DNA sequence data became an integral part of species characterization and identification. Still, specimens associated with a particular DNA sequence must be identified by the use of traditional morphology-based analysis and correct linking of sequence and identification must be ensured. Only a small part of DNA sequences of the genus Diplostomum (Diplostomidae) is based on adult isolates which are essential for accurate identification. In this study, we provide species identification with an aid of morphological and molecular (cox1, ITS-5.8S-ITS2 and 28S) characterization of adults of Diplostomum baeri Dubois, 1937 from naturally infected Larus canus Linnaeus in Karelia, Russia. Furthermore, we reveal that the DNA sequences of our isolates of D. baeri are identical with those of the lineage Diplostomum sp. clade Q , while other sequences labelled as the ‘D. baeri’ complex do not represent lineages of D. baeri. Our new material of cercariae from Radix balthica (Linnaeus) in Ireland is also linked to Diplostomum sp. clade Q. We reveal that D. baeri is widely distributed in Europe; as first intermediate hosts lymnaeid snails (Radix auricularia (Linnaeus), R. balthica) are used; metacercariae occur in eye lens of cyprinid fishes. In light of the convoluted taxonomy of D. baeri and other Diplostomum spp., we extend the recommendations of Blasco-Costa et al. (2016, Systematic Parasitology 93, 295–306) for the ‘best practice’ in molecular approaches to trematode systematics. The current study is another step in elucidating the species spectrum of Diplostomum based on integrative taxonomy with well-described morphology of adults linked to sequences.

• Klíčová slova
• Ireland, Laridae, Lymnaeidae, Russia, nuclear and mitochondrial DNA, trematoda,
• MeSH
• Cyprinidae * MeSH
• fylogeneze MeSH
• infekce červy třídy Trematoda * parazitologie   veterinární   MeSH
• nemoci ryb * parazitologie   MeSH
• Trematoda * MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Evropa MeSH

BACKGROUND: Metacercariae of Diplostomum are important fish pathogens, but reliable data on their diversity in natural fish populations are virtually lacking. This study was conducted to explore the species diversity and host-parasite association patterns of Diplostomum spp. in a large riverine system in Europe, using molecular and morphological data. METHODS: Twenty-eight species of fish of nine families were sampled in the River Danube at Nyergesújfalu in Hungary in 2012 and Štúrovo in Slovakia in 2015. Isolates of Diplostomum spp. were characterised morphologically and molecularly. Partial sequences of the 'barcode' region of the cytochrome c oxidase subunit 1 (cox1) and complete sequences of the nicotinamide adenine dinucleotide dehydrogenase subunit 3 (nad3) mitochondrial genes were amplified for 76 and 30 isolates, respectively. The partial cox1 sequences were used for molecular identification of the isolates and an assessment of haplotype diversity and possible host-associated structuring of the most prevalent parasite species. New primers were designed for amplification of the mitochondrial nad3 gene. RESULTS: Only lens-infecting Diplostomum spp. were recovered in 16 fish species of five families. Barcoding of representative isolates provided molecular identification for three species/species-level genetic lineages, D. spathaceum, D. pseudospathaceum and 'D. mergi Lineage 2', and three single isolates potentially representing distinct species. Molecular data helped to elucidate partially the life-cycle of 'D. mergi Lineage 2'. Many of the haplotypes of D. spathaceum (16 in total), D. pseudospathaceum (15 in total) and 'D. mergi Lineage 2' (7 in total) were shared by a number of fish hosts and there was no indication of genetic structuring associated with the second intermediate host. The most frequent Diplostomum spp. exhibited a low host-specificity, predominantly infecting a wide range of cyprinid fishes, but also species of distant fish families such as the Acipenseridae, Lotidae, Percidae and Siluridae. The nad3 gene exhibited distinctly higher levels of interspecific divergence in comparison with the cox1 gene. CONCLUSIONS: This first exploration of the species diversity and host ranges of Diplostomum spp., in natural fish populations in the River Danube, provided novel molecular, morphological and host-use data which will advance further ecological studies on the distribution and host ranges of these important fish parasites in Europe. Our results also indicate that the nad3 gene is a good candidate marker for multi-gene approaches to systematic estimates within the genus.

• Klíčová slova
• Barcodes, Diplostomidae, Diplostomum, Europe, Freshwater fishes, Metacercariae, River Danube, cox1, nad3,
• MeSH
• fylogeneze MeSH
• genetická variace * MeSH
• mitochondriální DNA genetika   MeSH
• nemoci ryb parazitologie   MeSH
• oční čočka parazitologie   MeSH
• oční nemoci parazitologie   veterinární   MeSH
• řeky MeSH
• ryby parazitologie   MeSH
• taxonomické DNA čárové kódování * MeSH
• Trematoda genetika   izolace a purifikace   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Maďarsko epidemiologie   MeSH
• Slovenská republika epidemiologie   MeSH
• Názvy látek
• mitochondriální DNA MeSH

We analysed two novel databases containing 2,380 and 8,202 host-parasite-locality records for trematode parasites of molluscs and fishes, respectively, to assess the biodiversity of trematodes in their intermediate mollusc and fish hosts in the freshwater environment in Europe. The "mollusc" dataset covers large numbers of pulmonate (29 spp.), "prosobranch" (15 spp.) and bivalve (11 spp.) molluscs acting as first intermediate hosts for 171 trematode species of 89 genera and 35 families. Of these, 23 and 40 species utilise freshwater fishes as definitive and second intermediate hosts, respectively. The most frequently recorded families are the Echinostomatidae Looss, 1899, Diplostomidae Poirier, 1886 and Schistosomatidae Stilles & Hassal, 1898, and the most frequently recorded species are Diplostomum spathaceum (Rudolphi, 1819), D. pseudospathaceum Niewiadomska, 1984 and Echinoparyphium recurvatum (von Linstow, 1873). Four snail species harbour extremely rich trematode faunas: Lymnaea stagnalis (L.) (41 spp.); Planorbis planorbis (L.) (39 spp.); Radix peregra (O.F. Müller) (33 spp.); and R. ovata (Draparnaud) (31 spp.). The "fish" dataset covers 99 fish species of 63 genera and 19 families acting as second intermediate hosts for 66 species of 33 genera and nine families. The most frequently recorded families are the Diplostomidae Poirier, 1886, Strigeidae Railliet, 1919 and Bucephalidae Poche, 1907, and the most frequently recorded species are Diplostomum spathaceum (Rudolphi, 1819), Tylodelphys clavata (von Nordmann, 1832) and Posthodiplostomum cuticola (von Nordmann, 1832). Four cyprinid fishes exhibit the highest species richness of larval trematodes: Rutilus rutilus (L.) (41 spp.); Abramis brama (L.) (34 spp.); Blicca bjoerkna (L.) (33 spp.); and Scardinius erythrophthalmus (L.) (33 spp.). Larval stages of 50 species reported in fish are also reported in freshwater molluscs, thus indicating a relatively good knowledge of the life-cycles of fish trematodes in Europe. We provide host-parasite lists for 55 species of molluscs with a European distribution comprising 413 host-parasite associations.

• MeSH
• biodiverzita * MeSH
• ekosystém * MeSH
• larva MeSH
• měkkýši parazitologie   MeSH
• ryby parazitologie   MeSH
• Trematoda klasifikace   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Geografické názvy
• Evropa MeSH

BACKGROUND: Recent molecular studies have discovered substantial unrecognised diversity within the genus Diplostomum in fish populations in Europe and North America including three species complexes. However, data from the first intermediate host populations are virtually lacking. This study addresses the application of an integrative taxonomic approach to the cryptic species diversity of Diplostomum spp. in natural lymnaeid snail populations in Europe with a focus on the 'D. mergi' species complex. METHODS: Totals of 1,909 Radix auricularia, 349 Radix peregra, 668 Stagnicola palustris and 245 Lymnaea stagnalis were sampled at five reservoirs of the Ruhr river system in Germany and screened for infections with Diplostomum spp. Cercariae were examined and identified alive, fixed and under scanning electron microscopy. Sequences from the barcode region of the cytochrome c oxidase subunit 1 (cox1) mitochondrial gene and from the internal transcribed spacer cluster (ITS1-5.8S-ITS2) of the rRNA gene were amplified for 51 and 13 isolates, respectively. RESULTS: Detailed morphological and molecular analyses provided evidence for three named species (Diplostomum spathaceum, D. pseudospathaceum and D. parviventosum), and a further four species-level lineages ('D. mergi Lineages 2-4' and 'Diplostomum sp. Clade Q' in the lymnaeid snail populations from the Ruhr river basin. The paper provides the first descriptions of molecularly identified cercariae of D. spathaceum and of the cercariae of D. parviventosum, three lineages of the 'D. mergi' species complex and of 'Diplostomum sp. Clade Q'. CONCLUSION: The integration of molecular and morphological evidence for Diplostomum spp. achieved in this study will serve as a baseline for species identification of these important parasites of snail and fish populations and thus advance further studies on the distribution of Diplostomum spp. in Europe.

• MeSH
• biodiverzita MeSH
• fylogeneze MeSH
• hlemýždi klasifikace   parazitologie   MeSH
• infekce červy třídy Trematoda parazitologie   veterinární   MeSH
• molekulární sekvence - údaje MeSH
• nemoci ryb parazitologie   MeSH
• ryby klasifikace   parazitologie   MeSH
• Trematoda klasifikace   genetika   izolace a purifikace   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Německo MeSH
• Severní Amerika MeSH