Ivica Králová-Hromadová

SEQUENCE ANALYSIS OF RIBOSOMAL AND MITOCHONDRIAL GENES OF THE GIANT LIVER FLUKE FASCIOLOIDES MAGNA (TREMATODA: FASCIOLIDAE): INTRASPECIFIC VARIATION AND DIFFERENTIATION FROM FASCIOLA HEPATICA

Complete sequences of ribosomal and mitochondrial genes of the giant liver fluke Fascioloides magna are presented. In particular, small subunit (18S) and internal transcribed spacers (ITS1 and ITS2) of the ribosomal gene (rDNA), as well as cytochrome c oxidase subunit I (cox1) and nicotinamide dehydrogenase subunit I (nad1) of the mitochondrial DNA (mtDNA), were analyzed. The 18S and ITS sequences were compared with previously published sequences of the liver fluke Fasciola hepatica. Fixed interspecific genetic differences were determined that allow molecular differentiation of F. magna and F. hepatica using either the PCR-RFLP method or PCR amplification of species-specific DNA regions. Additionally, intraspecific sequence polymorphism of the complete cox1 and nad1 mitochondrial genes in geographically distinct F. magna populations was determined. Based on the sequence divergences, short (<500 bp) variable regions suitable for broader biogeographical studies of giant liver fluke were designed.

Intra-individual internal transcribed spacer 1 (ITS1) and ITS2 ribosomal sequence variation linked with multiple rDNA loci: A case of triploid Atractolytocestus huronensis, the monozoic cestode of common carp

Complete sequences of the ribosomal internal transcribed spacers (ITS1 and ITS2) and karyological characters of the monozoic (unsegmented) tapeworm Atractolytocestus huronensis Anthony, 1958 (Cestoda: Caryophyllidea) from Slovakia were analysed, revealing considerable intra-genomic variability and triploidy in all analysed specimens. Analysis of 20 sequences of each ITS1 and ITS2 spacer yielded eight and 10 different sequence types, respectively. In individual tapeworms, two to four ITS1 and three to four ITS2 sequence types were found. Divergent intra-genomic ITS copies were mostly induced by nucleotide substitutions and different numbers of short repetitive motifs within the sequence. In addition, triploidy was found to be a common feature of A. huronensis. The karyotype of Slovakian A. huronensis possesses three sets of chromosomes (3n=24, n=4m+3st+1minute chromosome), similar to the previously described triploidy in conspecific tapeworms from North America. Fluorescent in situ hybridisation (FISH) with a ssrDNA probe revealed two distinct rDNA clusters for each homologue of the triplet number 2. To date, A. huronensis is the only cestode species in which intra-individual ITS sequence variants were found in parallel with its triploid nature and multiple rDNA loci. Some of these molecular and genetic features were observed in several other species of basal or nearly basal tapeworms of the orders Caryophyllidea and Diphyllobothriidea, which indicates that the phenomena may be characteristic for evolutionarily lower tapeworms and deserve more attention in future studies.

Determination of ribosomal internal transcribed spacer 2 (ITS2) interspecific markers in Fasciola hepatica, Fascioloides magna, Dicrocoelium dendriticum and Paramphistomum cervi (Trematoda), parasites of wild and domestic ruminants

SummaryThe species-specific ribosomal internal transcribed spacer 2 (ITS2) markers were designed for PCR-based molecular differentiation of Fasciola hepatica, Fascioloides magna, Dicrocoelium dendriticum and Paramphistomum cervi, liver and stomach flukes of domestic and free living ruminants. Complete ITS2 sequences were obtained for D. dendriticum and P. cervi, for the later species, ITS2 structure was determined for the first time. Intraspecific variation within geographically distant populations was found to be either very low (F. hepatica; D. dendriticum) or even absent (F. magna; P. cervi). ITS2 regions with the absence of intraspecific polymorphisms but with interspecific sequence heterogeneity were applied for design of speciesspecific primers. The specificity of developed primers was tested on genomic DNA isolated from adult individuals of studied fluke species. Application of the primers is of particular value for molecular differentiation of morphologically hardly distinguishable F. hepatica, F. magna and P. cervi eggs after coprological examinations.

Multiple origins of European populations of the giant liver fluke Fascioloides magna (Trematoda: Fasciolidae), a liver parasite of ruminants ☆

The giant liver fluke, Fascioloides magna, a liver parasite of free-living and domestic ruminants of Europe and North America, was analysed in order to determine the origin of European populations and to reveal the biogeography of this originally North American parasite on the European continent. The variable fragments of the mitochondrial cytochrome c oxidase subunit I (cox1; 384bp) and nicotinamide dehydrogenase subunit I (nad1; 405bp) were used. Phylogenetic trees and haplotype networks were constructed and the level of genetic structuring was evaluated using population genetic tools. In F. magna individuals originating from all European foci of infection (Italy, Czech Republic and Danube floodplain forests involving the territories of Slovakia, Hungary and Croatia) and from four of five major North American enzootic areas, 16 cox1 and 18 nad1 haplotypes were determined. The concatenated sequence set produced 22 distinct haplotypes. The European fluke populations were less diverse than those from North America in that they contained proportionately fewer haplotypes (eight), while a more substantial level of genetic diversity and a greater number of haplotypes (15) were recorded in North America. Only one haplotype was shared between the European (Italy) and North American (USA/Oregon and Canada/Alberta) flukes, supporting a western North American origin of the Italian F. magna population. Haplotypes found in Italy were distinct from those determined in the remaining European localities which indicates that introduction of F. magna to the European continent occurred more than once. In the Czech focus of infection, a south-eastern USA origin was revealed. Identical haplotypes, common to parasites from the Czech Republic and from an expanding focus in Danube floodplain forests, implies that the introduction of F. magna to the Danube region came from an already established Czech focus of infection.

Substitution saturation and nuclear paralogs of commonly employed phylogenetic markers in the Caryophyllidea, an unusual group of non-segmented tapeworms (Platyhelminthes)

Caryophyllidean cestodes (Platyhelminthes) represent an unusual group of tapeworms lacking serially repeated body parts that potentially diverged from the common ancestor of the Eucestoda prior to the evolution of segmentation. Here we evaluate the utility of two nuclear and two mitochondrial molecular markers (ssrDNA and lsrDNA, nad3 and cox1) for use in circumscribing generic boundaries and estimating interrelationships in the group. We show that these commonly employed markers do not contain sufficient signal to infer well-supported phylogenetic estimates due to substitution saturation. Moreover, we detected multiple trnK+nad3+trnS+trnW+cox1 haplotypes within individuals, indicating a history of gene exchange between the mitochondrial and nuclear genomes. The presence of such nuclear paralogs (i.e. numts), to our knowledge described here in cestodes for the first time, together with the results of phylogenetic, saturation and split-decomposition analyses all suggest that finding informative markers for estimating caryophyllidean evolution is unusually problematic in comparison to other major lineages of tapeworms.

Application of a Reverse Line Blot hybridisation assay for the species-specific identification of cyathostomins (Nematoda, Strongylida) from benzimidazole-treated horses in the Slovak Republic.

Five horse farms located in eastern Slovakia were investigated for the presence of benzimidazole-resistant strongyles by faecal egg count reduction test and egg hatch assay. Coprocultures were prepared for each farm from faecal samples taken pre- and post-treatment and harvested larvae were molecularly examined with a Reverse Line Blot assay. Faecal egg count reduction values ranged from 0 to 52.5% and all farms were positive for benzimidazole-resistant cyathostomins. Seven benzimidazole-resistant cyathostomin species were molecularly identified on farms before and also after treatment. These data demonstrate that resistance to benzimidazoles is well established in cyathostomin populations from horse farms in the Slovak Republic and that the molecular assay was able to determine the species-specific distribution of resistant cyathostomins under field conditions.

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 (Beyşehir Lake), which are described herein as a new species. Paracaryophyllaeus vladkae Scholz, Oros and Aydoğdu 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.

Development and characterization of multiplex panels of polymorphic microsatellite loci in giant liver fluke Fascioloides magna (Trematoda: Fasciolidae), using next-generation sequencing approach

The microsatellite markers were designed for the giant liver fluke, Fascioloides magna, veterinary important liver parasite of free-living and domestic ruminants. Due to its geographic distribution (five enzootic regions across USA and Canada, three permanent European foci) and invasive character, F. magna is an interesting model for population genetics. Out of 667 amplicon candidates generated after NGS, 118 provided the best resolution and were tested with PCR analysis. In total, 56 yielded PCR products of expected size and in 36 of them the declared repetitive motif was identified by Sanger sequencing. After fragment analysis, 12 loci were proved to be polymorphic in individuals from one tested European and four North American populations. These loci were selected for setup of multiplex STR assays and utilized in genotyping of larger sample cohort. The outputs of statistical analyses indicate further global application of 11 conclusive loci in population genetics of the parasite.

Molecular characterization of Fascioloides magna (Trematoda: Fasciolidae) from south-western Poland based on mitochondrial markers

Abstract The giant liver fluke, Fascioloides magna, is a veterinary important liver parasite of free living and domestic ruminants. This originally North American parasite was introduced along with its cervid hosts to Europe where it has established three permanent natural foci - in northern Italy, central and southern parts of the Czech Republic and the Danube floodplain forests. The first record on fascioloidosis in Poland originated from the Lower Silesian Forest in south-western Poland and since then an occurrence of F. magna in this country has not been documented. Recently, the parasitological examination of red deer (Cervus elaphus elaphus) from the Lower Silesian Wilderness (south-western Poland) revealed the presence of F. magna eggs. In order to determine the genetic interrelationships of the Polish giant liver fluke individuals, they were molecularly analyzed by mitochondrial cytochrome c oxidase subunit I (cox1) and nicotinamide dehydrogenase subunit I (nad1) and compared with haplotypes of so far studied European populations of the parasite. The study revealed the genetic uniformity of F. magna specimens from Poland with part of individuals from the Czech natural focus. Note: Nucleotide sequence data reported in this paper are available in the GenBankTM, EMBL and DDBJ databases under the accession numbers KP635008-9.

Ribosomal ITS2 structure in Caryophyllaeus laticeps and Caryophyllaeus brachycollis (Cestoda: Caryophyllidea), parasites of cyprinid fish

SummaryRibosomal internal transcribed spacer 2 (ITS2) structure was studied in monozoic tapeworms Caryophyllaeus laticeps (Pallas, 1781) from freshwater bream Abramis brama (L.) and white-eyed bream Ballerus sapa (Pallas) and Caryophyllaeus brachycollis Janiszewska, 1951 from A. brama and Mediterranean barbel Barbus meridionalis (Risso). Homologous intragenomic ITS2 structure (474 bp) was determined for C. brachycollis from both fish hosts and for C. laticeps from A. brama (486 bp). Contrary to this, divergent intragenomic ITS2 copies (ITS2 paralogues) were detected in C. laticeps from B. sapa. They were mostly induced by different numbers of short repetitive motif (TA)n within the sequences, allowing their assortment into two ITS2 variants (457 and 467 bp). Current data represent first information on ITS2 structure/ITS paralogues in the caryophyllidean family Caryophyllaeidae with focus on their applicability in the molecular taxonomy of the genus Caryophyllaeus.