); ex A. akyndynos (QM G237564).Site in host: Intestine.Prevalence: Ex A. curacao (in 5 of 71 fish examined); ex A. akyndynos (in 1 of 7 fish examined).Representative DNA sequences: ITS2 rDNA, three identical replicates (two submitted to GenBank MH730002–03); cox1 mtDNA, three identical replicates ; 28S rDNA, one sequence (submitted to GenBank MH730017).ZooBank registration: To comply with the regulations set out in article 8.5 of the amended 2012 version of the International Code of Zoological Nomenclature , large-scaled leatherjacket.Type-locality: Off Heron Island, Great Barrier Reef, Australia.Prevalence: In 2 of 42 fish examined.L. xanthichthydis, the vitellarium reaches to the oral sucker. These worms differ in cirrus-sac length, the lack or presence of uterine coils in the forebody, and egg-length  but all have been uninfected. Total prevalence now stands at two of 42. We suspect that this species is oioxenous to C. grandisquamis, but the low prevalence detected is puzzling. This species is clearly morphologically distinct from the only other species of Lepotrema we have found in a monacanthid, L. amansis n. sp.The original description of Lepotrema monileBray & Cribb, 1998Lepocreadium sp. from Pomacentrus cf. wardi of Bray et al. , Ward’s damsel.Type-locality: Off Heron Island, Great Barrier Reef, Australia.New materialHosts: Pomacentrus amboinensis Bleeker, Ambon damsel; Pomacentrus chrysurus Cuvier, whitetail damsel; Stegastes apicalis (De Vis), Australian Gregory .Localities: Off Lizard Island  (ex P. amboinensis and P. chrysurus); off Heron Island .Voucher material: Ex P. amboinensis (QM G237567); ex P. chrysurus (QM G237565–6); ex S. apicalis (QM G237568).Prevalence: Ex P. amboinensis (in 1 of 54 fish examined); ex P. chrysurus (in 1 of 26 fish examined); ex S. apicalis (in 2 of 27 fish examined).Representative DNA sequences: ITS2 rDNA, two identical replicates (one submitted to GenBank MH730009); cox1 mtDNA, two identical replicates (both submitted to GenBank MH730048–49); 28S rDNA, one sequence (submitted to GenBank MH730024).Lepotrema species for which molecular data are available. New measurements are given in Table This species is distinct in that it lacks a prominent folded muscular pad at the distal metraterm, having a reduced muscular sphincter. It differs genetically from all other Lepotrema clavatum is larger, with a longer forebody, a shorter pre-vitelline distance, a longer cirrus-sac, a shorter ventral sucker to ovary distance, slightly larger gonads, a shorter post-testicular region and longer caeca. Lepotrema adlardi is narrower and mostly larger, with a longer forebody, slightly smaller suckers, a distinctly longer prepharynx, a longer oesophagus and pre-vitelline distance, a shorter ventral sucker to ovary distance and a slightly smaller ovary. Lepotrema acanthochromidis n. sp. has a slightly longer intestinal bifurcation to ventral sucker distance, a longer cirrus-sac and a shorter ventral sucker to ovary distance. Lepotrema amansis n. sp. has a slightly longer forebody, larger suckers and pharynx, a longer intestinal bifurcation to ventral sucker distance, a shorter pre-vitelline distance, a longer cirrus-sac, a shorter ventral sucker to ovary distance and post-testicular region and possibly slightly shorter eggs. Lepotrema amblyglyphidodonis n. sp. has a less distinct pre-oral lobe and a shorter ventral sucker to ovary distance. Lepotrema cirripectis n. sp. has a less distinct pre-oral lobe, a longer intestinal bifurcation to ventral sucker distance and cirrus-sac and possibly slightly longer caeca. Lepotrema hemitaurichthydis n. sp. has a slightly longer forebody, slightly larger suckers, a longer intestinal bifurcation to ventral sucker distance, a longer cirrus-sac, shorter ventral sucker to ovary distance and post-testicular region, longer caeca and possibly slightly longer eggs. Lepotrema justinei n. sp. is broader, with a distinct pre-oral lobe, a shorter prepharynx, a larger pharynx, shorter pre-vitelline distance and ventral sucker to ovary distances, larger gonads and shorter caeca. Lepotrema melichthydis n. sp. has a longer pre-oral lobe, a shorter prepharynx, a longer pharynx and intestinal bifurcation to ventral sucker distance, a shorter pre-vitelline distance, a longer cirrus-sac, a shorter ventral sucker to ovary distance, a smaller ovary and possibly slightly shorter eggs. Lepotrema moretonense n. sp. has a shorter prepharynx, a larger pharynx, a longer cirrus-sac, a shorter ventral sucker to ovary distance, a larger posterior testis and a shorter post-testicular region.It also differs from other similar species in the following characteristics. L. monile in seven of 61 P. wardi examined at Heron Island  from off Lizard Island, in each case the identification is based of morphology only. This species is interpreted as predominantly stenoxenous to the genus Pomacentrus. The low recorded prevalence in two of the species of Pomacentrus suggests that this species might well be found in more species of Pomacentrus given sufficient sampling.We have collected Lepotremasp. 1Host: Rhinecanthus aculeatus (Linnaeus) , white-banded triggerfish.Locality: Off Lizard Island , Great Barrier Reef, Australia.Site in host: Intestine.Voucher material: QM G237569.L. justinei n. sp., but the folded muscular pad on the metraterm is far smaller. Dyer et al.  or distinct. We have examined 38 individuals of this fish from off Lizard Island and another 23 from other sites in the region, but apart from Lepotrema sp. 2 (see below), only the single infection has been detected.One specimen is available. Measurements of the specimen are given in Table r et al.  reportedLepotremasp. 2Host: Rhinecanthus aculeatus (Linnaeus) , white-banded triggerfish.Locality: Off Palau .Site in host: Intestine.Prevalence: In 1 of 2 fish examined.Voucher material: QM G237570–1.Lepotrema sp. 1) reported from the same host at Lizard Island. One of two individuals of R. aculeatus examined from off Palau was infected.Two specimens are available Table ; Fig. 32Lepotremasp. 3Lepotrema clavatum of Bray & Cribb (Syn.  & Cribb  in partHost: Sufflamen chrysopterum (Bloch & Schneider) , halfmoon triggerfish.Locality: Off Heron Island , Great Barrier Reef, Australia.Site in host: Intestine.Prevalence: In 1 of 67 fish examined.Voucher material: QM G212867.S. chrysopterum at Heron Island with only one infection detected. This species is clearly different from Lepotrema justinei n. sp., the other form found in Sufflamen. It is a smaller, much narrow worm with almost tandem testes and the ovary is distinctly separated from the ventral sucker.Only one specimen is available Table ; Fig. 33Lepotremasp. 4Lepotrema clavatum of Bray et al. , banded Parma.Locality: Off Heron Island , Great Barrier Reef, Australia.Site in host: Intestine.Prevalence: In 2 of 6 fish examined.Voucher material: QM GL 14773–4; BM(NH) 1992.10.5.6.P. polylepis examined from off Heron Island. This pomacentrid is not common at the sites we have surveyed on the GBR, and we have not detected this fish there since 1992.Three adult specimens were collected, measurements of one are given in Table Lepotremasp. 5Host: Ctenochaetus striatus (Quoy & Gaimard) (Perciformes: Acanthuridae), striated surgeonfish.Locality: Off Heron Island , Great Barrier Reef, Australia.Site in host: Intestine.Prevalence: In 1 of 42 fish examined.Voucher material: Hologenophore QM G237572.Representative DNA sequences: ITS2 rDNA, one sequence (submitted to GenBank MH730010); cox1 mtDNA, one sequence (submitted to GenBank MH730050); 28S rDNA, one sequence (submitted to GenBank MH730022).Lepotrema specimen was found in 119 specimens of this host, 42 of which were from off Heron Island. This specimen  Bray & Cribb, 1996Lepocreadium incisum Hanson, 1955Syn. Type-host: Melichthys niger (Bloch) (as buniva) , black triggerfish.Type-locality: Off Hawaii.RemarksMelichthys buniva, but according to Randall  Bray & Cribb, 1996Lepocreadium xanthichthydis Yamaguti, 1970Syn. Type-host: Xanthichthys ringens (Linnaeus) , sargassum triggerfish.Type-locality: Off Hawaii.Important differentiating characters include the forebody length, the anterior position of the bifurcation and the vitelline extent, the cirrus-sac not reaching into the hindbody, the uterine coil in the forebody and the egg length (46–56)  n. comb.Preptetos cylindricus Wang, 1989Syn. Type-host: Thamnaconus septentrionalis (Günther) (first host listed) (Tetraodontiformes: Monacanthidae), drab leatherjacket.Other host: Monacanthus chinensis (Osbeck) (Monacanthidae), fan-bellied leatherjacket.Type-locality: Off Pingtan County, Fujian, China.Preptetos, this species is consistent with Lepotrema and we formally propose the new combination here. It is much bigger than any other described species of Lepotrema  n. comb.Lepocreadium navodoni Shen, 1986Syn. Type-host: Thamnaconus modestus (Günther) (Tetraodontiformes: Monacanthidae), Korean black scraper.Type-locality: Off Nongbo, Zhejiang, China.Lepocreadium, this species is consistent with Lepotrema and we formally propose the new combination here. It appears to be of an unusual pyriform shape with the narrower part anterior and the cirrus-sac beside the ventral sucker  and its frequency among cox1 sequences (6 of 9 species for which at least two sequences were obtained). The topologies from the two analyses were substanially different  having identical sequences in the final dataset; they differ by a single indel in the partial 28S sequence alignment. Preliminary analysis that included all available lepocreadoid taxa showed that all Lepotrema species sequenced formed a single well-supported clade. Thus, due to the limited number of differences in the Lepotrema dataset, a reduced analysis relative to three closely related genera was conducted to limit the loss of informative characters. The phylogram produced by Bayesian inference analysis of this dataset is shown in Fig. Lepotrema as a genus was again well-supported, as was a clade containing all Lepotrema species to the exclusion of L. melichthydis n. sp. Lepotrema acanthochromidis n. sp., L. hemitaurichthydis n. sp. and L. moretonense n. sp. formed a stongly-supported clade, sister to a clade consisting of L. amblyglyphidodonis n. sp. and Lepotrema sp. 5. These five species formed a strongly-supported clade, sister to the poorly-supported clade of L. adlardi, L. amansis n. sp., L. cirripectis n. sp. and L. monile.Neighbour-joining phylograms produced for the ITS2 and Recognition of speciesL. adlardi  and L. amansis n. sp. . For almost all the other forms, distinction is not immediately obvious and is critically dependent on the capacity to examine multiple specimens and on corroborative indications from molecular data and host-specificity. In this respect, many of the species can be considered ‘cryptic’ in the broad sense of the term, if not in the strictest sense.We have taken an integrative approach to the recognition of species here. By this we mean that we have been influenced by evidence from morphology, genetics and host-specificity. Overall, we have found morphology to be inadequate for the convincing recognition of many species. Clear exceptions are Hemitaurichthys , all distinct genotypes in the ITS2 and clades in cox1 analyses were ultimately interpreted as relating to distinct species. The distinctions in ITS2 rDNA sequences were often small (as low as 1 bp), but the distinctions always correlated with morphological and host distributional distinctions. In addition, several of the combinations were in sympatry . Thus, four taxa  that all occur on the GBR, each differ from one another by only 2 bp in the ITS2 region. Differences in the 28S data for L. amansis n. sp., L. cirripectis n. sp. and L. monile were also low (0–2 bp); the two genotypes that were identical in the final dataset did differ by a single indel. However, these differences are entirely consistent with and supported by greater differences in the cox1 dataset (40–56 bp differences for the same four species). We thus have no hesitation in considering these small ITS2 and 28S differences as informative.With the exception of the forms associated with species of L. cirripectis n. sp., L. monile and L. moretonense n. sp.) (stenoxenous specificity). This pattern is consistent with the overall pattern of trematode specificity in coral reef fishes recognised by Miller et al. (Lepotrema clavatum as one of only four trematode species reported from multiple orders of GBR fishes. The doubt about the breadth of host-specificity of that species expressed in that paper has been supported here. The restriction of many Lepotrema species is quite remarkable given the context of the examination (reported for species of Lepotrema above) of often large numbers of closely-related fish species. Thus, four species are recognised in Pomacentridae; L. adlardi is found in only one species of Abudefduf, L. acanthochromidis n. sp. in only the single species of Acanthochromis, L. monile in only three species of Pomacentrus and Stegastes apicalis, and L. amblyglyphidodonis n. sp. only in single species each of Amblyglyphidodon and Amphiprion. This pattern of host-specificity leads to the suspicion that the form reported rarely from Parma polylepis may represent a further undescribed species. The pattern of oioxenous or stenoxenous host-specificity is not perfect. The clear example is that of L. moretonense n. sp. which occurs frequently in two species of Prionurus (Acanthuridae) and rarely  in Selenotoca multifasciata. Such rarities serve to emphasise the importance of finding multiple specimens and infected hosts to allow confidence that infections are not uninformative “stragglers”. The rarity of infections, such as the single specimens detected in one of 67 Sufflamen chrysopterus and one of 42 Ctenochaetus striatus at Heron Island, may well be an indication that the species concerned typically infect another fish species. However, the single specimen of Lepotrema from C. striatus is genetically unique, so it may be simply rare or localised in that host and the same may apply to the form from S. chrysopterum.The final component of our integrated analysis was the nature of the host-specificity in the system. In general, the species recognised here were found consistently in just one fish species (oioxenous specificity) or in multiple congeners , and the ITS2 and cox1 sequences differ by 1 bp (in addition to a single indel) and 14 bp, respectively. Importantly, the intraspecific cox1 variation is greater than that for any ‘good’ species whereas it is considerably less than the interspecific variation between any combination of recognised species. In the face of this somewhat conflicting information, we propose a conservative approach, interpreting the forms from H. polylepis as a single species that demonstrates geographical genetic variation. This approach (interpreting low-level genetic differences in worms from the same or very similar hosts over geographic range as intra-specific variation) has been adopted for several trematode taxa of late [Cryptogonimidae: Miller et al. , Acanthuridae (3%) and Pomacanthidae (1.7%) are also represented, and the Blenniidae, which occurs frequently on coral reefs, has 5% of records. Records in the Paralichthyidae (1.7%) and the Scatophagidae (1.7%) indicate that the genus is not solely reef-associated. Nevertheless, it is clearly mostly associated with fishes on coral reefs. Two orders predominate with the Tetraodontiformes (50% of records) and the Perciformes (with 48%). The single report from a pleuronectiform appears anomolous and may be accidental, although the worms are described as ovigerous, but relatively small  and Isopora sp. from the intestine of specimens of A. scopas collected from off Heron Island. It seems possible, therefore, that species of this genus may have a predilection for cnidarians, both polyps and medusae, as second intermediate hosts. Regardless of the range of second intermediate hosts for species of this genus, the apparent randomness of distribution among apparently comparable fishes remains baffling.This host distribution can best be considered in the context of the recent report that metacercariae of a et al.  demonstr of Arai  and Ates of Arai , just twHost-parasite listOrder PerciformesFamily AcanthuridaePrionurus maculatus OgilbyLepotrema moretonense n. sp.Prionurus microlepidotus LacépèdeLepotrema moretonense n. sp.Ctenochaetus striatus (Quoy & Gaimard)Lepotrema sp. 5Family BlenniidaeCirripectes filamentosus (Alleyne & Macleay)Lepotrema cirripectis n. sp.Cirripectes chelomatus Williams & MaugéLepotrema cirripectis n. sp.Cirripectes stigmaticus Strasburg & SchultzLepotrema cirripectis n. sp.Family ChaetodontidaeHemitaurichthys polylepis (Bleeker)Lepotrema hemitaurichthydis n. sp.Hemitaurichthys thompsoni FowlerLepotrema hemitaurichthydis n. sp.Hemitaurichthys zoster (Bennett)Lepotrema clavatum (s.l.)Family PomacanthidaeGenicanthus semifasciatus (Kamohara)Lepotrema clavatum (s.l.)Family PomacentridaeAbudefduf bengalensis (Bloch)Lepotrema adlardi Acanthochromis polyacanthus (Bleeker)Lepotrema acanthochromidis n. sp.Amblyglyphidodon curacao (Bloch)Lepotrema amblyglyphidodonis n. sp.Amphiprion akindynosLepotrema amblyglyphidodonis n. sp.Dascyllus albisella GillLepotrema clavatum (s.l.)Parma polylepis GüntherLepotrema sp. 4Pomacentrus amboinensis BleekerLepotrema monile Bray & Cribb, 1998Pomacentrus chrysurus CuvierLepotrema monile Bray & Cribb, 1998Pomacentrus wardi WhitleyLepotrema monile Bray & Cribb, 1998Stegastes apicalis (De Vis)Lepotrema monile Bray & Cribb, 1998Family ScatophagidaeSelenotoca multifasciata (Richardson)Lepotrema moretonense n. sp.Order PleuronectiformesFamily ParalichthyidaePseudorhombus cinnamoneus (Temminck & Schlegel)Lepotrema clavatum (s.l.)Order TetraodontiformesFamily BalistidaeMelichthys niger (Bloch)Lepotrema clavatum (s.l.)Lepotrema incisum Melichthys vidua (Richardson)Lepotrema clavatum (s.l.)Lepotrema melichthydis n. sp.Odonus niger (Rüppell)Lepotrema clavatum (s.l.)Rhinecanthus aculeatus (Linnaeus)Lepotrema clavatum (s.l.)Lepotrema sp. 1Lepotrema sp. 2Sufflamen chrysopterum (Bloch & Schneider)Lepotrema sp. 3Sufflamen fraenatum (Latreille)Lepotrema justinei n. sp.Xanthichthys ringens (Linnaeus)Lepotrema xanthichthydis Family MonacanthidaeAmanses scopas (Cuvier)Lepotrema amansis n. sp.Cantheschenia grandisquamis HutchinsLepotrema canthescheniae Bray & Cribb, 1996Monacanthus chinensis (Osbeck)Lepotrema cylindricum  n. comb.Stephanolepis cirrhifer (Temminck & Schlegel)Lepotrema clavatum (s.s.)Thamnaconus modestus (Günther)Lepotrema clavatum (s.l.)Lepotrema navodonis  n. comb.Thamnaconus septentrionalis (Günther)Lepotrema cylindricum  n. comb.