An examination of nervous system revealed unexpected immunoreactivity of both secretory apparatus and excretory canals in plerocercoids of two broad tapeworms (Cestoda: Diphyllobothriidea).

Dibothriocephalus ditremus and Dibothriocephalus latus are diphyllobothriidean tapeworms autochthonous to Europe. Their larval stages (plerocercoids) may seriously alter health of their intermediate fish hosts (D. ditremus) or cause intestinal diphyllobothriosis of the final human host (D. latus). Despite numerous data on the internal structure of broad tapeworms, many aspects of the morphology and physiology related to host­parasite co-existence remain unclear for these 2 species. The main objective of this work was to elucidate functional morphology of the frontal part (scolex) of plerocercoids, which is crucial for their establishment in fish tissues and for an early attachment in final hosts. The whole-mount specimens were labelled with different antibodies and examined by confocal microscope to capture their complex 3-dimensional microanatomy. Both species exhibited similar general pattern of immunofluorescent signal, although some differences were observed. In the nervous system, FMRF amide-like immunoreactivity (IR) occurred in the bi-lobed brain, 2 main nerve cords and surrounding nerve plexuses. Differences between the species were found in the structure of the brain commissures and the size of the sensilla. Synapsin IR examined in D. ditremus occurred mainly around FMRF amide-like IR brain lobes and main cords. The unexpected finding was an occurrence of FMRF amide-like IR in terminal reservoirs of secretory gland ducts and excretory canals, which has not been observed previously in any tapeworm species. This may indicate that secretory/excretory products, which play a key role in host­parasite relationships, are likely to contain FMRF amide-related peptide/s.

Unique genetic structure of the human tapeworm Dibothriocephalus latus from the Alpine lakes region - a successful adaptation?

Dibothriocephalus latus is the most frequent causative agent of fish-borne zoonosis (diphyllobothriosis) in Europe, where it is currently circulating mainly in the Alpine lakes region (ALR) and Russia. Three mitochondrial genes (cox1, cob and nad3) and 6 microsatellite loci were analysed to determine how is the recently detected triploidy/parthenogenesis in tapeworms from ALR displayed at the DNA level. A geographically distant population from the Krasnoyarsk Reservoir in Russia (RU-KR) was analysed as a comparative population. One or 2 alleles of each microsatellite locus was detected in plerocercoids from RU-KR, corresponding to the microsatellite pattern of a diploid organism. In contrast, 1­3 alleles were observed in tapeworms from ALR, in accordance with their triploidy. The high diversity of mitochondrial haplotypes in D. latus from RU-KR implied an original and relatively stable population, but the identical structure of mitochondrial genes of tapeworms from ALR was probably a consequence of a bottleneck typical of introduced populations. These results indicated that the diploid/sexually reproducing population from RU-KR was ancestral, located within the centre of the distribution of the species, and the triploid/parthenogenetically reproducing subalpine population was at the margin of the distribution. The current study revealed the allelic structure of the microsatellite loci in the triploid tapeworm for the first time.

Mitochondrial and microsatellite data show close genetic relationships between Dibothriocephalus latus from South America (Argentina) and Europe (the Alpine lakes region).

The most frequent causative agent of diphyllobothriosis, a fish-borne parasitic zoonosis, is the broad fish tapeworm Dibothriocephalus latus distributed mainly throughout the Holarctic region. The larval stages of the tapeworm were also detected in native and introduced freshwater fish in several lakes in South America, particularly in the north-western Patagonia in Argentina. The main objective of the present study was to determine the genetic structure of D. latus from rainbow trout (Oncorhynchus mykiss), brown trout (Salmo trutta), and brook trout (Salvelinus fontinalis) from Gutiérrez Lake and Alicura Reservoir in Argentina using the sequences of three mitochondrial genes (cox1, cob, and nad3) and six polymorphic microsatellite loci. The results were compared with the corresponding molecular data of D. latus from Europe (Switzerland and Italy; the Alpine lakes region) and Asia (Siberia, Russia). Only one concatenated haplotype identical with the con-Ha1 specific for D. latus from the Alpine lakes region was detected in all individuals from Argentina. Three different alleles were detected in five out of six analysed microsatellite loci, indicating the presence of three sets of chromosomes. The same structure of microsatellite loci was recently observed in D. latus from Switzerland and Italy, in which triploidy was previously confirmed by cytogenetic study. The data on the mitochondrial genes, the allelic structure of microsatellite loci, and the principal coordinate analysis showed close genetic relationships between D. latus from Argentina and the Alpine lakes region, thus supporting the hypothesis of the European origin of the broad fish tapeworm from South America.

The first records of Spirometra erinaceieuropaei (Cestoda: Diphyllobothriidae), a causative agent of human sparganosis, in Latvian wildlife.

Diphyllobothriid tapeworms of the genus Spirometra are causative agents of sparganosis, food-borne zoonotic parasitic disease. They have been recorded in broad spectrum of hosts, including humans, in all continents except Antarctica. Spirometra tapeworms have been intensively studied in several Asian countries; however, they have been rather neglected in Europe. The aim of this study was to provide a pilot screening of Spirometra spp. in Latvia, where data on sparganosis are not available. Tapeworms morphologically identified as diphyllobothriid species were isolated from grey wolves Canis lupus and Eurasian lynxes Lynx lynx from Latvia during the hunting periods 2013-2019. The parasites were subjected to molecular genotyping using sequences of the partial large (LSU rDNA; 615 bp) and small (SSU rDNA; 720 bp) subunits of the nuclear ribosomal RNA gene and complete (1566 bp) cytochrome c oxidase subunit I gene of the mitochondrial DNA (cox1 mtDNA). Analyses of both ribosomal subunits of 13 tapeworms revealed no intraspecific variation within the respective rDNA subunits. On the other hand, sequence analysis of mitochondrial cox1 revealed intraspecific polymorphism displayed by 12 cox1 haplotypes. Comparison of the current data with sequences of the corresponding DNA regions deposited in the GenBank revealed 99.3-99.5% (LSU rDNA), 99.2% (SSU rDNA) and 99.6-100% (cox1 mtDNA) identity of studied tapeworms with Spirometra erinaceieuropaei, which provided the first confirmation of this diphyllobothriid tapeworm in Latvia. Since S. erinaceieuropaei is probably prevalent in Latvian wildlife and may also occur in other potential host species, further studies are needed in order to acquire complex data on its geographic distribution and transmission in the natural environment of Latvia, as well as on the spectrum of its intermediate, paratenic, and definitive hosts.

Comparative analysis of monozoic fish tapeworms Caryophyllaeus laticeps (Pallas, 1781) and recently described Caryophyllaeus chondrostomi Barcák, Oros, Hanzelová, Scholz, 2017, using microsatellite markers.

The monozoic tapeworm Caryophyllaeus laticeps has been characterized by five markedly different morphotypes largely corresponding to different fish hosts. Recently, the most distinct morphotype 4 from the common nase Chondrostoma nasus was studied in more details resulting in description of a new species Caryophyllaeus chondrostomi. The molecular study based on mitochondrial cox1 and ribosomal lsrDNA did not reveal any interspecific differences between C. laticeps and C. chondrostomi and did not provide any molecular support for recognition of these two species. In the current study, six polymorphic microsatellite markers were applied in order to detect molecular differences between the two species and to provide molecular evidence of validity of C. chondrostomi. While all six microsatellite loci were amplified in different geographic populations of C. laticeps, only two of them provided the amplification product in C. chondrostomi. Results on the Bayesian analysis assigned C. chondrostomi and all geographic populations of C. laticeps to distinct clusters. Neither any close relationships among C. laticeps populations nor specific position of C. chondrostomi were revealed. Contrary, the results of the principal coordinate analysis revealed striking genetic separation of C. chondrostomi with no overlaps with any of the C. laticeps population or morphotype. Caryophyllaeus chondrostomi very probably underwent morphological divergence as a result of ongoing speciation, but this process has not yet been accompanied by sufficient genetic divergence. In this particular case, microsatellites were proved to be better molecular discriminative markers than rDNA and mtDNA.

Host Switching of Zoonotic Broad Fish Tapeworm (Dibothriocephalus latus) to Salmonids, Patagonia.

Diphyllobothriosis is a reemerging zoonotic disease because of global trade and increased popularity of eating raw fish. We present molecular evidence of host switching of a human-infecting broad fish tapeworm, Dibothriocephalus latus, and use of salmonids as intermediate or paratenic hosts and thus a source of human infection in South America.

Asian fish tapeworm, Khawia japonensis (Yamaguti, 1934), has expanded its European invasive range.

The invasive fish tapeworm, Khawia japonensis (Yamaguti, 1934) originally described in Japan, is reported for the first time in Slovakia. The tapeworm was found in farmed common carp (Cyprinus carpio L.) from East-Slovakian breeding fish pond. This finding is registered 4 years after the first announcement of this parasite in Europe (Po River Basin in Italy) in 2010 and increases its distribution area within this continent. Morphological characterization of K. japonensis supplemented with DNA sequences of cox1 and ribosomal lsrDNA genes is provided. Specimens from Slovakia phenotypically corresponded with those from feral and farmed carps from China, Vietnam, and Italy. Moreover, 100 and 98.7% identity of partial ribosomal lsrDNA gene and mitochondrial cox1 genes, respectively, were detected with K. japonensis from Japan. The invasive and pathogenic potential of K. japonensis in commercial breeding fisheries and its possible further spread in natural habitats is difficult to estimate for now. As yet, K. japonensis appears to be without a major impact on commercial breeding fisheries, but calls for more attention to the problem of biological invasions.

Cytogenetics of Aspidogaster limacoides (Trematoda, Aspidogastrea): karyotype, spermatocyte division, and genome size.

A detailed cytogenetic analysis of the aspidogastrean fluke Aspidogaster limacoides revealed a karyotype consisting of six medium-sized chromosome pairs. The first and the last pairs were two-armed while four remaining were one-armed; 2n = 12, n = 1 m + 1 m - sm + 4a. Fluorescence in situ hybridization with 18S ribosomal DNA (rDNA) probe detected a single cluster of ribosomal genes (NOR) located in pericentromeric regions of the long arms of the third chromosome pair in a site of secondary constriction apparent in meiotic prophase, especially in diplotene. The silver nitrate staining showed only a single active NOR site on one of homologous chromosomes in the majority of spermatogonia and spermatocyte divisions. A course of meiosis corresponded to standard schemes. The nucleolus was apparent in early meiotic spermatocytes and disintegrated by the end of pachytene. For the first time in Aspidogastrea, the genome size was determined. The flow cytometry showed 1.21 pg DNA per haploid nucleus in A. limacoides which is in accordance with relatively low genome sizes of other flukes and tapeworms (Neodermata). A comparison of cytogenetic data available to date in the fluke sister groups Aspidogastrea and Digenea suggests that the lower chromosome number of Aspidogastrea might represent an ancestral condition and their split might have been accompanied by an increase in chromosome number via either chromosome fissions or paleopolyploidy.

Development of microsatellite markers in Caryophyllaeus laticeps (Cestoda: Caryophyllidea), monozoic fish tapeworm, using next-generation sequencing approach.

Caryophyllaeus laticeps (Pallas 1781) (Cestoda: Caryophyllidea) is a monozoic tapeworm of cyprinid fishes with a distribution area that includes Europe, most of the Palaearctic Asia and northern Africa. Broad geographic distribution, wide range of definitive fish hosts and recently revealed high morphological plasticity of the parasite, which is not in an agreement with molecular findings, make this species to be an interesting model for population biology studies. Microsatellites (short tandem repeat (STR) markers), as predominant markers for population genetics, were designed for C. laticeps using a next-generation sequencing (NGS) approach. Out of 165 marker candidates, 61 yielded PCR products of the expected size and in 25 of the candidates a declared repetitive motif was confirmed by Sanger sequencing. After the fragment analysis, six loci were proved to be polymorphic and tested for heterozygosity, Hardy-Weinberg equilibrium and the presence of null alleles on 59 individuals coming from three geographically widely separated populations (Slovakia, Russia and UK). The number of alleles in particular loci and populations ranged from two to five. Significant deficit of heterozygotes and the presence of null alleles were found in one locus in all three populations. Other loci showed deviations from Hardy-Weinberg equilibrium and the presence of null alleles only in some populations. In spite of relatively low polymorphism and the potential presence of null alleles, newly developed microsatellites may be applied as suitable markers in population genetic studies of C. laticeps.

Conflict between morphology and molecular data: a case of the genus Caryophyllaeus (Cestoda: Caryophyllidea), monozoic tapeworms of cyprinid fishes.

Molecular analysis of an extensive specimen collection of morphologically distinct tapeworms of the genus Caryophyllaeus Gmelin, 1790 (Cestoda: Caryophyllidea), parasites of cyprinid fishes in the Palaearctic Region, brought evidence of host-related plasticity in critical morphological characters widely used for species circumscription and classification of these tapeworms. The results obtained here do not support the traditionally used morphological concept of species-defining characters of the order Caryophyllidea Carus, 1863, especially due to high morphological plasticity of the scolex and the anterior end of the body. Several morphotypes within both Caryophyllaeus laticeps (Pallas, 1781) and Caryophyllaeus brachycollis Janiszewska, 1953 generally corresponding to different hosts and geographical regions, were recognised likely suggesting host- and geography-related intraspecific morphological variability. Genetic data confirmed euryxenous host specificity of both species, most profoundly C. laticeps, but did not support the existence of cryptic species. In fact, some of the fish hosts may harbour both of the congeneric species including several of their respective morphotypes. The pattern of morphological and genetic divergence observed in both cestode species studied indicates a scenario of possible host range extensions and subsequent parasite diversification. If molecular sequence variability and host-related morphological polymorphism are confirmed in other groups of monozoic cestodes, it may complicate species identification and straightforward classification of these parasites.

The origin of the giant liver fluke, Fascioloides magna (Trematoda: Fasciolidae) from Croatia determined by high-resolution melting screening of mitochondrial cox1 haplotypes.

The high-resolution melting (HRM) method, recently optimized as a reliable technique for population study of the European Fascioloides magna populations, was applied to determine an origin of F. magna individuals from Croatia. The structure and frequency of mitochondrial cytochrome c oxidase subunit I (439 bp; cox1) haplotypes of 200 Croatian flukes coming from 19 red deer (Cervus elaphus elaphus) livers were screened and compared with recently determined reference samples of F. magna from all European foci-Italy, Czech Republic, and Danube floodplain forests. While the reference haplotypes Ha1 and Ha2 were specific for flukes from the first European focus of fascioloidosis, the Natural Park La Mandria in Italy, the remaining three haplotypes (Ha3, Ha4, and Ha5) represented parasites from the second focus, Czech Republic. Besides, Ha3 and Ha4 were found also in the third, latest, and still expanding European focus, the Danube floodplain forests. The HRM screening of cox1 haplotypes of Croatian F. magna individuals resulted in classification of samples into the two mitochondrial haplogroups characterized by well-distinguished melting curves. They corresponded to Ha3 and Ha4 reference haplotypes that confirmed the Danube origin of F. magna from Croatia. The results support the theory that the Danube floodplain forests population of F. magna represents uniform genetic pool of the parasite. The spread of F. magna alongside the Danube River down to Croatia was possible due to suitable ecological conditions for definitive and intermediate hosts present in this unique biotope.

The tapeworm Atractolytocestus tenuicollis (Cestoda: Caryophyllidea)--a sister species or ancestor of an invasive A. huronensis?

Atractolytocestus tenuicollis (Li, 1964) Xi, Wang, Wu, Gao et Nie, 2009 is a monozoic, non-segmented tapeworm of the order Caryophyllidea, parasitizing exclusively common carp (Cyprinus carpio L.). In the current work, the first molecular data, in particular complete ribosomal internal transcribed spacer 2 (ITS2) and partial mitochondrial cytochrome c oxidase subunit I (cox1) on A. tenuicollis from Niushan Lake, Wuhan, China, are provided. In order to evaluate molecular interrelationships within Atractolytocestus, the data on A. tenuicollis were compared with relevant data on two other congeners, Atractolytocestus huronensis and Atractolytocestus sagittatus. Divergent intragenomic copies (ITS2 paralogues) were detected in the ITS2 ribosomal spacer of A. tenuicollis; the same phenomenon has previously been observed also in two other congeners. ITS2 structure of A. tenuicollis was very similar to that of A. huronensis from Slovakia, USA and UK; overall pairwise sequence identity was 91.7-95.2%. On the other hand, values of sequence identity between A. tenuicollis and A. sagittatus were lower, 69.7-70.9%. Cox1 sequence, analysed in five A. tenuicollis individuals, were 100 % identical and no intraspecific variation was observed. Comparison of A. tenuicollis cox1 with respective sequences of two other Atractolytocestus species showed that the mitochondrial haplotype found in Chinese A. tenuicollis is structurally specific (haplotype 4; Ha4) and differs from all so far determined Atractolytocestus haplotypes (Ha1 and Ha2 for A. huronensis; Ha3 for A. sagittatus). Pairwise sequence identity between A. tenuicollis cox1 haplotype and remaining three haplotypes followed the same pattern as in ITS2. The nucleotide and amino acide (aa) sequence comparison with A. huronensis Ha1 and Ha2 revealed higher sequence identity, 90.3-90.8% (96.9% in aa), while lower values were achieved between A. tenuicollis haplotype and Ha3 of Japanese A. sagittatus-75.2 % (81.9 % in aa). The phylogenetic analyses using cox1, ITS2 and combined cox1 + ITS2 sequences revealed close genetic interrelationship between A. tenuicollis and A. huronensis. Independently of a type of analysis and DNA region used, the topology of obtained trees was always identical; A. tenuicollis formed separate clade with A. huronensis forming a closely related sister group.

Ups and downs of infections with the broad fish tapeworm Dibothriocephalus latus in Europe (Part II) and Asia from 1900 to 2020.

The broad fish tapeworm, Dibothriocephalus latus (Diphyllobothriidea), is the most important causative agent of diphyllobothriosis, a fish-borne zoonosis, in Europe. Part I of this review focused on the occurrence of D. latus in northwestern and central Europe, particularly in Fennoscandia, the Baltic, the Alpine lakes and Danube River regions during 1900-2020. Part II summarises data on D. latus from the European and Asian parts of Russia and from Asian countries. The tapeworm has occurred throughout Russia, with the most important foci in (i) the Republic of Karelia in the northwest of European Russia, (ii) the Volga River basin in the central and southern parts of European Russia, (iii) the Ob-Irtysh rivers region in the Ural region, (iv) the Yenisei-Lena rivers region in Siberia, and (v) the Lake Baikal basin in Siberia. The incidence of diphyllobothriosis has declined in recent decades, especially in European Russia, but zoonosis is still prevalent in some regions of Siberia. Cases reported from Arctic regions, the region around Lake Baikal, and the Pacific coast, including the Amur basin, however, were probably misidentifications with D. dendriticus and/or D. nihonkaiensis. No other Asian country where D. latus findings represented either imported cases or misidentifications had natural focus of diphyllobothriosis. Patterns of distribution of D. latus occurrence were similar in all Eurasian foci between 1900 and 2020. The numbers of records were associated with historical and epidemiological milestones of particular time periods.

Bird Louse Flies Ornithomya spp. (Diptera: Hippoboscidae) as Potential Vectors of Mammalian Babesia and Other Pathogens.

Background: Birds and mammals share various ectoparasites, which are responsible for the transmission of a wide range of pathogens. The louse flies (family Hippoboscidae) are ectoparasitic dipterans feeding strictly on the blood of mammals and birds. Both sexes of the louse flies are obligatory hematophagous and are known to act as the vectors of infectious agents. Materials and Methods: A total of 20 specimens of Ornithomya sp. were collected by hand on birds caught in nets or by hand from humans in two localities in Eastern Slovakia in 2021. The DNA samples were individually screened by species-specific PCRs for the presence of selected vector-borne pathogens. Results: Taxonomic identification folowed by molecular analyses revealed two louse fly species of Ornithomya spp. (O. avicularia and O. biloba). The molecular screening provided negative PCR results for Anaplasma phagocytophilum, Borrelia burgdorferi s.l., Rickettsia spp., Bartonella spp., and Hepatozoon canis. In contrast, positive PCR results were obtained for Babesia spp., Wolbachia spp., and Trypanosoma corvi. Conclusions: Of epidemiological importance is that the louse flies can presumably spread Babesia and other pathogens by host switching which facilitates the transmission and spread of numerous pathogens.

Molecular characterization of Atractolytocestus sagittatus (Cestoda: Caryophyllidea), monozoic parasite of common carp, and its differentiation from the invasive species Atractolytocestus huronensis.

Sequence structure of complete internal transcribed spacer 1 and 2 (ITS1 and ITS2) of the ribosomal DNA region and partial mitochondrial cytochrome c oxidase subunit I (cox1) gene sequences were studied in the monozoic tapeworm Atractolytocestus sagittatus (Kulakovskaya et Akhmerov, 1965) (Cestoda: Caryophyllidea), a parasite of common carp (Cyprinus carpio carpio L.). Intraindividual sequence diversity was observed in both ribosomal spacers. In ITS1, a total number of 19 recombinant clones yielded eight different sequence types (pairwise sequence identity, 99.7-100%) which, however, did not resemble the structure typical for divergent intragenomic ITS copies (paralogues). Polymorphism was displayed by several single nucleotide mutations present exclusively in single clones, but variation in the number of short repetitive motifs was not observed. In ITS2, a total of 21 recombinant clones yielded ten different sequence types (pairwise sequence identity, 97.5-100%). They were mostly characterized by a varying number of (TCGT)(n) repeats resulting in assortment of ITS2 sequences into two sequence variants, which reflected the structure specific for ITS paralogues. The third DNA region analysed, mitochondrial cox1 gene (669 bp) was detected to be 100% identical in all studied A. sagittatus individuals. Comparison of molecular data on A. sagittatus with those on Atractolytocestus huronensis Anthony, 1958, an invasive parasite of common carp, has shown that interspecific differences significantly exceeded intraspecific variation in both ribosomal spacers (81.4-82.5% in ITS1, 74.4-75.2% in ITS2) as well as in mitochondrial cox1, which confirms validity of both congeneric tapeworms parasitic in the same fish host.

Sequence structure and intragenomic variability of ribosomal ITS2 in monozoic tapeworms of the genus Khawia (Cestoda: Caryophyllidea), parasites of cyprinid fish.

The sequence structure of the ribosomal internal transcribed spacer 2 (ITS2) was determined for six species of Khawia (Cestoda: Caryophyllidea), parasites of cyprinid fish in the Holarctic Region. Homologous intragenomic ITS2 structure was found in Khawia armeniaca, Khawia baltica, and Khawia rossittensis; whereas divergent intragenomic ITS2 copies were detected in Chinese, Japanese, and Slovak isolates of Khawia sinensis and in Khawia japonensis, both parasitic in common carp, and in Khawia saurogobii, recently described from Chinese lizard gudgeon in China. Despite distinct morphological differences between K. saurogobii and K. sinensis, both species display very high level of molecular homogeneity. Variation in number of short repetitive motifs [(GCCT)(n) (GCCC)(n)], [(GTG)(n)], [(ATAC)(n)], [ACGTGT (TCGTGT)(n)], [(GT)(n)], [(GT)(n)], and [(ACCT)(n) (GCCT)(n)] resulted in assortment of ITS2 sequences in four ITS2 variants in K. saurogobii from China, three in Chinese and Japanese isolates of K. sinensis, and five ITS2 variants in K. sinensis from Slovakia. In K. japonensis, the structure and arrangement of microsatellites was different from those of K. sinensis and K. saurogobii. The heterogeneity in the number of two microsatellite regions [(TG)(n); (TTG)(n)] divided ITS2 clones into two variants-first ITS2 variant (472 bp) with (TG)(5) and (TTG)(6), and second variant with (TG)(7) and (TTG)(2) (465 bp). Sequence identity of K. saurogobii with all but one (K. sinensis) congeneric species ranged between 49.5 and 69.2%, which corresponds to the interspecific differences. In contrast, sequence identity of K. saurogobii and K. sinensis (87.6-95.0%) failed into the range of intraspecific variation determined for K. sinensis samples. This close genetic similarity indicates that recently described K. saurogobii may have undergone morphological divergence as a result of ongoing sympatric speciation by host switching.

Genetic interrelationships of Spirometra erinaceieuropaei (Cestoda: Diphyllobothriidea), the causative agent of sparganosis in Europe.

The geographic distribution of Spirometra erinaceieuropaei (Cestoda: Diphyllobothriidea), the causative agent of food/water-borne sparganosis, is restricted to Europe, where infected canids, felids, mustelids, suids, and reptiles have been documented from Poland, Ukraine, Belarus, Russia, Serbia, Estonia, Latvia, and Finland. The main objective of the current study was to map the molecular divergence of S. erinaceieuropaei from Finland using the complete sequences of the mitochondrial cytochrome c oxidase subunit 1 gene (cox1 mtDNA). Seven cox1 haplotypes were determined in 15 tapeworms from Eurasian lynx (Lynx lynx) from three localities in southern Finland. In addition, the first inter-population study of S. erinaceieuropaei based on currently obtained data on cox1 from Finland and previously published data from Finland, Latvia, Ukraine, and Poland, was performed. The haplotype network showed a star-like pattern without specific subdivision of lineages according to the locality. Samples from Finland, Latvia, and Poland shared several haplotypes and formed the common Baltic lineage. The haplotype of S. erinaceieuropaei from Ukraine was unique and placed on a separate mutational pathway, suggesting a different lineage of the parasite.

TITLE: Interrelations génétiques de Spirometra erinaceieuropaei (Cestoda, Diphyllobothriidea), l'agent causal de la sparganose en Europe. ABSTRACT: La distribution géographique de Spirometra erinaceieuropaei (Cestoda : Diphyllobothriidea), l'agent causal de la sparganose d'origine alimentaire/hydrique, est limitée à l'Europe, où des canidés, félidés, mustélidés, suidés et reptiles infectés ont été documentés en Pologne, Ukraine, Biélorussie, Russie, Serbie, Estonie, Lettonie et Finlande. L'objectif principal de la présente étude était de cartographier la divergence moléculaire de S. erinaceieuropaei de Finlande à l'aide des séquences complètes du gène mitochondrial de la sous-unité 1 de la cytochrome c oxydase (ADNmt cox1). Sept haplotypes cox1 ont été déterminés chez quinze cestodes du Lynx d'Eurasie (Lynx lynx) de trois localités du sud de la Finlande. En outre, la première étude inter-populationnelle de S. erinaceieuropaei basée sur les données actuellement obtenues sur cox1 de Finlande et sur des données précédemment publiées de Finlande, Lettonie, Ukraine et Pologne, a été réalisée. Le réseau d'haplotypes a montré un motif en étoile sans subdivision spécifique des lignées selon la localité. Des échantillons de Finlande, Lettonie et Pologne partagent plusieurs haplotypes et forment la lignée commune de la Baltique. L'haplotype de S. erinaceieuropaei d'Ukraine est unique et placé sur une voie de mutation distincte suggérant une lignée différente du parasite.

Development of high-resolution melting (HRM) analysis for population studies of Fascioloides magna (Trematoda: Fasciolidae), the giant liver fluke of ruminants.

The high-resolution melting (HRM) technique was successfully optimized as fast and effective method for population study of digenetic fluke, Fascioloides magna (Trematoda: Fasciolidae), originally North American liver parasite of free-living and domestic ruminants. Previously selected variable region (439 bp) of mitochondrial cytochrome c oxidase subunit I (cox1) of 249 fluke individuals from enzootic European and North American regions were sequenced and mutually compared. The sequence analysis of partial cox1 revealed presence of seven structurally different haplotypes. Based on the sequence structure and alignments of six of them (Ha1-Ha6), three internal probes were designed and applied in HRM-based haplotype determination of all F. magna specimens. HRM analysis, performed with three designed probes, resulted in classification of samples into the seven haplogroups, equally with their assortment according to the sequence analysis. The representative of the haplotype, which was not involved in probe design (Ha7), was characterized by a unique melting curve shape as well. This provided an evidence of optimally settled conditions in HRM assay and indicated a probability of successful discrimination of novel haplotypes in future population studies on F. magna. The successful optimization of HRM method stands for an opportunity of detection of genetically unknown North American variants of F. magna and promises its application as fast and cheap screening technique for phylogeography studies of the giant liver fluke on its original continent.

A comparative study of karyotypes and chromosomal location of rDNA genes in important liver flukes Fasciola hepatica and Fascioloides magna (Trematoda: Fasciolidae).

Chromosomal characteristics, i.e., number, size, morphology, and location of ribosomal DNA (rDNA) clusters were examined in two medically important liver flukes, Fasciola hepatica and Fascioloides magna (Fasciolidae), using conventional Giemsa staining and fluorescent in situ hybridization (FISH) with ribosomal 18S rDNA probe. A comparison of F. magna and F. hepatica karyotypes confirmed significant differences in all chromosomal features. Whilst the karyotype of F. hepatica comprised ten pairs of chromosomes (one metacentric and nine medium-sized subtelocentrics and submetacentrics; 2n = 20, n = 1 m + 5 sm + 4 st; TCL = 49.9 µm), the complement of F. magna was composed of 11 pairs of medium-sized subtelocentrics and submeta-metacentrics (2n = 22, n = 9 st + 1 sm + 1 sm-m; TCL = 35.2 µm). Noticeable differences were found mainly in length and morphology of first chromosome pair. It was metacentric and 9.0 µm long in F. hepatica while subtelocentric and 4.7 µm long in F. magna. Although FISH with rDNA probe revealed a single cluster of ribosomal genes in both species, conspicuous interspecific differences were displayed by chromosomal location of ribosomal loci (i.e., NORs). The signals were found on short arms of fifth homologous pair in F. hepatica; however, they were detected in pericentromeric regions of the long arms of tenth pair in F. magna. The observed cytogenetic differences were interpreted in terms of karyotype evolution of fasciolid flukes; F. hepatica may be regarded phylogenetically younger than F. magna. The present paper provides a pilot study on molecular cytogenetics within a group of hermaphroditic digenetic flukes.

Population study of Atractolytocestus huronensis (Cestoda: Caryophyllidea), an invasive parasite of common carp introduced to Europe: mitochondrial cox1 haplotypes and intragenomic ribosomal ITS2 variants.

The invasive monozoic tapeworm Atractolytocestus huronensis, a specific parasite of common carp, was originally found and described in the North American continent. It has been introduced to Europe and reported in several countries in the last 15 years, as well. In the current study, tapeworms from one North American (USA) and five European localities (United Kingdom/UK, Slovakia, Hungary, Croatia, and Romania) were subjected to molecular analyses in order to determine the level of intrapopulation and intraspecific molecular variation and to assess interrelationships among American and European populations of the parasite. Partial sequences (672 bp) of the mitochondrial cytochrome c oxidase subunit I (cox1) revealed the presence of only two cox1 haplotypes, in accordance with the nonnative character of the populations. The first haplotype was common for all tapeworms from the Continental Europe (Slovakia, Hungary, Croatia, and Romania); no differences were determined either within or among respective A. huronensis populations. The second cox1 haplotype was characterized in all individuals from the USA and UK, indicating their close genetic relationship. Both haplotypes differed in three nucleotide positions (99.6% identity) which did not change the amino acid sequence. The cox1 data imply that introduction of the parasite to Europe was probably the result of two independent events directed to the UK and Continental Europe. The very close genetic relationship between British and American A. huronensis was reflected also by similar ribosomal internal transcribed spacer 2 (ITS2) sequence structure; considerable intragenomic ITS2 variability was detected in all individuals of both geographic populations. Divergent ITS2 copies were mostly induced by different numbers of short repetitive motifs within the sequences, allowing their assortment into two ITS2 variants.