Phylogenetic position of Ancyrocephalus (sensu lato) curtus Achmerov, 1952 (Monopisthocotylea, Dactylogyridae), a parasite of fish Perccottus glenii Dybowski, 1877(Gobiiformes: Odontobutidae).

The genus Ancyrocephalus sensu lato is a large assemblage of species of dactylogyrid monopisthocotyleans without clear taxonomic boundaries. Despite an urgent need for revision, only three representatives of this taxon have been molecularly characterised so far. We found specimens of Ancyrocephalus curtus, a previously non-genotyped species, in gills of Perccottus glenii caught in the River Syumnyur, Amur Basin, Russia. The aim of this study was to assess the phylogenetic position of this parasite using partial sequences of 28S rRNA gene. In the phylogenetic tree, A. curtus appeared as a sister taxon to the dactylogyrine genus Gobioecetes. The new molecular evidence supports the hypothesis about the non-monophyletic status of Ancyrocephalus sensu lato.

Unraveling the Unusual Subgenomic Organization in the Neopolyploid Free-Living Flatworm Macrostomum lignano.

Whole genome duplication (WGD) is an evolutionary event resulting in a redundancy of genetic material. Different mechanisms of WGD, allo- or autopolyploidization, lead to distinct evolutionary trajectories of newly formed polyploids. Genome studies on such species are important for understanding the early stages of genome evolution. However, assembling neopolyploid is a challenging task due to the presence of 2 homologous (or homeologous) chromosome sets and therefore the existence of the extended paralogous regions in its genome. Post-WGD evolution of polyploids includes cytogenetic diploidization leading to the formation of species, whose polyploid origin might be hidden by disomic inheritance. Earlier we uncovered the hidden polyploid origin of the free-living flatworms of the genus Macrostomum (Macrostomum lignano, M. janickei, and M. mirumnovem). Cytogenetic diploidization in these species is accompanied by intensive chromosomal rearrangements including chromosomes fusions. In this study, we unravel the M. lignano genome organization through generation and sequencing of 2 sublines of the commonly used inbred line of M. lignano (called DV1) differing only in a copy number of the largest chromosome (MLI1). Using nontrivial assembly free comparative analysis of their genomes, we deciphered DNA sequences belonging to MLI1 and validated them by sequencing the pool of microdissected MLI1. Here we presented the uncommon mechanism of genome rediplodization of M. lignano, which consists of (i) presence of 3 subgenomes, which emerged via formation of large fused chromosomes and its variants, and (ii) sustaining their heterozygosity through inter- and intrachromosomal rearrangements.

Genome assemblies of the simultaneously hermaphroditic flatworms Macrostomum cliftonense and Macrostomum hystrix.

The free-living, simultaneously hermaphroditic flatworms of the genus Macrostomum are increasingly used as model systems in various contexts. In particular, Macrostomum lignano, the only species of this group with a published genome assembly, has emerged as a model for the study of regeneration, reproduction, and stem-cell function. However, challenges have emerged due to M. lignano being a hidden polyploid, having recently undergone whole-genome duplication and chromosome fusion events. This complex genome architecture presents a significant roadblock to the application of many modern genetic tools. Hence, additional genomic resources for this genus are needed. Here, we present such resources for Macrostomum cliftonense and Macrostomum hystrix, which represent the contrasting mating behaviors of reciprocal copulation and hypodermic insemination found in the genus. We use a combination of PacBio long-read sequencing and Illumina shot-gun sequencing, along with several RNA-Seq data sets, to assemble and annotate highly contiguous genomes for both species. The assemblies span ∼227 and ∼220 Mb and are represented by 399 and 42 contigs for M. cliftonense and M. hystrix, respectively. Furthermore, high BUSCO completeness (∼84-85%), low BUSCO duplication rates (8.3-6.2%), and low k-mer multiplicity indicate that these assemblies do not suffer from the same assembly ambiguities of the M. lignano genome assembly, which can be attributed to the complex karyology of this species. We also show that these resources, in combination with the prior resources from M. lignano, offer an excellent foundation for comparative genomic research in this group of organisms.

Monogeneans and chubs: Ancient host-parasite system under the looking glass.

Host-parasite coevolution is one of the fundamentals of evolutionary biology. Due to the intertwined evolutionary history of two interacting species and reciprocal coadaptation processes of hosts and parasites, we can expect that studying parasites will shed more light onto the evolutionary processes of their hosts. Monogenea (ectoparasitic Platyhelminthes) and their cyprinoid fish hosts represent one of the best models for studying host-parasite evolutionary relationships using a cophylogenetic approach. These parasites have developed remarkably high host specificity, where each host species often serves as a potential host for its own host-specific monogenean species. Here, the cophylogenetic relationships in the Dactylogyrus-Squalius system was investigated, as Squalius is one of several cyprinoid genera with puzzling phylogeography and inhabits all four major peri-Mediterranean peninsulas. Of 29 endemic Squalius species examined for the presence of Dactylogyrus parasites, a total of 13 Dactylogyrus species were collected from the gills of 20 Squalius species across a wide range of distribution. Phylogenetic reconstruction revealed a polyphyletic origin for Dactylogyrus species parasitizing congeneric Squalius, with four major clades being recognized. On the basis of the delimitation of host specificity, strict specialists parasitizing single host species, geographic specialists parasitizing congeners in a limited geographical region, and true generalists parasitizing congeners in various geographical regions were recognized in Dactylogyrus species parasitizing Squalius. The phylogenetic reconstruction of Squalius hosts revealed two major clades, the first encompassing only peri-Mediterranean species and the second including species from other Euro-Asian regions. Distance-based cophylogenetic methods did not reveal a statistically significant global cophylogenetic structure in the studied system; however, several host-parasite links among Iberian endemic species contributed significantly to the overall structure. The widest host range and associated genetic variability were recorded for D. folkmanovae, parasitizing nine Squalius species, and D. vistulae, parasitizing 13 Squalius species. Two different dispersion mechanisms and morphological adaptations to Squalius hosts were clearly reflected in the contrasting cophylogenetic patterns for these two species with different levels of host specificity. While host-parasite cospeciation plays an important role in diversification within D. folkmanovae, diversification within D. vistulae is driven mainly by host switching.

Revision of the systematics of the Polystomoidinae (Platyhelminthes, Monogenea, Polystomatidae) with redefinition of Polystomoides Ward, 1917 and Uteropolystomoides Tinsley, 2017.

Polystomatids are platyhelminth parasites that infect mainly amphibians and freshwater turtles. For more than seven decades, chelonian polystomes were classified into three genera according to the number of hamuli, i.e. absent for Neopolystoma, one pair for Polystomoidella and two pairs for Polystomoides. Following re-examination of morphological characters, seven new genera were erected the past six years, namely Apaloneotrema, Aussietrema, Fornixtrema, Manotrema, Pleurodirotrema, Uropolystomoides and Uteropolystomoides. However, the polyphyly of Neopolystoma and Polystomoides on the one hand, and the nested position of Uteropolystomoides within a clade encompassing all Neopolystoma and Polystomoides spp. on the other, still raised questions about the validity of these genera. We therefore re-examined several types, paratypes and voucher specimens, and investigated the molecular phylogeny of polystomes sampled from the oral cavity of North American turtles to re-evaluate their systematic status. We show that all Polystomoides Ward, 1917, sensu Du Preez et al., 2022, Neopolystoma Price, 1939, sensu Du Preez et al., 2022 and Uteropolystomoides Tinsley, 2017 species, display vaginae that are peripheral and extend well beyond the intestine. We thus reassign all species of the clade to Polystomoides and propose nine new combinations; however, although Uteropolystomoides is nested within this clade, based on its unique morphological features, we propose to keep it as a valid taxon. Polystomoides as redefined herein groups all polystome species infecting either the oral cavity or the urinary bladder of cryptodires, with peripheral vaginae and with or without two pairs of small hamuli. Uteropolystomoides nelsoni (Du Preez & Van Rooyen 2015), originally described from Pseudemys nelsoni Carr is now regarded as Uteropolystomoides multifalx (Stunkard, 1924) n. comb. infecting three distinct Pseudemys species of North America.

Title: Révision de la systématique des Polystomoidinae (Plathelminthes, Monogenea, Polystomatidae) avec redéfinition des genres Polystomoides Ward, 1917 et Uteropolystomoides Tinsley, 2017. Abstract: Les Polystomatidés sont des plathelminthes parasites qui infestent principalement les amphibiens et les tortues d'eau douce. Pendant plus de sept décennies, les polystomes de chéloniens ont été classés en trois genres selon le nombre d'hamuli, absents pour Neopolystoma, une paire pour Polystomoidella et deux paires pour Polystomoides. Suite au réexamen des caractères morphologiques, sept nouveaux genres ont été érigés ces six dernières années, à savoir Apaloneotrema, Aussietrema, Fornixtrema, Manotrema, Pleurodirotrema, Uropolystomoides et Uteropolystomoides. Cependant, la polyphylie de Neopolystoma et Polystomoides d'une part, et la position imbriquée d'Uteropolystomoides au sein d'un clade englobant toutes les espèces de Neopolystoma et Polystomoides d'autre part, soulèvent encore des questions sur la validité de ces trois genres. Nous avons donc réexaminé plusieurs types, paratypes et vouchers et étudié la phylogénie moléculaire de polystomes prélevés dans la cavité buccale de tortues d'Amérique du Nord pour réévaluer leur statut systématique. Nous montrons que toutes les espèces de Polystomoides Ward, 1917, sensu Du Preez et al., 2022, Neopolystoma Price, 1939, sensu Du Preez et al., 2022 et Uteropolystomoides Tinsley, 2017, présentent des vagins périphériques qui s'étendent bien au-delà de l'intestin. Nous réattribuons ainsi toutes les espèces du clade à Polystomoides et proposons neuf nouvelles combinaisons; cependant, nous proposons de conserver Uteropolystomoides sur la base de ses caractéristiques morphologiques exceptionnelles, bien que son espèce soit imbriquée au sein de ce clade. Polystomoides tel que redéfini ici regroupe toutes les espèces de polystomes infectant soit la cavité buccale, soit la vessie des cryptodires, avec des vagins périphériques, et deux paires de petits hamuli ou sans hamuli. Uteropolystomoides nelsoni (Du Preez & Van Rooyen 2015), l'unique espèce décrite à l'origine à partir de Pseudemys nelsoni Carr est maintenant considérée comme Uteropolystomoides multifalx (Stunkard, 1924) n. comb., qui infecte trois espèces distinctes de Pseudemys d'Amérique du Nord.

First Record of a Polystome from Alligator Snapping Turtle, Macrochelys temminckii (Cryptodira: Chelydridae) or Mississippi; with Comments on "Neopolystoma orbiculare (Stunkard, 1916)" and Its Junior Subjective Synonyms.

Herein, we describe several newly-collected specimens of Neopolystoma cf. orbiculare from the urinary bladder of 2 alligator snapping turtles, Macrochelys temminckii (Troost in Harland, 1835) (Cryptodira: Chelydridae Gray, 1831) from Comet Lake (30°35'46.94″N, 88°36'3.12″W), Pascagoula River, Mississippi. Our specimens differed from all previous descriptions of N. orbiculare and its junior subjective synonyms by the combination of having intestinal ceca adorned with triangular pockets and that terminate dorsal to the haptor, distinctive hooklets each having a handle and guard of approximately equal length and having a much longer and curved blade, 16 genital coronet spines that each possess 1-2 flanges per spine, pre-testicular vaginal pores, and vaginal ducts that are anterior to the junction of the oviduct and genito-intestinal canal. Some of our specimens were enantiomorphic (4 and 3 had a dextral and sinistral ovary, respectively). Nucleotide sequences (large subunit ribosomal DNA [28S], small subunit ribosomal DNA [18S], and cytochrome oxidase subunit 1 mitochondrial gene [COI]) for our specimens were most similar to GenBank sequences ascribed to N. orbiculare. Single-gene and concatenated phylogenetic analyses confirmed that NeopolystomaPrice, 1939 is polyphyletic and that our isolates share a recent common ancestor with those ascribed to N. orbiculare. This is the first record of a polystomatid from Mississippi, from the Pascagoula River, and from the alligator snapping turtle (and only the second species of Neopolystoma reported from any snapping turtle).

Developmental scRNAseq Trajectories in Gene- and Cell-State Space-The Flatworm Example.

Single-cell RNA sequencing has become a standard technique to characterize tissue development. Hereby, cross-sectional snapshots of the diversity of cell transcriptomes were transformed into (pseudo-) longitudinal trajectories of cell differentiation using computational methods, which are based on similarity measures distinguishing cell phenotypes. Cell development is driven by alterations of transcriptional programs e.g., by differentiation from stem cells into various tissues or by adapting to micro-environmental requirements. We here complement developmental trajectories in cell-state space by trajectories in gene-state space to more clearly address this latter aspect. Such trajectories can be generated using self-organizing maps machine learning. The method transforms multidimensional gene expression patterns into two dimensional data landscapes, which resemble the metaphoric Waddington epigenetic landscape. Trajectories in this landscape visualize transcriptional programs passed by cells along their developmental paths from stem cells to differentiated tissues. In addition, we generated developmental "vector fields" using RNA-velocities to forecast changes of RNA abundance in the expression landscapes. We applied the method to tissue development of planarian as an illustrative example. Gene-state space trajectories complement our data portrayal approach by (pseudo-)temporal information about changing transcriptional programs of the cells. Future applications can be seen in the fields of tissue and cell differentiation, ageing and tumor progression and also, using other data types such as genome, methylome, and also clinical and epidemiological phenotype data.

The evolution of TNF signaling in platyhelminths suggests the cooptation of TNF receptor in the host-parasite interplay.

BACKGROUND: The TNF signaling pathway is involved in the regulation of many cellular processes (such as apoptosis and cell proliferation). Previous reports indicated the effect of human TNF-α on metabolism, physiology, gene expression and protein phosphorylation of the human parasite Schistosoma mansoni and suggested that its TNF receptor was responsible for this response. The lack of an endogenous TNF ligand reinforced the idea of the use of an exogenous ligand, but also opens the possibility that the receptor actually binds a non-canonical ligand, as observed for NGFRs. METHODS: To obtain a more comprehensive view, we analyzed platyhelminth genomes deposited in the Wormbase ParaSite database to investigate the presence of TNF receptors and their respective ligands. Using different bioinformatics approaches, such as HMMer and BLAST search tools we identified and characterized the sequence of TNF receptors and ligand homologs. We also used bioinformatics resources for the identification of conserved protein domains and Bayesian inference for phylogenetic analysis. RESULTS: Our analyses indicate the presence of 31 TNF receptors in 30 platyhelminth species. All platyhelminths display a single TNF receptor, and all are structurally remarkably similar to NGFR. It suggests no events of duplication and diversification occurred in this phylum, with the exception of a single species-specific duplication. Interestingly, we also identified TNF ligand homologs in five species of free-living platyhelminths. CONCLUSIONS: These results suggest that the TNF receptor from platyhelminths may be able to bind canonical TNF ligands, thus strengthening the idea that these receptors are able to bind human TNF-α. This also raises the hypothesis that an endogenous ligand was substituted by the host ligand in parasitic platyhelminths. Moreover, our analysis indicates that death domains (DD) may be present in the intracellular region of most platyhelminth TNF receptors, thus pointing to a previously unreported apoptotic action of such receptors in platyhelminths. Our data highlight the idea that host-parasite crosstalk using the TNF pathway may be widespread in parasitic platyhelminths to mediate apoptotic responses. This opens up a new hypothesis to uncover what might be an important component to understand platyhelminth infections.

Alternative splicing of coq-2 controls the levels of rhodoquinone in animals.

Parasitic helminths use two benzoquinones as electron carriers in the electron transport chain. In normoxia, they use ubiquinone (UQ), but in anaerobic conditions inside the host, they require rhodoquinone (RQ) and greatly increase RQ levels. We previously showed the switch from UQ to RQ synthesis is driven by a change of substrates by the polyprenyltransferase COQ-2 (Del Borrello et al., 2019; Roberts Buceta et al., 2019); however, the mechanism of substrate selection is not known. Here, we show helminths synthesize two coq-2 splice forms, coq-2a and coq-2e, and the coq-2e-specific exon is only found in species that synthesize RQ. We show that in Caenorhabditis elegans COQ-2e is required for efficient RQ synthesis and survival in cyanide. Importantly, parasites switch from COQ-2a to COQ-2e as they transit into anaerobic environments. We conclude helminths switch from UQ to RQ synthesis principally via changes in the alternative splicing of coq-2.

Copper and cadmium administration induce toxicity and oxidative stress in the marine flatworm Macrostomum lignano.

The contamination of coastal regions with different toxicants, including heavy metal ions such as copper and cadmium jeopardize health and survival of organisms exposed to this habitat. To study the effects of high copper and cadmium concentrations in these marine environments, we used the flatworm Macrostomum lignano as a model. This platyhelminth lives in shallow coastal water and is exposed to high concentrations of all toxicants that accumulate in these sea floors. We could show that both, cadmium and copper show toxicity at higher concentrations, with copper being more toxic than cadmium. At concentrations below acute toxicity, a reduced long-term survival was observed for both metal ions. The effects of sublethal doses comprise reduced physical activities, an increase in ROS levels within the worms, and alterations of the mitochondrial biology. Moreover, cell death events were substantially increased in response to sublethal concentrations of both metal ions and stem cell activity was reduced following exposure to higher cadmium concentrations. Finally, the expression of several genes involved in xenobiotic metabolism was substantially altered by this intervention. Taken together, M. lignano has been identified as a suitable model for marine toxicological studies as it allows to quantify several relevant life-history traits as well as of physiological and behavioral read-outs.

Genome and Karyotype Reorganization after Whole Genome Duplication in Free-Living Flatworms of the Genus Macrostomum.

The genus Macrostomum represents a diverse group of rhabditophoran flatworms with >200 species occurring around the world. Earlier we uncovered karyotype instability linked to hidden polyploidy in both M. lignano (2n = 8) and its sibling species M. janickei (2n = 10), prompting interest in the karyotype organization of close relatives. In this study, we investigated chromosome organization in two recently described and closely related Macrostomum species, M. mirumnovem and M. cliftonensis, and explored karyotype instability in laboratory lines and cultures of M. lignano (DV1/10, 2n = 10) and M. janickei in more detail. We revealed that three of the four studied species are characterized by karyotype instability, while M. cliftonensis showed a stable 2n = 6 karyotype. Next, we performed comparative cytogenetics of these species using fluorescent in situ hybridization (FISH) with a set of DNA probes (including microdissected DNA probes generated from M. lignano chromosomes, rDNA, and telomeric DNA). To explore the chromosome organization of the unusual 2n = 9 karyotype discovered in M. mirumnovem, we then generated chromosome-specific DNA probes for all chromosomes of this species. Similar to M. lignano and M. janickei, our findings suggest that M. mirumnovem arose via whole genome duplication (WGD) followed by considerable chromosome reshuffling. We discuss possible evolutionary scenarios for the emergence and reorganization of the karyotypes of these Macrostomum species and consider their suitability as promising animal models for studying the mechanisms and regularities of karyotype and genome evolution after a recent WGD.

Mitochondrial Genomes of Two Thaparocleidus Species (Platyhelminthes: Monogenea) Reveal the First rRNA Gene Rearrangement among the Neodermata.

Phylogenetic framework for the closely related Ancylodiscoidinae and Ancyrocephalinae subfamilies remains contentious. As this issue was never studied using a large molecular marker, we sequenced the first two Ancylodiscoidinae mitogenomes: Thaparocleidus asoti and Thaparocleidus varicus. Both mitogenomes had two non-coding regions (NCRs) that contained a number of repetitive hairpin-forming elements (RHE). Due to these, the mitogenome of T. asoti (16,074 bp) is the longest among the Monogenea; especially large is its major NCR, with 3500 bp, approximately 1500 bp of which could not be sequenced (thus, the total mitogenome size is ≈ 17,600 bp). Although RHEs have been identified in other monopisthocotyleans, they appear to be independently derived in different taxa. The presence of RHEs may have contributed to the high gene order rearrangement rate observed in the two mitogenomes, including the first report of a transposition of rRNA genes within the Neodermata. Phylogenetic analyses using mitogenomic dataset produced Dactylogyrinae embedded within the Ancyrocephalinae (paraphyly), whereas Ancylodiscoidinae formed a sister-group with them. This was also supported by the gene order analysis. 28S rDNA dataset produced polyphyletic Dactylogyridae and Ancyrocephalinae. The phylogeny of the two subfamilies shall have to be further evaluated with more data.

Lineage-specific expansion and loss of tyrosinase genes across platyhelminths and their induction profiles in the carcinogenic oriental liver fluke, Clonorchis sinensis.

Tyrosinase provides an essential activity during egg production in diverse platyhelminths by mediating sclerotization of eggshells. In this study, we investigated the genomic and evolutionary features of tyrosinases in parasitic platyhelminths whose genomic information is available. A pair of paralogous tyrosinases was detected in most trematodes, whereas they were lost in cyclophyllidean cestodes. A pseudophyllidean cestode displaying egg biology similar to that of trematodes possessed an orthologous gene. Interestingly, one of the paralogous tyrosinases appeared to have been multiplied into three copies in Clonorchis sinensis and Opisthorchis viverrini. In addition, a fifth tyrosinase gene that was minimally transcribed through all developmental stages was further detected in these opisthorchiid genomes. Phylogenetic analyses demonstrated that the tyrosinase gene has undergone duplication at least three times in platyhelminths. The additional opisthorchiid gene arose from the first duplication. A paralogous copy generated from these gene duplications, except for the last one, seemed to be lost in the major neodermatans lineages. In C. sinensis, tyrosinase gene expressions were initiated following sexual maturation and the levels were significantly enhanced by the presence of O2 and bile. Taken together, our data suggest that tyrosinase has evolved lineage-specifically across platyhelminths related to its copy number and induction mechanism.

Rapid identification of nine species of diphyllobothriidean tapeworms by pyrosequencing.

The identification of diphyllobothriidean tapeworms (Cestoda: Diphyllobothriidea) that infect humans and intermediate/paratenic hosts is extremely difficult due to their morphological similarities, particularly in the case of Diphyllobothrium and Spirometra species. A pyrosequencing method for the molecular identification of pathogenic agents has recently been developed, but as of yet there have been no reports of pyrosequencing approaches that are able to discriminate among diphyllobothriidean species. This study, therefore, set out to establish a pyrosequencing method for differentiating among nine diphyllobothriidean species, Diphyllobothrium dendriticum, Diphyllobothrium ditremum, Diphyllobothrium latum, Diphyllobothrium nihonkaiense, Diphyllobothrium stemmacephalum, Diplogonoporus balaenopterae, Adenocephalus pacificus, Spirometra decipiens and Sparganum proliferum, based on the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene as a molecular marker. A region of 41 nucleotides in the cox1 gene served as a target, and variations in this region were used for identification using PCR plus pyrosequencing. This region contains nucleotide variations at 12 positions, which is enough for the identification of the selected nine species of diphyllobothriidean tapeworms. This method was found to be a reliable tool not only for species identification of diphyllobothriids, but also for epidemiological studies of cestodiasis caused by diphyllobothriidean tapeworms at public health units in endemic areas.

Pipeline for the identification and classification of ion channels in parasitic flatworms.

BACKGROUND: Ion channels are well characterised in model organisms, principally because of the availability of functional genomic tools and datasets for these species. This contrasts the situation, for example, for parasites of humans and animals, whose genomic and biological uniqueness means that many genes and their products cannot be annotated. As ion channels are recognised as important drug targets in mammals, the accurate identification and classification of parasite channels could provide major prospects for defining unique targets for designing novel and specific anti-parasite therapies. Here, we established a reliable bioinformatic pipeline for the identification and classification of ion channels encoded in the genome of the cancer-causing liver fluke Opisthorchis viverrini, and extended its application to related flatworms affecting humans. METHODS: We built an ion channel identification + classification pipeline (called MuSICC), employing an optimised support vector machine (SVM) model and using the Kyoto Encyclopaedia of Genes and Genomes (KEGG) classification system. Ion channel proteins were first identified and grouped according to amino acid sequence similarity to classified ion channels and the presence and number of ion channel-like conserved and transmembrane domains. Predicted ion channels were then classified to sub-family using a SVM model, trained using ion channel features. RESULTS: Following an evaluation of this pipeline (MuSICC), which demonstrated a classification sensitivity of 95.2 % and accuracy of 70.5 % for known ion channels, we applied it to effectively identify and classify ion channels in selected parasitic flatworms. CONCLUSIONS: MuSICC provides a practical and effective tool for the identification and classification of ion channels of parasitic flatworms, and should be applicable to a broad range of organisms that are evolutionarily distant from taxa whose ion channels are functionally characterised.

Major acid endopeptidases of the blood-feeding monogenean Eudiplozoon nipponicum (Heteronchoinea: Diplozoidae).

In parasitic flatworms, acid endopeptidases are involved in crucial processes, including digestion, invasion, interactions with the host immune system, etc. In haematophagous monogeneans, however, no solid information has been available about the occurrence of these enzymes. Here we aimed to identify major cysteine and aspartic endopeptidase activities in Eudiplozoon nipponicum, an invasive haematophagous parasite of common carp. Employing biochemical, proteomic and molecular tools, we found that cysteine peptidase activities prevailed in soluble protein extracts and excretory/secretory products (ESP) of E. nipponicum; the major part was cathepsin L-like in nature supplemented with cathepsin B-like activity. Significant activity of the aspartic cathepsin D also occurred in soluble protein extracts. The degradation of haemoglobin in the presence of ESP and worm protein extracts was completely inhibited by a combination of cysteine and aspartic peptidase inhibitors, and diminished by particular cathepsin L, B and D inhibitors. Mass spectrometry revealed several tryptic peptides in ESP matching to two translated sequences of cathepsin L genes, which were amplified from cDNA of E. nipponicum and bioinformatically annotated. The dominance of cysteine peptidases of cathepsin L type in E. nipponicum resembles the situation in, e.g. fasciolid trematodes.

The first multi-gene phylogeny of the Macrostomorpha sheds light on the evolution of sexual and asexual reproduction in basal Platyhelminthes.

The Macrostomorpha-an early branching and species-rich clade of free-living flatworms-is attracting interest because it contains Macrostomum lignano, a versatile model organism increasingly used in evolutionary, developmental, and molecular biology. We elucidate the macrostomorphan molecular phylogeny inferred from both nuclear (18S and 28S rDNA) and mitochondrial (16S rDNA and COI) marker genes from 40 representatives. Although our phylogeny does not recover the Macrostomorpha as a statistically supported monophyletic grouping, it (i) confirms many taxa previously proposed based on morphological evidence, (ii) permits the first placement of many families and genera, and (iii) reveals a number of unexpected placements. Specifically, Myozona and Bradynectes are outside the three classic families (Macrostomidae, Microstomidae and Dolichomacrostomidae) and the asexually fissioning Myomacrostomum belongs to a new subfamily, the Myozonariinae nov. subfam. (Dolichomacrostomidae), rather than diverging early. While this represents the first evidence for asexuality among the Dolichomacrostomidae, we show that fissioning also occurs in another Myozonariinae, Myozonaria fissipara nov. sp. Together with the placement of the (also fissioning) Microstomidae, namely as the sister taxon of Dolichomacrostomidae, this suggests that fissioning is not basal within the Macrostomorpha, but rather restricted to the new taxon Dolichomicrostomida (Dolichomacrostomidae+Microstomidae). Furthermore, our phylogeny allows new insights into the evolution of the reproductive system, as ancestral state reconstructions reveal convergent evolution of gonads, and male and female genitalia. Finally, the convergent evolution of sperm storage organs in the female genitalia appears to be linked to the widespread occurrence of hypodermic insemination among the Macrostomorpha.

Comparative analysis of cystatin superfamily in platyhelminths.

The cystatin superfamily is comprised of cysteine proteinase inhibitors and encompasses at least 3 subfamilies: stefins, cystatins and kininogens. In this study, the platyhelminth cystatin superfamily was identified and grouped into stefin and cystatin subfamilies. The conserved domain of stefins (G, QxVxG) was observed in all members of platyhelminth stefins. The three characteristics of cystatins, the cystatin-like domain (G, QxVxG, PW), a signal peptide, and one or two conserved disulfide bonds, were observed in platyhelminths, with the exception of cestodes, which lacked the conserved disulfide bond. However, it is noteworthy that cestode cystatins had two tandem repeated domains, although the second tandem repeated domain did not contain a cystatin-like domain, which has not been previously reported. Tertiary structure analysis of Taenia solium cystatin, one of the cestode cystatins, demonstrated that the N-terminus of T. solium cystatin formed a five turn α-helix, a five stranded ß-pleated sheet and a hydrophobic edge, similar to the structure of chicken cystatin. Although no conserved disulfide bond was found in T. solium cystatin, the models of T. solium cystatin and chicken cystatin corresponded at the site of the first disulfide bridge of the chicken cystatin. However, the two models were not similar regarding the location of the second disulfide bridge of chicken cystatin. These results showed that T. solium cystatin and chicken cystatin had similarities and differences, suggesting that the biochemistry of T. solium cystatin could be similar to chicken cystatin in its inhibitory function and that it may have further functional roles. The same results were obtained for other cestode cystatins. Phylogenetic analysis showed that cestode cystatins constituted an independent clade and implied that cestode cystatins should be considered to have formed a new clade during evolution.

On the systematic position of Collyricloides massanae Vaucher, 1969 (Platyhelminthes: Digenea) with notes on distribution of this trematode species.

The systematic position of the Collyricloides massanae, a rare cyst-dwelling parasite, located on intestinal wall of European birds and rodents, have always been controversial. Based on newly obtained sequences of the 28 sDNA of C. massanae from avian and rodent host from Central Europe, and on the previously published sequences of several genera and families among Microphalloidea, we evaluate its taxonomic position and the phylogenetic relationships within the genera Collyriclum Kossack, 1911 and Collyricloides Vaucher, 1969 which form the family Collyriclidae Ward, 1917. In the cladogram, C. massanae appears among the Pleurogenidae, forming a clade with Gyrabascus amphoraeformis (Modlinger, 1930) and Cortrema magnicaudata (Bykhovskaya-Pavlovskaya, 1950). We reject the commonly accepted placement of Collyricloides as the sister genus to Collyriclum within the Collyriclidae. Besides, we present and discuss the unusual records of C. massanae in the bank vole Myodes glareolus from northeastern Poland.

Molecular cloning and characterization of SL3: a stem cell-specific SL RNA from the planarian Schmidtea mediterranea.

Spliced leader (SL) trans-splicing is a biological phenomenon, common among many metazoan taxa, consisting in the transfer of a short leader sequence from a small SL RNA to the 5' end of a subset of pre-mRNAs. While knowledge of the biochemical mechanisms driving this process has accumulated over the years, the functional consequences of such post-transcriptional event at the organismal level remain unclear. In addition, the fact that functional analyses have been undertaken mainly in trypanosomes and nematodes leaves a somehow fragmented picture of the possible biological significance and evolution of SL trans-splicing in eukaryotes. Here, we analyzed the spatial expression of SL RNAs in the planarian flatworm Schmidtea mediterranea, with the goal of identifying novel developmental paradigms for the study of trans-splicing in metazoans. Besides the previously identified SL1 and SL2, S. mediterranea expresses a third SL RNA described here as SL3. While, SL1 and SL2 are collectively expressed in a broad range of planarian cell types, SL3 is highly enriched in a subset of the planarian stem cells engaged in regenerative responses. Our findings provide new opportunities to study how trans-splicing may regulate the phenotype of a cell.