Description of Crenosoma vismani n. sp., parasitic in the lungs of Lynx lynx (L.) (Carnivora: Felidae), with identification key to the species of the genus Crenosoma Molin, 1861 (Nematoda: Crenosomatidae).

A new species of Crenosoma Molin, 1861 is described from the lungs of the Eurasian lynx, Lynx lynx (L.) (Carnivora: Felidae). Crenosoma vismani n. sp. differs from other species of the genus in the morphology of the male copulatory organs (two ventral rays originating from a common stem; three lateral rays with common stem; gubernaculum 79-92 µm long, well developed, spoon-shaped; spicules 232-253 µm long, slightly curved, with robust dorsal appendage) and a vulva with rounded, prominent vulvar cuticular appendage is located in middle region of body in females. An analysis of the morphology and life-cycle data for Crenosoma petrowi Morozov, 1939 from Ursus americanus Pallas indicate that this nematode should be described as a new species. The validity of Crenosoma schulzi Gagarin, 1958 is resurrected. A new identification key for the species of Crenosoma is provided. Published records of the genus Crenosoma from definitive hosts and patterns of host specificity are presented. Morphological data revealed that the genus Crenosoma should contain 15 species (14 with valid names) with different host specificity, ranging from oioxenous to euryxenous. However, a review of the current genetic data indicates that the actual number of Crenosoma spp. is greater, and the host range is still unknown, even in Europe.

Phyllodistomum kupermani n. sp. from the European perch, Perca fluviatilis L. (Perciformes: Percidae), and redescription of Phyllodistomum macrocotyle (Lühe, 1909) with notes on the species diversity and host specificity in the European Phyllodistomum spp. (Trematoda: Gorgoderidae).

BACKGROUND: European species of the large genus Phyllodistomum Braun, 1899 had historically been erected based solely on morphological characters. Unfortunately, many of them are still poorly known and inadequately described. Molecular approaches are critical to delineate species which were impossible to differentiate based on morphology alone. METHODS: New samples of adult Phyllodistomum spp. were collected from the urinary bladder and/or ureters of European freshwater fishes and fixed to conduct a light and scanning electron microscopy study, and to obtain sequences of nuclear (ITS2 spacer and 28S rRNA gene), to be analysed in the context of a molecular phylogeny. RESULTS: Based on new findings, a new species of Phyllodistomum from the urinary bladder of the European perch, Perca fluviatilis, in Volga River basin, Russia, is described. Additionally, new data on the morphology and tegumental surface topography of P. macrocotyle (Lühe, 1909) Odhner, 1911 from ureters of the common rudd, Scardinius erythrophthalmus, is presented. The host range of P. folium, confirmed by DNA analysis, is extended to other cyprinid fish species. CONCLUSIONS: The present study has again shown that species of the genus Phyllodistomum are in dire need of revision based on both molecular analysis and detailed morphological redescriptions of the forms attributed to the genus. Morphologically, P. kupermani n. sp. most closely resembles P. pseudofolium, a highly host-specific parasite of Gymnocephalus cernuus (L.), but molecular phylogenetic analyses based on ITS2 and 28S rDNA sequences showed that these species are distantly related. Phyllodistomum kupermani n. sp. was found to be phylogenetically most closely related to the type-species of Phyllodistomum, P. folium. Phylogenetic analyses revealed that Phyllodistomum kupermani n. sp. and P. folium formed a clade with other freshwater species for which cystocercous cercariae develop in bivalves of the family Sphaeriidae. The micromorphology and tegumental surface topography of P. macrocotyle revealed in the present study provide a valuable taxonomic criterion for congeneric species differentiation.

Diversity and phylogenetic relationships of European species of Crepidostomum Braun, 1900 (Trematoda: Allocreadiidae) based on rDNA, with special reference to Crepidostomum oschmarini Zhokhov & Pugacheva, 1998.

BACKGROUND: Within the genus Crepidostomum Braun, 1900, identification of species and taxonomic decisions made only on the basis of adult morphology have resulted in great problems associated with evaluating actual diversity and validity of species. Life-cycle data, while equal in importance to adult characters, are scarce, controversial or incomplete for most Crepidostomum spp. In this study, rDNA sequences generated from adult and larval Crepidostomum spp. and some other allocreadiid species were analysed to reveal the diversity and phylogenetic relationships of the species and their host range. Detailed morphological description based on light microscopy, SEM tegumental surface topography and genetic data are provided for the poorly known trematode C. oschmarini Zhokhov & Pugacheva, 1998 found in the intestine of two teleost fish species, Barbatula barbatula (L.) and Cottus gobio L. RESULTS: We characterized 27 isolates of adult and larval parasites. Based on newly obtained 28S and ITS1-5.8S-ITS2 rDNA sequences, new intermediate and final hosts were ascertained, and life-cycles clarified for some allocreadiids. New knowledge on the diversity and phylogenetic relationships of European Crepidostomum spp. was gained. The validity of C. oschmarini was verified based on comparative sequence analysis. Ophthalmoxiphidiocercariae of C. oschmarini were recorded in sphaeriid bivalves Pisidium (Euglesa) casertanum (Poli). Additionally, morphological differences between gravid specimens of C. oschmarini and other related species were observed. CONCLUSIONS: Species of the Allocreadiidae parasitizing fishes in Europe are distributed among two monophyletic genera, Allocreadium and Bunodera, and two paraphyletic Crepidostomum clades. A complex of Crepidostomum metoecus (syn. C. nemachilus), C. oschmarini and Crepidostomum sp. 2 clustered in one clade, and a complex of C. farionis, Crepidostomum sp. 1 and, probably, C. wikgreni in the other. Molecular data indicated that C. oschmarini and Crepidostomum sp. 2 presumably have a wide geographical distribution in Europe. The new data provided evidence that Crepidostomum is a more diverse genus than can be judged from morphological data and host switching in this genus may occur independently of fish-host phylogeny.

Environmental genotoxicity and cytotoxicity levels in herring (Clupea harengus), flounder (Platichthys flesus) and cod (Gadus morhua) inhabiting the Gdansk Basin of the Baltic Sea.

Frequencies of eight nuclear abnormalities (NAs) reflecting environmental genotoxicity and cytotoxicity, were examined in 739 specimens of herring (Clupea harengus), flounder (Platichthys flesus) and cod (Gadus morhua) collected between 2009 and 2017 at 50 study stations located in the Gulf of Gdansk and the southern part of the Gdansk Deep (the Baltic Sea). The highest levels of geno-cytotoxicity were recorded in fish caught at stations located in close proximity to chemical and conventional munitions dumping sites or in zones polluted by Chemical Warfare Agents (CWAs). The values of NAs were significantly higher (up to 51-fold compared with the reference level) in herring caught at seven stations and in cod from one station located close to the munitions dumping sites. Exceptionally high total genotoxicity (∑Gtox) risk was found for flounder collected from 18 (72.0%) stations, herring caught at 12 (38.7%) stations and cod caught at four (17.4%) studied stations.

Comments on species divergence in the genus Sphaerium (Bivalvia) and phylogenetic affinities of Sphaerium nucleus and S. corneum var. mamillanum based on karyotypes and sequences of 16S and ITS1 rDNA.

Chromosome, 16S and ITS1 rDNA sequence analyses were used to obtain reliable diagnostic characters and to clarify phylogenetic relationships of sphaeriid bivalves of the genus Sphaerium. The species studied were found to be diploid, with modal number 2n = 28 in S. nucleus and 2n = 30 in S. corneum var. mamillanum. Small, biarmed, C- negative B chromosomes were found in all studied populations of both species. Karyological and molecular markers revealed no differences between S. corneum s. str. and S. corneum var. mamillanum. No intraspecific differences were found in the basic karyotype of S. nucleus. Molecular analyses, however, uncovered three genetically distinct ITS1 lineages: one comprised of samples from Lithuania, Slovakia, and Russia, another from Czech, and a third from Ukraine. Additionally to known 16S haplotype from Ukraine, three new 16S haplotypes of S. nucleus were detected: one in the samples from Lithuania and Russia, one in Slovakian and one in Czech population. In the ITS1 phylogenetic tree, all branches of S. nucleus clustered in one clade. In the 16S phylogenetic tree, however, the haplotype of Czech S. nucleus formed a separate branch, distant from three other haplotypes of S. nucleus. Molecular results indicate that in the context of the Evolutionary Species Concept the S. nucleus morphospecies may represent a complex of separate taxa, however referring on the Biological Species Concept the genetic lineages could represent the intraspecific variability.

Host specificity, molecular phylogeny and morphological differences of Phyllodistomum pseudofolium Nybelin, 1926 and Phyllodistomum angulatum Linstow, 1907 (Trematoda: Gorgoderidae) with notes on Eurasian ruffe as final host for Phyllodistomum spp.

BACKGROUND: Host-specificity patterns are not well-defined for trematodes of the genus Phyllodistomum Braun, 1899. The Eurasian ruffe, Gymnocephalus cernuus L., has been recorded as a definitive host for Phyllodistomum folium (Olfers, 1816), P. angulatum Linstow, 1907 and P. megalorchis Nybelin, 1926 and as the type-host for P. pseudofolium Nybelin (1926). A wide range of other host fishes have been recorded for these species as well. All present host records have been based on light microscopy and the life-cycles of P. pseudofolium, P. angulatum and P. megalorchis are unknown. The validity of P. pseudofolium and P. megalorchis require verification. In this study, rDNA sequences generated from adult Phyllodistomum spp., as well as from larval stages developing in Pisidium amnicum Müller, were analysed to establish the real number of Phyllodistomum species utilizing G. cernuus, and to associate larvae with the corresponding adult forms. RESULTS: Phylogenetic analyses of adult and larval stages of Phyllodistomum spp. based on ITS2 and partial 28S rDNA data allowed the confirmation of the validity of P. pseudofolium. A macrocercous cercaria, known as Phyllodistomum sp. from P. amnicum is genetically identical to adult P. pseudofolium. Phyllodistomum megalorchis obtained from its type-host, Lota lota L., showed no genetic differences from P. angulatum parasitizing Sander lucioperca L. In our analysis, P. pseudofolium, P. angulatum and P. macrocotyle formed a highly supported clade despite the fact that these species appear to be associated with distinct patterns of first intermediate host identity and cercarial morphology. Some morphological differences between gravid specimens of P. pseudofolium and P. angulatum were observed and their SEM tegumental surface topography is described. CONCLUSIONS: The results lead us to the perception that macroevolutionary host switching in the genus Phyllodistomum is independent of host phylogeny. This study suggests strict host-specificity (oioxeny) for P. pseudofolium using one first intermediate host species (P. amnicum) and one definitive host species (G. cernuus). Phyllodistomum megalorchis is to be regarded as a synonym of P. angulatum. The close phylogenetic relatives, P. pseudofolium and P. angulatum, can be differentiated by morphological traits, the micromorphology and tegumental surface topography of these two species is intended to provide useful data for their identification and support the use of such features as a valuable taxonomic criterion. Molecular data showed that G. cernuus is a definitive host for two species: the oioxenous P. pseudofolium and the euryxenous P. folium.

Environmental genotoxicity assessment along the transport routes of chemical munitions leading to the dumping areas in the Baltic Sea.

The frequencies of micronuclei (MN), nuclear buds (NB) and nuclear buds on filament (NBf) were examined in 660 specimens of herring (Clupea harengus) collected in 2009-2014 at 65 study stations located mainly along the chemical munition transport routes in the Baltic Sea. The frequency of nuclear abnormalities was strongly increased in herring caught at four stations located close to chemical munition dumping sites, or CWAs - substances (chemical warfare agents) in sediments. Significant increase of MN, NB and NBf was observed in fish caught November 2010-2013 compared to 2009. The most significantly increased genotoxicity responses were recorded in fish caught at stations along CW (chemical weapons) transport routes, close to the Bornholm CW dumping area, in zones with CWAs in sediments and with oil-gas platforms.

Phylogenetic relationships of some species of the family Echinostomatidae Odner, 1910 (Trematoda), inferred from nuclear rDNA sequences and karyological analysis.

The family Echinostomatidae Looss, 1899 exhibits a substantial taxonomic diversity, morphological criteria adopted by different authors have resulted in its subdivision into an impressive number of subfamilies. The status of the subfamily Echinochasminae Odhner, 1910 was changed in various classifications. Genetic characteristics and phylogenetic analysis of four Echinostomatidae species - Echinochasmus sp., Echinochasmuscoaxatus Dietz, 1909, Stephanoprorapseudoechinata (Olsson, 1876) and Echinoparyphiummordwilkoi Skrjabin, 1915 were obtained to understand well enough the homogeneity of the Echinochasminae and phylogenetic relationships within the Echinostomatidae. Chromosome set and nuclear rDNA (ITS2 and 28S) sequences of parthenites of Echinochasmus sp. were studied. The karyotype of this species (2n=20, one pair of large bi-armed chromosomes and others are smaller-sized, mainly one-armed, chromosomes) differed from that previously described for two other representatives of the Echinochasminae, Echinochasmusbeleocephalus (von Linstow, 1893), 2n=14, and Episthmiumbursicola (Creplin, 1937), 2n=18. In phylogenetic trees based on ITS2 and 28S datasets, a well-supported subclade with Echinochasmus sp. and Stephanoprorapseudoechinata clustered with one well-supported clade together with Echinochasmusjaponicus Tanabe, 1926 (data only for 28S) and Echinochasmuscoaxatus. These results supported close phylogenetic relationships between Echinochasmus Dietz, 1909 and Stephanoprora Odhner, 1902. Phylogenetic analysis revealed a clear separation of related species of Echinostomatoidea restricted to prosobranch snails as first intermediate hosts, from other species of Echinostomatidae and Psilostomidae, developing in Lymnaeoidea snails as first intermediate hosts. According to the data based on rDNA phylogeny, it was supposed that evolution of parasitic flukes linked with first intermediate hosts. Digeneans parasitizing prosobranch snails showed higher dynamic of karyotype evolution provided by different chromosomal rearrangements including Robertsonian translocations and pericentric inversions than more stable karyotype of digenean worms parasitizing lymnaeoid pulmonate snails.

Molecular and karyological identification and morphological description of cystocercous cercariae of Phyllodistomum umblae and Phyllodistomum folium (Digenea, Gorgoderidae) developing in European sphaeriid bivalves.

Two cystocercous gorgoderid cercariae of the genus Phyllodistomum Braun, 1899, Phyllodistomum umblae (Fabricius, 1780) and Phyllodistomum folium (Olfers, 1816), developing in sphaeriid bivalves from Norway and Lithuania, were studied and compared. Our previous molecular studies revealed very close phylogenetic relation of these two species and proved that cystocercous cercariae of P. folium develop in sporocysts in the gills of sphaeriid bivalves. In the present study morphological descriptions are given for the two cercariae, together with karyological and molecular characteristics. Comparative karyological and sequence analysis using ITS2 and 28S rDNA revealed the conspecificity of cercariae emitted from Pisidium hibernicum and Sphaerium corneum with adult P. umblae from Thymallus thymallus and Coregonus albula. The cercariae of P. folium and P. umblae are very similar morphologically with main differences in the structure of the tail. The two species clearly differ in karyotype structure. The diploid set of P. folium is composed of 18 chromosomes, but 16 chromosomes with one pair of large metacentrics were found in mitotic cells of P. umblae. Interspecific karyotypic difference presumably arose from Robertsonian fusion of two uni-armed chromosomes.

The taxonomic identity and phylogenetic relationships of Cercaria pugnax and C. helvetica XII (Digenea: Lecitho-dendriidae) based on morphological and molecular data.

The present study analysed the taxonomic status and phylogenetic relationships of two species of xiphidiocercariae of the 'microcotylae' group, Cercaria pugnax La Valette St. George, 1855, from Viviparus viviparus (Linnaeus) in the Ukraine and Cercaria helvetica XII Dubois, 1928 from Bithynia tentaculata (Linnaeus) in Lithuania. Molecular phylogenetic analyses based on sequences of the ITS2 region and partial 28S gene of the nuclear rDNA revealed that both these xiphidiocercariae belong to the Lecithodendriidae Lühe, 1901 and represent larval stages of lecithodendriids parasitic in bats. Cercaria helvetica XII clustered with the typical representatives of the genus Lecithodendrium Looss, 1896, being very close, but not identical, to Lecithodendrium linstowi Dollfus, 1931. Sequences of C. pugnax matched exactly the sequences of adult Paralecithodendrium chilostomum (Mehlis, 1831). Morphological descriptions of the cercariae are included; these represent the first report of non-virgulate xiphidiocercariae belonging to the family Lecithodendriidae. Until now, the presence of glandular virgula organ in the region of the oral sucker was considered a robust synapomorphy for the Lecithodendriidae and several closely related families. Our results have shown that the relative importance of this character is in need of a re-assessment.

Rhipidocotyle fennica (Digenea: Bucephalidae) from Anodonta anatina and pike Esox lucius in Lithuania.

Ribosomal DNA sequences of Rhipidocotyle sp. adults from Esox lucius were shown to be identical to sequences of larval Rhipidocotyle fennica, occurring in Anodonta anatina in Lake Vilkoksnis, Lithuania. Morphological features and host specificity of this adult worm correspond with that, determinate in the first description of R. fennica in Finland. These data give the first evidence that a viable population of R. fennica exists in east central Europe. Bucephalus polymorphus which was reported in unionids in all previous publications is probably R. fennica.

Differentiation of European freshwater bucephalids (Digenea: Bucephalidae) based on karyotypes and DNA sequences.

Three species of bucephalid digeneans are known in European freshwater habitats. In this study parthenitae of Rhipidocotyle campanula (Dujardin, 1845) and R. fennica Gibson, Taskinen & Valtonen, 1992, infecting unionid bivalves, and adult Bucephalus polymorphus von Baer, 1827 from perch (Perca fluviatilis L.) were investigated using karyological analysis and DNA sequencing. Our previously published data on genetic characteristics of parthenitae of B. polymorphus from Dreissena polymorpha Pallas were used for comparative analysis. Ribosomal DNA sequences (ITS2 and 28S rDNA) were used to estimate the phylogenetic relationships of the three bucephalid species. Very close phylogenetic affinity between investigated species was revealed; the sequence difference between the two species of Rhipidocotyle Diesing, 1858 (3.78% based on 28S) was comparable with intergeneric differences observed in comparisons of B. polymorphus with R. campanula and R. fennica (3.43% and 4.49% based on 28S, respectively). A high degree of similarity was noted in karyotype structure of the two species of Rhipidocotyle. The diploid chromosome sets consist of 14 bi-armed chromosomes with the first pair of metacentric elements markedly larger than the remaining chromosomes. This chromosome set structure is also specific to B. polymorphus. One specimen of Anodonta anatina L. was infected with tetraploid R. fennica (4n = 28). On the basis of karyotype characters and molecular data, species of the genus Rhipidocotyle cannot be recognised as more closely related to each other than to B. polymorphus. Our findings of Lithuanian and Ukrainian populations of unionid mussels infected with R. fennica provide evidence that this species occurs not only in Finland but also in Central and Eastern Europe. Previous reports of B. polymorphus in unionids in these regions are equivocal because of possible confusion with R. fennica.

Comparison of the developmental stages of some European allocreadiid trematode species and a clarification of their life-cycles based on ITS2 and 28S sequences.

Genetic data were used to examine the diversity in some allocreadiid trematodes. Nuclear ribosomal DNA (ITS2 and partial sequences of 5.8S and 28S) was sampled from sexual adult and 'larval' stages. From these and previous reference datasets phylogenetic trees were constructed. The results uncovered genetically distinct lineages within Bunodera luciopercae (Müller, 1776), suggesting that the two Palaearctic subspecies, B. l. luciopercae and B. l. acerinae Roitman & Sokolov, 1999, and Nearctic B. luciopercae from Perca flavescens may represent distinct species with a restricted host-specificity. Identical rDNA was revealed for the sexual adult of B. l. acerinae and 'larval' B. luciopercae described by Wisniewski (1958). An unexpected match between the rDNA sequences of adult B. l. luciopercae and 'larval' Allocreadium isoporum (sensu Wisniewski, 1958) was also detected. The adult A. isoporum (Looss, 1894) differs significantly from the 'larval' A. isoporum, the level of rDNA sequence divergence between them (8.6 % for 5.8S-ITS2-28S sequences and 6.26% for 28S) being consistent with the level expected for intergeneric variation. These results revealed the possible existence of a cryptic species complex within the nominal species B. luciopercae and a clear need for reconsideration of some of the accepted, but largely untested, tenets regarding allocreadiid life-cycles.

Comparison of rDNA sequences from colchicine treated and untreated sporocysts of Phyllodistomum folium and Bucephalus polymorphus (Digenea).

The most frequently used antimitotic agent in cytogenetic studies is colchicine. We investigated whether the initial treatment of trematodes for karyological analysis with colchicine would have mutagenic or degradational effect on rDNA sequences. Dreissena polymorpha is the intermediate host of Phyllodistomum folium and Bucephalus polymorphus, and the sporocyst stage of these trematode species develop, respectively, in the gills and gonads of this mussel. Sporocysts of P. folium and B. polymorphus were obtained from D. polymorpha collected from waterbodies in Belarus and in Lithuania. 5.8S and 28S rDNA genes, ITS1 and ITS2 of P folium and B. polymorphus were sequenced and compared, and no nucleotide sequence differences between colchicine treated and untreated trematodes were found. Based on these results, we conclude that colchicine treatment for 3-5 h has no mutagenic or degradational effect on rDNA sequences. During the course of this investigation, two genetically different P. folium samples were noted in Belarus.