A Description of Echinochasmus pseudobeleocephalus n. sp. (Echinochasmidae) Based on Morphological and Molecular Data.

Adult individuals of Echinochasmus pseudobeleocephalus n. sp. were obtained during an experimental study on trematodes' life cycle. An analysis of the morphometric characteristics of the developmental stages and involvement of first intermediate hosts, snails of the genus Boreoelona, in their life cycle, revealed the identity of the obtained trematodes to the European species Echinochasmus beleocephalus previously discovered in the south of the Russian Far East. However, an analysis of molecular data, in particular sequences of the 28S rRNA gene, showed that the Far Eastern trematodes examined do not belong to European E. beleocephalus despite their morphological similarities. An analysis of phylogenetic relationships within the family Echinochasmidae supported the status of E. pseudobeleocephalus n. sp. as an independent species. Our new data confirmed that the individuals attributed to Echinochasmus can be subdivided into two groups on the basis of the number of head-collar spines and the tail length in cercariae on an intergeneric level.

Morpho-Molecular Features and Phylogenetic Relationships of Metorchis butoridi Oschmarin, 1963 (Trematoda: Opisthorchiidae) from East Asia.

Adult trematodes of the genus Metorchis were found in the gallbladders of ducklings that had been experimentally fed freshwater fishes of the genera Rhynchocypris and Rhodeus that were naturally infected by Metorchis metacercariae. Some of the trematodes were identified as Metorchis ussuriensis, whose morphology of developmental stages and molecular data had previously been described in detail. The other trematodes were confirmed as species Metorchis butoridi on the basis of morphological features: subterminal oral sucker, vitelline follicles with interrupted bands, and rosette-shaped testes. An analysis of phylogenetic relationships within Opisthorchiidae using nuclear and mitochondrial markers confirmed that the obtained trematodes were actually from the genus Metorchis. The morphological and molecular features indicated that a number of trematodes found in East Asia and described as Metorchis orientalis belong to M. butoridi. Also, the M. orientalis individuals from Europe are, in fact, representatives of another Metorchis species.

New species of Psilotrema and Sphaeridiotrema (Psilostomidae Odhner, 1913) in the east Asian region: Morphology of developmental stages and genetic data.

Morphological and genetic data have been obtained for five new East Asian species of the Psilostomatidae. The life cycle of Psilotrema limosum n. sp. was carried out using Parafossarulus manchouricus as the first intermediate host. Compared with the East Asian species of the genus, these worms differ in morphometric characters in both the cercarial and adult stages. Its validity was also confirmed by the 28S rRNA gene data. Data on the life cycle and morphology of developmental stages of Sphaeridiotrema ussuriensis n. sp. and Sphaeridiotrema aziaticus n. sp. were also obtained. Cercariae of these species are found in Parafossarulus and Boreoelona snails, respectively. Sphaeridiotrema ussuriensis n. sp., like Sphaeridiotrema monorchis in China, has one testis, while S. aziaticus n. sp. has two testes. In addition, S. monorchis from Vietnam and Sphaeridiotrema spinoacetabulum from the Russian southern Far East are justified as belonging to the new species named Sphaeridiotrema vietnamensis n. sp. and Sphaeridiotrema pyriforme n. sp., respectively. This proposition is based on the morphology of developmental stages, the list of the first intermediate hosts and the 28S rRNA gene data. Analysis of the phylogenetic relationships within Psilostomatidae revealed three clusters, including taxa with different life cycles strategies. Sphaeridiotrema was also divided into two groups, which combine species according to their intermediate hosts and geographical localisation.

Echinostoma chankensis nom. nov., other Echinostoma spp. and Isthmiophora hortensis in East Asia: morphology, molecular data and phylogeny within Echinostomatidae.

Life cycles, and morphological and molecular data were obtained for Echinostoma chankensis nom. nov., Echinostoma cinetorchis, Echinostoma miyagawai and Isthmiophora hortensis from East Asia. It was established that, based on both life cycle and morphology data, one of the trematodes is identical to the worms designated as Euparyphium amurensis. Genetic data showed that this trematode belongs to Echinostoma. The complex data on biological, morphological and genetic characterizations establish that the distribution of the morphologically similar species, I. hortensis and Isthmiophora melis, in the Old World are limited by the East Asian and European regions, respectively. Data on mature worms of East Asian E. miyagawai revealed morphological and genetic identity with E. miyagawai from Europe. However, E. miyagawai from Europe differs from E. miyagawai from the type locality (East Asia) in terms of reaching maturity and the morphology of cercariae. These data indicate that the European worm, designated E. miyagawai, does not belong to this species. An analysis of the phylogenetic relationships of Echinostomatidae was conducted based on the 28S, ITS2 and nad1 markers. Analysis using the nad1 gene for the known representatives of Echinostomatidae is carried out for the first time, showing that nuclear markers are ineffective separate from mitochondrial ones.

Three species of Exorchis Kobayashi, 1921 (Digenea: Cryptogonimidae) in the East-Asian region: morphological and molecular data.

New data have been obtained for three representatives of Exorchis; Exorchis convictus sp. n., Exorchis oviformis and Exorchis sp., from fish in the East-Asian region. For the first time, based on combined sequences of the ITS2 rDNA region and the 28S rRNA gene, Exorchis is confirmed to belong Cryptogonimidae. Based on analysis of a mitochondrial marker (cox1), the 'Japanese' and 'Russian' haplogroups are identified for E. oviformis isolated from Silurus asotus. One specimen of E. oviformis obtained in Japan is identical to the 'Russian' haplotype. Haplotype patterns are also observed for metacercariae of Exorchis sp. from Tanakia lanceolata and Carassius sp. fish in Kyushu Island (Japan).

Paragonimus heterotremus Chen et Hsia, 1964 (Digenea: Paragonimidae): species identification based on the biological and genetic criteria, and pathology of infection.

As a result of the experimental infection of rats with metacercariae of Paragonimus heterotremus Chen et Hsia, 1964 from crabs (Potamiscus tannanti) caught in Yen Bai province, Vietnam, it was found that worms migrated into the lungs, to the liver and less frequently to the tissue that lines body cavities of the hosts, where they reached the adult stage, but in the muscles, worms stayed at the larval stage. Studies have shown that for P. heterotremus, rats can simultaneously play the role of the final and paratenic host; herewith, an infection with the trematode of this species can lead to the development of three forms of paragonimiasis: pulmonary, hepatic and muscular. Eggs from the adult worms localised in the liver, unlike eggs from the adult worms localised in the lungs, were not excreted into the external environment, but accumulated inside the organ. Histology and description of changes, which take place on the external surface of organs affected with P. heterotremus, are given in this study. Based on the behavioural characteristics of worms during rat infection and molecular genetic data, we established that worms from Vietnam and India should be assigned to different species of Paragonimus. P. heterotremus distribution is limited to the territory of the Southeast China, Northern Vietnam, Laos and Thailand.

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.

First description of genetic diversity for the genus Metagonimus using the complete cox1 gene sequence.

The complete cox1 gene sequence was analysed for Metagonimus suifunensis from eight localities in the Russian southern Far East, and the level of variability was compared with that of Clonorchis sinensis from the same territory of Russia. These species belong to the superfamily Opisthorchioidea, have a similar distribution in the Russian southern Far East and share second intermediate and definitive hosts, but are distinguished by their first intermediate hosts belonging to different orders of caenogastropods. The data obtained showed that the nucleotide sequence variability of the M. suifunensis cox1 gene was significantly lower. This fact is considered in connection with a recent bottleneck passage for the M. suifunensis population, in contrast to C. sinensis, which could be due to the features of the Metagonimus life cycle under seasonal freezing temperatures, as well as historical geological and climatic changes in the Russian Far East. These factors could influence the microevolutionary processes and lead to a decrease in the level of variability in the M. suifunensis population. Based on the combination of genetic data and historical geo-processes in the region, the probable route of M. suifunensis expansion from the northern part of its current area in the Amur River basin to the southern territories of the Russian Far East is justified.

Correction to: Morphometric and molecular analyses of Carassotrema koreanum park 1938 and Elonginurus mugilus Lu 1995 (Digenea: Haploporidae) Srivastava, 1937 from the Russian Far East and Vietnam.

The original version of this article contained mistake in the accession number found in Table 1. Correct accession numbers are presented here.

Description of a new species, Cryptocotyle lata sp. nov., and discussion of the phylogenetic relationships in Opisthorchioidea.

Adult Cryptocotyle lata sp. nov. worms were obtained from experimental studies. In the Russian southern Far East, the life cycle of this parasite is carried out using freshwater snails (Boreoelona ussuriensis), freshwater fish, and birds as the first intermediate, second intermediate, and definitive hosts, respectively. The morphological indices of C. lata sp. nov. are closest to Cryptocotyle concava; however, these two species differ in terms of their sizes of body, oral and ventral suckers, eggs, and the shape of their testes and ovaries. Analysis of the life cycles of the Cryptocotyle representatives suggested that C. concava were at least two cryptic species, one of which circulates using brackish water Hydrobia snails, and the other using freshwater Amnicola snails as the first intermediate hosts. Molecular data (i.e., the 28S gene and the second internal transcribed spacer (ITS2) of rDNA) were used to analyze the phylogenetic relationships of C. lata sp. nov. and other representatives of Opisthorchioidea. The long repeats and secondary structure of the ITS1 region were studied. Representatives of the Opisthorchiidae and several species from Heterophyidae (including the genus Cryptocotyle) were found to have molecular features that suggested that these species belonged to Opisthorchiidae. At the same time, the genetic relatedness of worms, which are united in common clusters on phylogenetic trees, is consistent with the use of the first intermediate hosts from different taxonomic groups in their life cycles; namely, snails of the Truncatelloidea are hosts of trematodes from a cluster with Opisthorchiidae and a number species of the family Heterophyidae, while snails of the Cerithioidea are hosts of worms from a cluster that includes only the Heterophyidae. In addition, the results of genetic studies indicate that Clonorchis sinensis, Metorchis ussuriensis, Metorchis bilis, Metorchis xanthosomus, and Metorchis orientalis should be included in the genus Opisthorchis.

Morphometric and molecular analyses of Carassotrema koreanum Park 1938 and Elonginurus mugilus Lu 1995 (Digenea: Haploporidae) Srivastava, 1937 from the Russian Far East and Vietnam.

Adult worms that belong to Carassotrema Park 1938 and Elonginurus Lu, 1955 were found in the intestine of Carassius gibelio Bloch, 1782 from the southern Russian Far East and Mugil cephalus Linnaeus, 1758 from northern Vietnam, respectively. Morphometric parameters, geographic location and host species composition of these worms correspond to Carassotrema koreanum Park 1938, which is a known parasite of cyprinid fish in Korea, Japan and China, and Elonginurus mugilus Lu, 1955, first described from M. cephalus in China. The validity of Carassotrema ginezinskajae Kulakova, Ha Ky, 1976, a synonym of C. koreanum, first described from Spinibarbichthys denticulatus Oshima, 1926 in Vietnam, supported the morphometric data. Phylogenetic analysis based on combined ITS2 ribosomal DNA (rDNA) and 28S rRNA indicated that C. koreanum and E. mugilus belong to the subfamily Waretrematinae and are closely related to the genera Skrjabinolecithum and Parasaccocoelium, respectively. Species similarity, revealed through molecular analysis, agreed with the generic diagnoses for Parasaccocoelium and Elonginurus, as well as for Carassotrema and Skrjabinolecithum.

Developmental stages of Notocotylus magniovatus Yamaguti, 1934, Catatropis vietnamensis n. sp., Pseudocatatropis dvoryadkini n. sp., and phylogenetic relationships of Notocotylidae Lühe, 1909.

Data on the life cycles and morphology of the developmental stages of Notocotylus magniovatus, Catatropis vietnamensis n. sp., and Pseudocatatropis dvoryadkini n. sp. were obtained. The Pseudocatatropis genus was restored based on our results. For the studied trematodes, the snails Parajuga spp., Helicorbis sujfunensis (Russia), and Melanoides tuberculata (Vietnam) serve as first intermediate hosts. It has been established that C. vietnamensis n. sp. differs from Catatropis harwoodi and Catatropis pakistanensis in the length of the ridge and metraterm and the location of the anterior papillae. In the life cycle of P. dvoryadkini n. sp., as in Pseudocatatropis joyeuxi, cercariae do not leave the first intermediate host. Both species are very similar in morphometric features, despite the fact that they share no common first intermediate hosts in their life cycles, and the areas of the European and Asian populations of flukes do not overlap. In phylogenetic trees and genetic distances based on the nucleotide sequences of the 28S gene and the ITS2 region of ribosomal DNA, Notocotylus attenuatus, Notocotylus intestinalis, and Notocotylus magniovatus are combined into one systematic group, while C. vietnamensis n. sp. and Catatropis indicus form another group. A third group includes members of different genera: P. dvoryadkini n. sp., and Notocotylus malhamensis, as well as three unclassified Notocotylus spp. The presence in the last group of flukes with three rows of papillae and a median ridge and lateral papillae indicates that these systematic criteria are not determinant in establishing membership of the parasitic worms to one or another genus of Notocotylidae.

Stephanoprora chasanensis n. sp. (Digenea: Echinochasmidae): Morphology, life cycle, and molecular data.

Echinostomatoidea is a large, globally distributed and heterogeneous group of hermaphroditic digeneans that parasite, as adults, vertebrate hosts of all classes. Species of this group have received attention from researchers as they can cause diseases in wildlife and humans. Here we describe the biological and molecular phylogenetic characteristics of Stephanoprora chasanensis n. sp. (Digenea: Echinochasmidae). The life cycle of this fluke was experimentally completed by the use of hosts, i.e. Stenothyra recondite Lindholm, 1929 snail (the 1st intermediate), Rhynchocypris percnurus mantschuricus (Berg, 1907) freshwater fish (the 2nd intermediate) and Gallus gallus chicken (the definitive host). In the adult worms, vitelline follicles were distributed anteriorly to the mid-level of the ventral sucker in our specimens whereas they did not reach the level of anterior testis in other species of Stephanoprora previously reported. Phylogenetic analysis based on 28S rDNA revealed that Stephanoprora and Echinochasmus with 20-22 collar spines grouped together in a single cluster. In addition, we showed that Stephanoprora chasanensis n. sp. was closely related to Echinochasmus milvi Yamaguti, 1939. Cercariae of these two echinostomes commonly have a long tail.

Trematode diversity in freshwater fishes of the Globe I: 'Old World'.

In this paper, we review, continent by continent, the trematode fauna of freshwater fishes of the 'Old World', a vast area consisting of the Palaearctic, Ethiopian, Oriental and Australasian zoogeographical regions. Knowledge of this fauna is highly uneven and clearly incomplete for almost all regions, sometimes dramatically so. Although the biggest problem remains the completion of the 'first pass' of alpha taxonomy, there are in addition great problems relating to biogeography and elucidation of life-cycles. For the latter, molecular data, i.e. matching DNA sequences of larval stages and corresponding adults, may represent a powerful tool that should be used in future studies. Another challenging problem represents the existence of cryptic species and, in particular, considerable decrease of experts in taxonomy and life-cycles of trematodes.

Hemozoin is a product of heme detoxification in the gut of the most medically important species of the family Opisthorchiidae.

Many species of trematodes such as Schistosoma spp., Fasciola hepatica and Echinostoma trivolvis are blood-feeding parasites. Nevertheless, there is no consensus on the feeding habits of the family Opisthorchiidae (Opisthorchis felineus, Opisthorchis viverrini and Clonorchis sinensis). Previously, histological studies of O. felineus and C. sinensis revealed some dark stained material in their gut lumen. In this study we conducted a comprehensive analysis of the gut contents of three members of the family Opisthorchiidae (O. felineus, O. viverrini and C. sinensis). Using transmission electron microscopy, we demonstrated for the first known time the presence of disintegrating blood cells in the gut of O. felineus as well as electron-dense crystals in the gut of O. felineus and C. sinensis. Electron energy loss spectroscopy revealed iron atoms in these crystals, and mass spectrometry of the purified pigment demonstrated the presence of heme. Fourier-transform infrared spectroscopy identified the signature peaks of the common iron-carboxylate bond characteristic in crystals isolated from O. felineus and C. sinensis. Scanning electron microscopy showed layered ovoid crystals of various sizes from 50 nm to 2 µm. Morphological, chemical and paramagnetic properties of these crystals were similar to those of hemozoin from Schistosoma mansoni. Crystal formation occurs on the surface of lipid droplets in O. felineus and C. sinensis guts. Our results suggest that the diet of O. felineus and C. sinensis includes blood. Detoxification of the free heme produced during the digestion proceeds via formation of insoluble crystals that contain iron and heme dimers, i.e. crystals of hemozoin. Furthermore, we believe that biocrystallisation of hemozoin takes place on the surface of the lipid droplets, similar to S. mansoni. Hemozoin was not detected in the closely related species O. viverrini.

Molecular evidence of cryptic diversity in Paracaryophyllaeus (Cestoda: Caryophyllidea), parasites of loaches (Cobitidae) in Eurasia, including description of P. vladkae n. sp.

Molecular phylogenetic analysis of an extensive collection of monozoic tapeworms of the genus Paracaryophyllaeus Kulakovskaya, 1961 (Cestoda: Caryophyllidea), parasites of loaches (Cypriniformes: Cobitidae) in Eurasia, has revealed cryptic species diversity within this long-time monotypic genus, especially in the Paracaryophyllaeus gotoi (Motomura, 1927) species complex [syn. Paracaryophyllaeus dubininorum (Kulakovskaya, 1961); type species]. Three independent, well-supported clades were discovered on the basis of molecular data: (i) specimens from Misgurnus anguillicaudatus and Cobitis lutheri from China, Russian Far East and Japan - called herein P. cf. gotoi 1, which may be conspecific with P. gotoi (Motomura, 1927), although in the absence of sequence data for P. gotoi from its type locality (basin of the River Kumkan in Korea), no certain inferences about their identity can currently be made; (ii) specimens from M. anguillicaudatus from China and Japan - P. cf. gotoi 2, which are morphologically indistinguishable from those of P. cf. gotoi 1; and (iii) morphologically distinct tapeworms from the endemic loach Cobitis bilseli from southwestern Turkey (Beysehir Lake), which are described herein as a new species. Paracaryophyllaeus vladkae Scholz, Oros and Aydogdu n. sp. differs from the remaining species of the genus in the following characteristics: the testes begin anterior to the first vitelline follicles (versus posterior), the body is short and robust (versus more elongate and slender), and the scolex is wide, rounded or apically tapered (versus claviform to truncate). Species composition of the genus, host specificity of species and geographical distribution are briefly discussed.

Genetic diversity of Clonorchis sinensis (Trematoda: Opisthorchiidae) in the Russian southern Far East based on mtDNA cox1 sequence variation.

We examined the phylogeography and the variation of the mitochondrial DNA (mtDNA) cytochrome c oxidase subunit 1 gene (cox1) of the Chinese liver fluke Clonorchis sinensis (Cobbold, 1875) in two geographic localities in the Russian southern Far East and compared them with those from different geographical regions (China, Korea, Japan and Vietnam). The Russian samples differed from those of the other regions in haplotype frequencies, haplotype and nucleotide diversities, and AT/GC ratios. Only 4 of the 18 haplotypes were common to Russian and Chinese samples, and two haplotypes were common to Russia and other regions. The intraspecific genetic distances ranged from 0 to 1.58% for the entire dataset studied and from 0 to 1.25% among the samples from Russia. Phylogenetic trees revealed no significant genealogical clades of samples corresponding to sampling localities and no strong isolation by distance was estimated with Mantel test. Neutrality test analysis suggested a relatively recent population expansion for C. sinensis, whereas goodness-of-fit tests indicated deviation from the strict model of uniform expansion. Therefore, the sequences of the mtDNA cox1 gene provide useful genetic markers for evaluating intraspecific diversity and generating phylogeographic reconstructions for this fish-borne trematode.

New data on the morphology of some Far-Eastern species of Rhabdochona (Nematoda: Rhabdochonidae), as revealed by SEM observations.

Specimens of the nematode genus Rhabdochona Railliet, 1916 (Rhabdochonidae) were collected during helminthological examination of four species of cyprinid fishes in two rivers of the Amur River basin in the Russian Far East (Primorsky Region) in June 2011. Detailed light microscopical (LM) and scanning electron microscopical (SEM) examinations (the latter used for the first time for the reported nematode species) of the available material revealed the presence of three inadequately described nominal species of this genus: R. (Rhabdochona) denudata (Dujardin, 1845) from the spotted steed Hemibarbus maculatus Bleeker (Gobioninae), and R. (Rhabdochona) longispicula Belous in Roytman, 1963 and R. (Globochonoides) coronacauda Belous, 1965 from Culter alburnus Basilewsky (Cultrinae) in the Ilistaya River. Detailed morphological study of these worms, especially SEM examination, made it possible to reveal some previously unreported morphological features (e.g., the presence of sublabia or the character of ventral precloacal ridges) and to confirm other taxonomically important characters such as the shape of deirids, number of anterior prostomal teeth, number and situation of lateral preanal and postanal papillae or the detailed structure of the crown-like formation on the female tail tip in R. coronacauda. Unidentifiable Rhabdochona (Rhabdochona) gravid females were recorded from the humpback Chanodichthys dabryi (Bleeker) (Cultrinae) in the Ilistaya River and from the Amur minnow Rhynchocypris lagowskii (Dybowski) (Leuciscinae) in the Komissarovka River.

The life cycle of Asymphylodora perccotti sp. n. (Trematoda: Lissorchiidae) in the Russian Southern Far East.

Specimens of Asymphylodora perccotti sp. n. (Trematoda: Lissorchidae) were found in the esophagus of the freshwater fish Perccottus glenii (Odobantidae) taken from the Bolshaya Ussurka River Basin (Primorsky Region, Russian Southern Far East). The first intermediate host of this trematode is a gastropod, Parafossarulus manchouricus, and the secondary hosts are the same mollusk and Boreoelona ussuriensis. Specimens of the new species are similar to A. amnicolae identified by Stunkard in 1959, but the mature worms have larger suckers and shorter ceca. The cercariae of these species are distinguished by body, suckers and pharynx size. These organs in A. perccotti sp. n. are more than one-third larger than what is observed in A. amnicolae. In addition, the new species lacks the capacity for progenesis. Finally, the new species is unusual in that it resides in the fish esophagus instead of the intestine, as is common for most Asymphylodora species. Partial ribosomal DNA sequences and phylogenetic reconstruction sequence data indicate that these worms represent a new digenean species.