Human growth factor-mediated signalling through lipid rafts regulates stem cell proliferation, development and survival of Schistosoma mansoni.

Although the mechanisms by which schistosomes grow and develop in humans are poorly defined, their unique outer tegument layer, which interfaces with host blood, is considered vital to homeostasis of the parasite. Here, we investigated the importance of tegument lipid rafts to the biology of Schistosoma mansoni in the context of host-parasite interactions. We demonstrate the temporal clustering of lipid rafts in response to human epidermal growth factor (EGF) during early somule development, concomitant with the localization of anteriorly orientated EGF receptors (EGFRs) and insulin receptors, mapped using fluorescent EGF/insulin ligand. Methyl-ß-cyclodextrin (MßCD)-mediated depletion of cholesterol from lipid rafts abrogated the EGFR/IR binding at the parasite surface and led to modulation of protein kinase C, extracellular signal-regulated kinase, p38 mitogen-activated protein kinase and Akt signalling pathways within the parasite. Furthermore, MßCD-mediated lipid raft disruption, and blockade of EGFRs using canertinib, profoundly reduced somule motility and survival, and attenuated stem cell proliferation and somule growth and development particularly to the fast-growing liver stage. These findings provide a novel paradigm for schistosome development and vitality in the host, driven through host-parasite interactions at the tegument, that might be exploitable for developing innovative therapeutic approaches to combat human schistosomiasis.

Identification of Cotylophoron cotylophorum (Fischoeder, 1901) in cattle on St. Kitts, West Indies and its relationship with African and Asian populations.

There is limited information about the species of rumen fluke (Family Paramphistomidae) in the Caribbean. However, knowledge of species distribution is needed to better understand disease risk and epidemiology. Morphological identification is challenging with more recent DNA sequencing enabling a better understanding of rumen fluke distribution. In this study, rumen fluke specimens, collected between 2015 and 2016 from cattle on the island of St. Kitts, West Indies, were analysed. The ribosomal internal transcribed spacer 2 (ITS-2) region of rDNA was amplified using generic trematode primers. Results from Sanger sequencing were compared to reference sequences in GenBank and indicated the species was Cotylophoron cotylophorum with 100% sequence identity and 91% query cover. The ITS2 sequences were then compared to previously published ITS2 sequences for the Cotylophoron genus. When all the St. Kitts C. cotylophorum ITS2 sequences were compared with all other Cotylophoron sequences from India, Kenya, and Zimbabwe, three variable nucleotide sites, resulting in five unique haplotypes, were identified. Nine ITS2 sequences shared haplotype 1, which included all those from St. Kitts and single representatives from India and Kenya, potentially indicating global movement of this species.

In silico design of a polypeptide as a vaccine candidate against ascariasis.

Ascariasis is the most prevalent zoonotic helminthic disease worldwide, and is responsible for nutritional deficiencies, particularly hindering the physical and neurological development of children. The appearance of anthelmintic resistance in Ascaris is a risk for the target of eliminating ascariasis as a public health problem by 2030 set by the World Health Organisation. The development of a vaccine could be key to achieving this target. Here we have applied an in silico approach to design a multi-epitope polypeptide that contains T-cell and B-cell epitopes of reported novel potential vaccination targets, alongside epitopes from established vaccination candidates. An artificial toll-like receptor-4 (TLR4) adjuvant (RS09) was added to improve immunogenicity. The constructed peptide was found to be non-allergic, non-toxic, with adequate antigenic and physicochemical characteristics, such as solubility and potential expression in Escherichia coli. A tertiary structure of the polypeptide was used to predict the presence of discontinuous B-cell epitopes and to confirm the molecular binding stability with TLR2 and TLR4 molecules. Immune simulations predicted an increase in B-cell and T-cell immune response after injection. This polypeptide can now be validated experimentally and compared to other vaccine candidates to assess its possible impact in human health.

Evolution of tetraspanin antigens in the zoonotic Asian blood fluke Schistosoma japonicum.

BACKGROUND: Despite successful control efforts in China over the past 60 years, zoonotic schistosomiasis caused by Schistosoma japonicum remains a threat with transmission ongoing and the risk of localised resurgences prompting calls for a novel integrated control strategy, with an anti-schistosome vaccine as a core element. Anti-schistosome vaccine development and immunisation attempts in non-human mammalian host species, intended to interrupt transmission, and utilising various antigen targets, have yielded mixed success, with some studies highlighting variation in schistosome antigen coding genes (ACGs) as possible confounders of vaccine efficacy. Thus, robust selection of target ACGs, including assessment of their genetic diversity and antigenic variability, is paramount. Tetraspanins (TSPs), a family of tegument-surface antigens in schistosomes, interact directly with the host's immune system and are promising vaccine candidates. Here, for the first time to our knowledge, diversity in S. japonicum TSPs (SjTSPs) and the impact of diversifying selection and sequence variation on immunogenicity in these protiens were evaluated. METHODS: SjTSP sequences, representing parasite populations from seven provinces across China, were gathered by baiting published short-read NGS data and were analysed using in silico methods to measure sequence variation and selection pressures and predict the impact of selection on variation in antigen protein structure, function and antigenic propensity. RESULTS: Here, 27 SjTSPs were identified across three subfamilies, highlighting the diversity of TSPs in S. japonicum. Considerable variation was demonstrated for several SjTSPs between geographical regions/provinces, revealing that episodic, diversifying positive selection pressures promote amino acid variation/variability in the large extracellular loop (LEL) domain of certain SjTSPs. Accumulating polymorphisms in the LEL domain of SjTSP-2, -8 and -23 led to altered structural, functional and antibody binding characteristics, which are predicted to impact antibody recognition and possibly blunt the host's ability to respond to infection. Such changes, therefore, appear to represent a mechanism utilised by S. japonicum to evade the host's immune system. CONCLUSION: Whilst the genetic and antigenic geographic variability observed amongst certain SjTSPs could present challenges to vaccine development, here we demonstrate conservation amongst SjTSP-1, -13 and -14, revealing their likely improved utility as efficacious vaccine candidates. Importantly, our data highlight that robust evaluation of vaccine target variability in natural parasite populations should be a prerequisite for anti-schistosome vaccine development.

Molecular epidemiological analyses reveal extensive connectivity between Echinostoma revolutum (sensu stricto) populations across Eurasia and species richness of zoonotic echinostomatids in England.

Echinostoma revolutum (sensu stricto) is a widely distributed member of the Echinostomatidae, a cosmopolitan family of digenetic trematodes with complex life cycles involving a wide range of definitive hosts, particularly aquatic birds. Integrative taxonomic studies, notably those utilising nad1 barcoding, have been essential in discrimination of E. revolutum (s.s.) within the 'Echinostoma revolutum' species complex and investigation of its molecular diversity. No studies, however, have focussed on factors affecting population genetic structure and connectivity of E. revolutum (s.s.) in Eurasia. Here, we used morphology combined with nad1 and cox1 barcoding to determine the occurrence of E. revolutum (s.s.) and its lymnaeid hosts in England for the first time, in addition to other echinostomatid species Echinoparyphium aconiatum, Echinoparyphium recurvatum and Hypoderaeum conoideum. Analysis of genetic diversity in E. revolutum (s.s.) populations across Eurasia demonstrated haplotype sharing and gene flow, probably facilitated by migratory bird hosts. Neutrality and mismatch distribution analyses support possible recent demographic expansion of the Asian population of E. revolutum (s.s.) (nad1 sequences from Bangladesh and Thailand) and stability in European (nad1 sequences from this study, Iceland and continental Europe) and Eurasian (combined data sets from Europe and Asia) populations with evidence of sub-population structure and selection processes. This study provides new molecular evidence for a panmictic population of E. revolutum (s.s.) in Eurasia and phylogeographically expands the nad1 database for identification of echinostomatids.

CaMKII regulates neuromuscular activity and survival of the human blood fluke Schistosoma mansoni.

Calcium/calmodulin dependant protein kinase II (CaMKII), an important transducer of Ca2+ signals, orchestrates multiple cellular functions in animals. Here we investigated the importance of CaMKII to Schistosoma mansoni, a blood parasite that causes human schistosomiasis. We demonstrate that phosphorylated (activated) CaMKII is present in cercariae, schistosomula and adult worms, and show that striking activation occurs in the nervous tissue of these parasite life-stages; CaMKII was also activated in the tegument and muscles of adult worms and the vitellaria of females. Exposure of worms to the anti-schistosomal drug praziquantel (PZQ) induced significant CaMKII activation and depletion of CaMKII protein/activation in adult worms resulted in hypokinesia, reduced vitality and death. At medium confidence (global score ≥ 0.40), S. mansoni CaMKII was predicted to interact with 51 proteins, with many containing CaMKII phosphorylation sites and nine mapped to phosphoproteome data including sites within a ryanodine receptor. The CaMKII network was functionally enriched with mitogen-activated protein kinase, Wnt, and notch pathways, and ion-transport and voltage-dependent channel protein domains. Collectively, these data highlight the intricacies of CaMKII signalling in S. mansoni, show CaMKII to be an active player in the PZQ-mediated response of schistosomes and highlight CaMKII as a possible target for the development of novel anti-schistosome therapeutics.

A reverse vaccinology approach identifies putative vaccination targets in the zoonotic nematode Ascaris.

Ascariasis is the most prevalent helminthic disease affecting both humans and pigs and is caused by the roundworms Ascaris lumbricoides and Ascaris suum. While preventive chemotherapy continues to be the most common control method, recent reports of anthelminthic resistance highlight the need for development of a vaccine against ascariasis. The aim of this study was to use a reverse vaccinology approach to identify potential vaccine candidates for Ascaris. Three Ascaris proteomes predicted from whole-genome sequences were analyzed. Candidate proteins were identified using open-access bioinformatic tools (e.g., Vacceed, VaxiJen, Bepipred 2.0) which test for different characteristics such as sub-cellular location, T-cell and B-cell molecular binding, antigenicity, allergenicity and phylogenetic relationship with other nematode proteins. From over 100,000 protein sequences analyzed, four transmembrane proteins were predicted to be non-allergen antigens and potential vaccine candidates. The four proteins are a Piezo protein, two voltage-dependent calcium channels and a protocadherin-like protein, are all expressed in either the muscle or ovaries of both Ascaris species, and all contained high affinity epitopes for T-cells and B-cells. The use of a reverse vaccinology approach allowed the prediction of four new potential vaccination targets against ascariasis in humans and pigs. These targets can now be further tested in in vitro and in vivo assays to prove efficacy in both pigs and humans.

Mitophylogenomics of the zoonotic fluke Echinostoma malayanum confirms it as a member of the genus Artyfechinostomum Lane, 1915 and illustrates the complexity of Echinostomatidae systematics.

The complete mitochondrial genome (mitogenome or mtDNA) of the trematode Echinostoma malayanum Leiper, 1911 was fully determined and annotated. The circular mtDNA molecule comprised 12 protein-coding genes (PCGs) (cox1 - 3, cob, nad1 - 6, nad4L, atp6), two mitoribosomal RNAs (MRGs) (16S or rrnL and 12S or rrnS), and 22 transfer RNAs (tRNAs or trn), and a non-coding region (NCR) rich in long and short tandem repeats (5.5 LRUs/336 bp/each and 7.5 SRUs/207 bp/each). The atp8 gene is absent and the 3' end of nad4L overlaps the 5' end of nad4 by 40 bp. Special DHU-arm missing tRNAs for Serine were found for both tRNASer1(AGN) and tRNASer2(UCN). Codons of TTT (for phenylalanine), TTG (for leucine), and GTT (for valine) were the most, and CGC (for Arginine) was the least frequently used. A similar usage pattern was seen in base composition, AT and GC skewness for PCGs, MRGs, and mtDNA* (coding cox3 to nad5) in E. malayanum and Echinostomatidae. The nucleotide use is characterized by (T > G > A > C) for PCGs/mtDNA*, and by (T > G ≈ A > C) for MRGs. E. malayanum exhibited the lowest genetic distance (0.53%) to Artyfechinostomum sufrartyfex, relatively high to the Echinostoma congeners (13.20-13.99%), higher to Hypoderaeum conoideum (16.18%), and the highest to interfamilial Echinochasmidae (26.62%); Cyclocoelidae (30.24%); and Himasthlidae (25.36%). Topology indicated the monophyletic position between E. malayanum/A. sufrartyfex and the group of Echinostoma caproni, Echinostoma paraensei, Echinostoma miyagawai, and Echinostoma revolutum, rendering Hypoderaeum conoideum and unidentified Echinostoma species paraphyletic. The strictly closed genomic/taxonomic/phylogenetic features (including base composition, skewness, codon usage/bias, genetic distance, and topo-position) reinforced Echinostoma malayanum to retake its generic validity within the Artyfechinostomum genus.

Toll like receptors and their evolution in the lymnaeid freshwater snail species Radix auricularia and Lymnaea stagnalis, key intermediate hosts for zoonotic trematodes.

One of the major evolutionarily conserved pathways in innate immunity of invertebrates is the toll-like receptor (TLR) pathway. However, little is known of the TLR protein family in gastropod molluscs despite their role in the transmission of human diseases, especially the common lymnaeid freshwater snail species Radix auricularia and Lymnaea stagnalis, key intermediate hosts of zoonotic trematodes. Using comparative genomics and gene prediction approaches utilising the freshwater snail Biomphalaria glabrata genome as a reference ten putative TLR proteins were identified in both R. auricularia and L. stagnalis. Phylogenetic analyses revealed that unlike other molluscs the lymnaeid species also possessed class 1 TLRs, previously thought to be unique to B. glabrata. Gene duplication events were also seen across the TLR classes in the lymnaeids with several of the genes appearing to exist as potential tandem elements in R. auricularia. Each predicted TLR was shown to possess the typical the leucine-rich repeat extracellular and TIR intracellular domains and both single cysteine clusters and multiple cysteine clusters TLRs were identified in both lymnaeid species. Principle component analyses of 3D models of the predicted TLRs showed that class 1 and 5 proteins did not cluster based on similarity of structure, suggested to be potential adaptation to a range of pathogens. This study provides the first detailed account of TLRs in lymnaeids and affords a platform for further research into the role of these proteins into susceptibility and compatibility of these snails with trematodes and their role in transmission.

Further evaluation and validation of HybridoMed Diff 1000 and its comparison to Basch medium for the cell-free culture of Schistosoma mansoni juvenile worm stages.

Schistosomules of the human parasite Schistosoma mansoni are vital for research focusing on the fundamental functional/developmental biology of schistosomes and many anti-schistosomal drug discovery programmes. Through the further evaluation and validation of a recently tested media, HybridoMed Diff 1000 (HM), for the cell-free culture of juvenile schistosomules, we show that while Basch medium was superior to HM for the survival/development of schistosomules, HM represents a viable and attractive alternative for somule culture, particularly to the early liver stage. Adoption of HM for schistosomule culture could facilitate more standardised approaches, which for drug screening should enable improved multi-centre target-hit evaluation.

DNA barcoding of Iranian radicine freshwater snails begins to untangle the taxonomy and phylogeography of intermediate hosts of schistosomiasis and fasciolosis from the Middle East and across Central Asia.

In the Middle East radicine snails are of considerable medical and veterinary importance acting as vectors of trematodes. In Iran, such snails are responsible for the transmission of the zoonotic trematodes Schistosoma turkestanicum and Fasciola gigantica. Historically, Radix gedrosiana has been incriminated as an important intermediate host for both trematodes, however, controversy remains over the snail's true taxonomic status. This species has been determined using morphological characters that has resulted in erroneous identification of species, affecting understanding of population biology, and ultimately affecting vector incrimination. In this current study DNA barcoding using cox1 and phylogenetic analyses revealed that snails identified as R. gedrosiana from Iran split into two separate species, Radix euphratica and Ampullaceana sp. The cox1 also provided useful insights into the evolutionary history of R. euphratica populations. Phylogeographic analyses indicated that R. euphratica had an Iraqi/Iranian origin approximately 3.3 MYA and exists as a large stable population across the Middle East and Central Asia, and a lack of genetic differentiation between geographical isolates. Such molecular barcoding techniques are crucial for the identification of radicine snails of Iran being invaluable for the monitoring of zoonotic flukes, understanding the distribution of infection and the accurate incrimination of snail vectors.

Molecular evidence of hybridization between pig and human Ascaris indicates an interbred species complex infecting humans.

Human ascariasis is a major neglected tropical disease caused by the nematode Ascaris lumbricoides. We report a 296 megabase (Mb) reference-quality genome comprised of 17,902 protein-coding genes derived from a single, representative Ascaris worm. An additional 68 worms were collected from 60 human hosts in Kenyan villages where pig husbandry is rare. Notably, the majority of these worms (63/68) possessed mitochondrial genomes that clustered closer to the pig parasite Ascaris suum than to A. lumbricoides. Comparative phylogenomic analyses identified over 11 million nuclear-encoded SNPs but just two distinct genetic types that had recombined across the genomes analyzed. The nuclear genomes had extensive heterozygosity, and all samples existed as genetic mosaics with either A. suum-like or A. lumbricoides-like inheritance patterns supporting a highly interbred Ascaris species genetic complex. As no barriers appear to exist for anthroponotic transmission of these 'hybrid' worms, a one-health approach to control the spread of human ascariasis will be necessary.

Characterization of the complete mitochondrial genome of Diplostomum baeri.

Despite Diplostomum baeri (Dubois, 1937) being one of the most widely distributed parasites of freshwater fish, there is no complete mitochondrial (mt) genome currently available. The complicated systematics presented by D. baeri has hampered investigations into the species distributions and infective dynamics of the species. Within this study we obtained complete mt genome sequences of D. baeri and assessed its phylogenetic relationship with other species of Digenea. The complete mitochondrial genome of D. baeri is 14,480 bp in length, containing 36 genes in total. The phylogenetic tree resulting from Bayesian inference of concatenated 12 protein coding gene sequences placed D. baeri alongside published mt genomes of Diplostomidae, with the overall taxonomic placement of the genus being a sister lineage of the order Plagiochiida The characterization of further mitochondrial genomes within the family Diplostomidae will help progress phylogenetic and epidemiological investigations as well as providing a framework for the analysis of diagnostic markers to be used in further monitoring of the parasite worldwide.

Morphological and molecular characterization of an African freshwater fish trypanosome, including its development in a leech vector.

Trypanosomes are ubiquitous blood parasites of fishes and at least 16 species were originally described infecting African freshwater fishes. This number was later reduced to six and in the late 1990s it was proposed that most records of freshwater fish trypanosomes across Africa are Trypanosoma mukasai Hoare, 1932. Recently, results from a molecular analysis of fish trypanosomes from the Okavango Delta, Botswana, reported the presence of at least two genotypic groups and concluded that the identification of T. mukasai remains problematic. The aims of the present study were thus to elucidate the life cycle of a freshwater fish trypanosome from southern Africa and to do a morphological and molecular characterization of this parasite from both the fish host and leech vector. To locate trypanosome stages, leeches were removed from fishes captured in the Phongolo River, South Africa, and fish blood films and leech squashes were Giemsa-stained and screened. To determine whether trypanosome stages in fishes and leeches were of the same genotype, DNA was extracted and fragments of the 18S rDNA gene were amplified and sequenced. Trypanosomes were detected in the fish families Cichlidae, Clariidae, Mochokidae and Schilbeidae. Sequence data showed that the trypanosome from one of the leeches, identified as Batracobdelloides tricarinata (Blanchard, 1897), was highly similar to those obtained from the plain squeaker, Synodontis zambezensis, with 0.7% difference recorded between them. From morphological and molecular data presented here, it is clear that the trypanosomes from Phongolo are closely related to those of the Okavango and should be considered as a single diverse species with genetic differentiation between 0.4-2.9%, under the 3-5% differences expected to be seen between true distinct species within the rRNA. Developmental stages of the trypanosome found in the leech B. tricarinata supports its status as the vector and the molecular evidence shows the relationship between the trypanosome in the fish and leech, but also illustrates the exceptional genetic and morphological diversity of a single species of trypanosome between host species. The work presented here provides us with clear information to take further steps in resolving the taxonomy and systematics of African freshwater fish trypanosomes.

Characterization and phylogenetic properties of the complete mitochondrial genome of Fascioloides jacksoni (syn. Fasciola jacksoni) support the suggested intergeneric change from Fasciola to Fascioloides (Platyhelminthes: Trematoda: Plagiorchiida).

Fascioloides jacksoni (syn. Fasciola jacksoni, Cobbold, 1869) (Platyhelminthes: Echinostomatoidea), is a liver fluke that causes severe morbidity and mortality of Asian elephants (Elephas maximus maximus). Understandings on molecular diagnosis, epidemiology, genetics and evolution of this flatworm are limited. In this study, we present the complete mitochondrial DNA (mt) sequence of 14,952 bp obtained from an individual fluke and comparative characterization of mitogenomic features with fasciolids, primarily, Fascioloides magna and other taxa in the superfamily Echinostomatoidea. Taxonomic relationship within and between Echinostomatoidea, Opisthorchioidea and Paramphistomoidea in the order Plagiorchiida, are also taxonomically considered. The complete circular mt molecule of Fas. jacksoni contained 12 protein-coding, two ribosomal RNA, 22 transfer RNA genes, and a non-coding region (NCR) rich in tandem repeat units. As common in digenean trematodes, Fas. jacksoni has the usual gene order, the absence of atp8 and the overlapped region by 40 bp between nad4L and nad4 genes. The NCR located between tRNAGlu (trnE) and cox3 contained nine nearly identical tandem repeat units (TRs of 113 bp each). Special DHU-arm missing tRNAs for Serine were found for both, tRNAS1(AGN) and tRNAS2(UCN). Base composition indicated that cox1 of Fas. jacksoni showed the lowest (11.8% to Fas. magna, 12.9 - 13.6% to Fasciola spp. and 18.1% to Fasciolopsis buski) and nad6 the highest divergence rate (19.2%, 23.8-26.5% and 27.2% to each fasciolid group), respectively. A clear bias in nucleotide composition, as of 61.68%, 62.88% and 61.54%, with a negative AT-skew of the corresponding values (-0.523, -0.225 and - 0.426) for PCGs, MRGs and mtDNA for Fas. jacksoni and likewise data for the fasciolids. Phylogenetic analysis confirmed the sister branch of Fas. jacksoni and Fas. magna with the nodal support of 100%, clearly separated from the taxonomically recognized Fasciola spp. With the previous studies, mitogenomic data presented in this study are strongly supportive for Fasciola jacksoni reappraisal as Fascioloides jacksoni in the Fascioloides genus.

Deep phosphoproteome analysis of Schistosoma mansoni leads development of a kinomic array that highlights sex-biased differences in adult worm protein phosphorylation.

Although helminth parasites cause enormous suffering worldwide we know little of how protein phosphorylation, one of the most important post-translational modifications used for molecular signalling, regulates their homeostasis and function. This is particularly the case for schistosomes. Herein, we report a deep phosphoproteome exploration of adult Schistosoma mansoni, providing one of the richest phosphoprotein resources for any parasite so far, and employ the data to build the first parasite-specific kinomic array. Complementary phosphopeptide enrichment strategies were used to detect 15,844 unique phosphopeptides mapping to 3,176 proteins. The phosphoproteins were predicted to be involved in a wide range of biological processes and phosphoprotein interactome analysis revealed 55 highly interconnected clusters including those enriched with ribosome, proteasome, phagosome, spliceosome, glycolysis, and signalling proteins. 93 distinct phosphorylation motifs were identified, with 67 providing a 'footprint' of protein kinase activity; CaMKII, PKA and CK1/2 were highly represented supporting their central importance to schistosome function. Within the kinome, 808 phosphorylation sites were matched to 136 protein kinases, and 68 sites within 37 activation loops were discovered. Analysis of putative protein kinase-phosphoprotein interactions revealed canonical networks but also novel interactions between signalling partners. Kinomic array analysis of male and female adult worm extracts revealed high phosphorylation of transformation:transcription domain associated protein by both sexes, and CDK and AMPK peptides by females. Moreover, eight peptides including protein phosphatase 2C gamma, Akt, Rho2 GTPase, SmTK4, and the insulin receptor were more highly phosphorylated by female extracts, highlighting their possible importance to female worm function. We envision that these findings, tools and methodology will help drive new research into the functional biology of schistosomes and other helminth parasites, and support efforts to develop new therapeutics for their control.

Divergence across mitochondrial genomes of sympatric members of the Schistosoma indicum group and clues into the evolution of Schistosoma spindale.

Schistosoma spindale and Schistosoma indicum are ruminant-infecting trematodes of the Schistosoma indicum group that are widespread across Southeast Asia. Though neglected, these parasites can cause major pathology and mortality to livestock leading to significant welfare and socio-economic issues, predominantly amongst poor subsistence farmers and their families. Here we used mitogenomic analysis to determine the relationships between these two sympatric species of schistosome and to characterise S. spindale diversity in order to identify possible cryptic speciation. The mitochondrial genomes of S. spindale and S. indicum were assembled and genetic analyses revealed high levels of diversity within the S. indicum group. Evidence of functional changes in mitochondrial genes indicated adaptation to environmental change associated with speciation events in S. spindale around 2.5 million years ago. We discuss our results in terms of their theoretical and applied implications.

Comparative mitogenomics of the zoonotic parasite Echinostoma revolutum resolves taxonomic relationships within the 'E. revolutum' species group and the Echinostomata (Platyhelminthes: Digenea).

The complete mitochondrial sequence of 17,030 bp was obtained from Echinostoma revolutum and characterized with those of previously reported members of the superfamily Echinostomatoidea, i.e. six echinostomatids, one echinochasmid, five fasciolids, one himasthlid, and two cyclocoelids. Relationship within suborders and between superfamilies, such as Echinostomata, Pronocephalata, Troglotremata, Opisthorchiata, and Xiphiditata, are also considered. It contained 12 protein-coding, two ribosomal RNA, 22 transfer RNA genes and a tandem repetitive consisting non-coding region (NCR). The gene order, one way-positive transcription, the absence of atp8 and the overlapped region by 40 bp between nad4L and nad4 genes were similar as in common trematodes. The NCR located between tRNAGlu (trnE) and cox3 contained 11 long (LRUs) and short repeat units (SRUs) (seven LRUs of 317 bp, four SRUs of 207 bp each), and an internal spacer sequence between LRU7 and SRU4 specifying high-level polymorphism. Special DHU-arm missing tRNAs for Serine were found for both tRNAS1(AGN) and tRNAS2(UCN). Echinostoma revolutum indicated the lowest divergence rate to E. miyagawai and the highest to Tracheophilus cymbius and Echinochasmus japonicus. The usage of ATG/GTG start and TAG/TAA stop codons, the AT composition bias, the negative AT-skewness, and the most for Phe/Leu/Val and the least for Arg/Asn/Asp codons were noted. Topology indicated the monophyletic position of E. revolutum to E. miyagawai. Monophyly of Echinostomatidae and Fasciolidae was clearly solved with respect to Echinochasmidae, Himasthlidae, and Cyclocoelidae which were rendered paraphyletic in the suborder Echinostomata.

Molecular evidence for resurrection of Plesiochorus elongatus (Digenea: Gorgoderidae): An urinary bladder parasite of sea turtles.

Trematodes of the genus Plesiochorus were recovered from the urinary bladder of a stranded female adult loggerhead sea turtle, Caretta caretta, on a beach in Rio de Janeiro State, Brazil. Morphological analysis of the specimens revealed characteristics resembling the sub-species Plesiochorus cymbiformis elongatus rather than the recently synonymised Plesiochorus cymbiformis. Molecular phylogenetic analysis of the ITS2 region also showed that P. c. elongatus was distinct from P. cymbiformis and related taxa. Further analysis of the ITS2 revealed substantial differentiation between P. cymbiformis from the USA and Brazil and the newly sequenced P. c. elongatus from Brazil, while a previously unspecified Plesiochorus sp. from the USA closely related to the novel Brazilian P. c. elongatus was reconciled as a USA isolate of P. c. elongatus. Based on both the morphological and molecular data it is suggested that P. c. elongatus should be referred to as Plesiochorus elongatus and be considered as the second species in the genus.