Diversity of European lissorchiid trematodes from fish and snail hosts with comments on the validity of the genus Parasymphylodora Szidat, 1943.

Genetic markers, DNA sequences and karyotypes, of some European lissorchiid species from their intermediate and final hosts were obtained to clarify controversial data about their life cycles and taxonomy, and to reveal phylogenetic affinities. The life cycles of three species have been confirmed for the first time based on molecular data. Comparative analysis of internal transcribed spacer 2 (ITS2) and partial 28S rDNA sequences has undoubtedly proven that cercariaeum of type-species of the genus Asymphylodora, Asymphylodora tincae, develops in pulmonate snails, Anisus vortex and Stagnicola palustris, but not in the genus Bithynia. The faucet snail, Bithynia tentaculata, serves as the first intermediate host for Parasymphylodora (=Asymphylodora) markewitschi and Parasymphylodora parasquamosa; adults of both species were isolated from the common rudd, Scardinius erythrophthalmus. It has also been confirmed that B. tentaculata serves as the second intermediate host for P. parasquamosa. Phylogenetic analysis supports the validity of the genus Parasymphylodora. Two species, Parasymphylodora markewitschi and P. parasquamosa, with cercariaeum belonging to the Parasquamosum group, are closely related and are being recovered as a well-defined evolutionary lineage in phylogenetic trees. A significant divergence between Parasymphylodora spp. and Asymphylodora spp. was revealed. The diploid chromosome set of P. markewitschi is composed of 14 chromosomes and does not show similarities with karyotypes of other lissorchiid species. Asymphylodora progenetica and Asymphylodora tincae share the basal diploid value of the family, 2n = 20, and reveal very close morphology of the corresponding chromosome pairs. Karyotypic similarities of these species are in accordance with molecular phylogenetic data. Thus, the available molecular and cytogenetic data support the assignment of P. markewitschi and P. parasquamosa to a separate genus, meanwhile, the assignment of A. progenetica to the genus Parasymphylodora was not justified.

Exploring species diversity of lissorchiid trematodes (Digenea: Lissorchiidae) associated with the gravel snail, Lithoglyphus naticoides, in European freshwaters.

Comparative analysis using complete ITS2 and partial 28S rDNA sequence data revealed that cercariaeum developing in rediae in Lithoglyphus naticoides represent two different lissorchiid species. One morphotype of cercariaeum is conspecific with adult Palaeorchis incognitus from European roach, Rutilus rutilus. The other cercariaeum is attributable to the genus Asymphylodora, but the species identity is not yet determined. We also generate the first rDNA sequences for Asymphylodora progenetica based on new collections from Bithynia tentaculata from Lithuania. Phylogenetic analyses of the newly generated sequences, together with information for other lissorchiids available on GenBank, showed that all representatives of Lissorchiidae form a strongly supported clade. Three monophyletic lineages, Asymphylodora, Palaeorchis and Lissorchis, were recognized at the generic level. Karyological analysis of the chromosome set of larval P. incognitus revealed a diploid number of 2n = 20. Its karyotype with subtelocentric chromosomes prevailing can be regarded as comparatively 'primitive', which is consistent with the basal position of P. incognitus in the 28S tree relative to the representatives of the genus Asymphylodora. The present study adds significant new information for establishing species-specific markers for the confident characterization of different developmental stages of lissorchiid species and clarification of their life cycles.

Molecular data confirm the taxonomic position of Hymenolepis erinacei (Cyclophyllidea: Hymenolepididae) and host switching, with notes on cestodes of Palaearctic hedgehogs (Erinaceidae).

The cestode Hymenolepis erinacei is regarded as a widely distributed parasite in European hedgehogs of the genus Erinaceus, although the taxonomic position of this hymenolepidid has been debated for a considerable period of time. We present the first molecular data for this cestode, including partial DNA sequences of mitochondrial 16S and nuclear 28S ribosomal genes. Molecular phylogenetic analysis clusters H. erinacei in one clade together with representatives of the genus Hymenolepis from rodents. Characteristic morphological features, including the oval embryophore without filaments and shape of the embryonic hooks of H. erinacei are described. Features of these cestode eggs are proposed as a basis for non-invasive detection of parasitic infections in small mammal populations. The present study explores phylogenetic relationships within the genus Hymenolepis and the host switching related to H. erinacei. Cases of host switching in other genera of the family Hymenolepididae are reviewed. A short critical review of cestodes parasitizing hedgehogs in the Palaearctic is presented.

Erwinia typographi sp. nov., isolated from bark beetle (Ips typographus) gut.

Gram-negative-staining bacteria that were resistant to monoterpene myrcene (7-methyl-3-methylene-1.6-octadiene, C10H16, at concentrations of up to 10 µl ml(-1) in TSB) were isolated from the gut contents of adult bark beetles Ips typographus (Coleoptera, Scolytidae). The beetles were collected from the bark of Norway spruce (Picea abies) in Lithuania. Bark beetles feed on conifers, which produce myrcene among many other defensive compounds. It has been suggested that the micro-organisms present within the beetles' guts could be involved in their resistance towards this plant defensive compound. The most resistant bacterial strains were isolated and characterized by phenotypic assays as well as fatty acid analysis, 16S rRNA gene sequencing, multilocus sequence analyses (MLSA) based on the rpoB, atpD and infB genes and DNA-DNA hybridization. Biochemical characterization indicated that the bacteria belonged to the family Enterobacteriaceae. Phylogenetic analyses of the 16S rRNA gene sequences and MLSA of the novel strains revealed that they belonged to the genus Erwinia, but represented a novel species. The dominant cellular fatty acids were C16:0 and C17:0 cyclo. The DNA G+C content was 49.1 mol%. The results obtained in this study indicated that these bacteria from the bark beetle gut represented a novel species, for which the name Erwinia typographi sp. nov. is proposed, with the type strain DSM 22678T (=Y1T=LMG 25347T).

Clarification of the systematic position of Cercariaeum crassum Wesenberg-Lund, 1934 (Digenea), based on karyological analysis and DNA sequences.

Chromosome set and rDNA sequences of the larval digenean Cercariaeum crassum were analysed in order to clarify its systematic position and possible adult form. Parasites were obtained from the sphaeriid bivalve Pisidium amnicum, collected in Lithuanian and Finnish rivers. The karyotype is shown to consist of five pairs (2n = 10) of large, up to 14 µm, chromosomes. Complement, composed of a low diploid number of exclusively bi-armed elements, presumably arose through Robertsonian fusions of acrocentric chromosomes. Consistent with a Robertsonian-derived karyotype, one or two small, metacentric, mitotically stable B chromosomes were detected in the cells of parthenitae isolated from some host individuals. A phylogenetic analysis using rDNA internal transcribed spacer 2 (ITS2) and 28S sequences corroborates the allocation of C. crassum to the family Allocreadiidae. In neighbour-joining and maximum parsimony phylogenetic trees C. crassum clusters into one clade with Allocreadium spp., and is the closest sister group in relation to A. isoporum; the level of rDNA sequence divergence between them (2.67% for ITS2 and 1.16% for 28S) is consistent with the level expected for intrageneric variation. The present study adds significant information to a database for establishing species-specific characters for confident characterization of different developmental stages of allocreadiid species, clarification of their life cycles and evaluation of intra- and interspecific variability.

Cytogenetic and sequence comparison of adult Phyllodistomum (Digenea: Gorgoderidae) from the three-spined stickleback with larvae from two bivalves.

Due to the low informative value of available morphological characters, cytogenetic and molecular methods, based on rDNA sequencing, were used to characterize adult and larval stages of Phyllodistomum spp. Species studied have 18 chromosomes with comparable absolute and relative lengths. Conventional Giemsa staining and karyometric analysis revealed clear differences in chromosome morphology of larval Phyllodistomum spp. infecting two bivalve host species, Sphaerium corneum and Pisidium amnicum. However, karyotypes of adult P. folium from three-spined sticklebacks and larval stages from S. corneum appear almost identical both with respect to the relative lengths and centromeric indices of the corresponding chromosome pairs. The entire internal transcribed spacer (ITS) region (ITS-1, 5.8S and ITS-2) and the D1-D3 region of 28S gene were sequenced and compared. Again, sufficient differences were observed between larval Phyllodistomum spp., while adult P. folium and larvae from S. corneum showed a high level of similarity. So, both cytogenetic and molecular data support the suggestion that they represent developmental stages of the same species. The results were compared with published data obtained by cytogenetic and molecular studies on the other Phyllodistomum species. Differences revealed in karyotype and rDNA sequences leads to the conclusion that the cercariaeum of P. folium sensu Sinitsin, 1905 could not be regarded as the larva of adult P. folium from three-spined stickleback.