Morphological and molecular data for highly pathogenic avian parasite Erschoviorchis anuiensis sp. n. and phylogenetic relationships within the Opisthorchiidae (Trematoda).

Adult worms of Erschoviorchis anuiensis sp. n., parasites of the pancreas and liver of birds, were found by feeding the Muscovy ducks Cairina moschata dom. with freshwater fish (Phoxinus percnurus) from the Amur River basin (Russia). The trematodes obtained differ from the only previously known representative of the genus, E. lintoni by the large size of the ventral sucker, testes and ovary, the shape of the ovary (three-lobed vs irregular oval for E. lintoni), and the degree of vitellarium development (well-developed vitellarium with numerous follicles vs weakly developed vitelline fields for E. lintoni). In addition, genetic data were obtained for E. anuiensis sp. n., including nucleotide sequences of the ITS region and the 28S rRNA gene of nuclear DNA, and the mitochondrial сох1 gene. These data show that the genus Erschoviorchis is a sister group to the representatives of the genera Opisthorchis, Clonorchis, and Metorchis. At the same time, it did not cluster with species of Amphimerus, in which E. lintoni has sometimes been placed. The results of the study indicated that E. anuiensis sp. n., as well as E. lintoni, when it occurs in the pancreas, leads to significant associated pathological changes, manifested in an increase in size, changes of structure and tissue density.

Chromosome morphometry in opisthorchiid species (Platyhelminthes, Trematoda).

Few existing studies have dealt with cytogenetics in trematodes, largely due to the attendant technical difficulty of chromosome preparation. We performed a comparative analysis of chromosomes in five opistorchiid species, including Opisthorchis felineus Rivolta, 1884, Opisthorchis viverrini Poirier, 1886, Clonorchis sinensis Cobbold, 1875, Metorchis xanthosomus Creplin 1846, and Metorchis bilis (Braun, 1790) Odening, 1962. For some of these species, no detailed morphometric description of their karyotypes has yet been published; for the karyotype of Metorchis bilis this is the first-ever description. We found that opisthorchiids, like other trematodes, are characterized by karyotypic conservatism (N=6-7) and karyotype asymmetry, although comparison of chromosome morphometric traits did reveal differences between the karyotypes of the species. Moreover, to address certain a methodological issue in trematode chromosome preparation, we analyzed how the source of chromosomal material (partenitae or mature flukes) and the chromosome preparation techniques used (air-drying and cell suspension methods) affected chromosome spreading and size, concluding that the most reliable comparative method involves comparing relative parameters (relative length, arm ratio, centromeric index) of chromosomes prepared using the same technique.

Integrative taxonomy of European parasitic flatworms of the genus Metorchis Looss, 1899 (Trematoda: Opisthorchiidae).

Metorchis spp. are flukes (Platyhelminthes: Digenea) that infect vertebrates, including humans, dogs, cats, poultry and wild game, with cyprinid freshwater fish serving as typical second intermediate hosts. In their definitive hosts, the Metorchis spp. are difficult to identify to species. We provide and analyze sequences of two nuclear (18S rDNA and ITS2) and two mitochondrial (CO1 and ND1) DNA loci of four morphologically identified European species of the Metorchis, namely Metorchis albidus, Metorchis bilis, Metorchis crassiusculus and Metorchis xanthosomus, and of another opisthorchiid, Euamphimerus pancreaticus. DNA analysis suggests that the Metorchis specimens identified morphologically as M. albidus (from Lutra lutra), M. bilis (from Phalacrocorax carbo) and M. crassiusculus (from Aquila heliaca and Buteo rufinus) represent a single species. Thus, M. albidus (Braun, 1893) Loos, 1899 and M. crassiusculus (Rudolphi, 1809) Looss, 1899 are recognized as junior subjective synonyms of M. bilis (Braun, 1790) Odening, 1962. We also provide comparative measurements of the Central European Metorchis spp., and address their tissue specificity and prevalence based on the examination of extensive bird cohort from 1962 to 2015. M. bilis and M. xanthosomus can be morphologically diagnosed by measuring the extent of genitalia relative to body length and by the size ratio of their suckers. They also differ in their core definitive hosts, with ducks (Anas, Aythya) and coots (Fulica) hosting M. xanthosomus, and cormorants (Phalacrocorax), the birds of prey (Buteo, Aquila, etc.), piscivorous mammals (Lutra, Vulpes, Ursus, etc.) and humans hosting M. bilis. Previous reports on the Metorchis spp. contain numerous suspected misidentifications.

Telomeric DNA in chromosomes of five opisthorchid species.

The analysis of telomere repeat distribution in chromosomes of five opisthorchid species (Opisthorchis felineus (Rivolta, 1884), Opisthorchis viverrini (Poirier, 1886), Metorchis xanthosomus (Creplin, 1846), Metorchis bilis (Braun, 1890), Clonorchis sinensis (Cobbold, 1875)) was performed with fluorescent in situ hybridization (FISH) of labeled (TTAGGG)n DNA-probe and PNA telomere probe on mitotic and meiotic chromosomes of these species. It was shown that chromosome telomeres of all studied species contain large clusters of (TTAGGG)n telomeric repeats. Interstitial clusters of the (TTAGGG)n repeats have not been revealed in the chromosomes of any studied species even when FISH of PNA telomere probe on pachytene chromosomes was performed. Furthermore interstitial clusters of the (TTAGGG)n repeats have not been detected in the chromosomes of O. viverrini, one of chromosomes of this species is the result of a fusion of two ancestral opisthorchid chromosomes.

Distribution of repetitive DNA sequences in chromosomes of five opisthorchid species (Trematoda, Opisthorchiidae).

Genomes of opisthorchid species are characterized by small size, suggesting a reduced amount of repetitive DNA in their genomes. Distribution of repetitive DNA sequences in the chromosomes of five species of the family Opisthorchiidae (Opisthorchis felineus 2n = 14 (Rivolta, 1884), Opisthorchis viverrini 2n = 12 (Poirier, 1886), Metorchis xanthosomus 2n = 14 (Creplin, 1846), Metorchis bilis 2n = 14 (Braun, 1890), Clonorchis sinensis 2n = 14 (Cobbold, 1875)) was studied with C- and AgNOR-banding, generation of microdissected DNA probes from individual chromosomes and fluorescent in situ hybridization on mitotic and meiotic chromosomes. Small-sized C-bands were discovered in pericentric regions of chromosomes. Ag-NOR staining of opisthorchid chromosomes and FISH with ribosomal DNA probe showed that karyotypes of all studied species were characterized by the only nucleolus organizer region in one of small chromosomes. The generation of DNA probes from chromosomes 1 and 2 of O. felineus and M. xanthosomus was performed with chromosome microdissection followed by DOP-PCR. FISH of obtained microdissected DNA probes on chromosomes of these species revealed chromosome specific DNA repeats in pericentric C-bands. It was also shown that microdissected DNA probes generated from chromosomes could be used as the Whole Chromosome Painting Probes without suppression of repetitive DNA hybridization. Chromosome painting using microdissected chromosome specific DNA probes showed the overall repeat distribution in opisthorchid chromosomes.