Vladimíra Hanzelová

Preliminary phylogenetic analysis of subfamilies of the Proteocephalidea (Eucestoda)

Cladistic analysis based on comparative morphology was used to examine the subfamily-level relationships within the cestode order Proteocephalidea. A single most parsimonious tree (70 steps, CI = 0.571; RC = 0.295; HI = 0.471) is consistent with monophyly for the Proteocephalidea and showed a relatively high consistency at the family level with the diagnosis of two major subclades. Unambiguous support for a Proteocephalidae subclade, including the Corallobothriinae, Proteocephalinae, Gangesiinae and Sandonellinae, and a Monticelliidae subclade, including the Marsypocephalinae, Zygobothriinae, Monticelliinae, Rudolphiellinae, Ephedrocephalinae and Othinoscolecinae was evident. Two subfamilies, the Acanthotaeniinae (historically in the Proteocephalidae) and Nupeliinae (historically in the Monticelliidae), were however, basal to all other subfamilies, indicating that neither family as currently conceived is monophyletic. Trees one or two steps longer, however, would be consistent with monophyly for the Proteocephalidae and Monticelliidae (excluding Acanthotaeniinae) or would result in the monophyly for both families, including all respective subfamilies congruent with current concepts for systematics of the order. Zoogeographical analysis demonstrated a strong Gondwanan association; proteocephalideans originated in Africa, with subsequent development linking Africa and South America. Colonisation of the Northern Hemisphere by proteocephalid subfamilies, the Proteocephalinae, Corallobothriinae and Gangesiinae, was secondary. Analysis of parasite-host relationships indicated that the basal hosts for the Proteocephalidea are equivocal; siluriform teleosts were, however, the basal hosts for the Nupeliinae + the Monticelliidae and Proteocephalidae subclades and an extensive co-evolutionary history with this host group is postulated. Independent colonisation events of reptilians by species of the Proteocephalinae and Acanthotaeniinae, non-siluriform teleosts associated with the Sandonellinae and some Proteocephalinae, and amphibians by some Proteocephalinae, are recognised. Some points which should be considered for further development of the systematics of the group are proposed, with special emphasis given to thorough morphological descriptions and investigations of life-cycles.

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.

Re-establishment of the fish parasite fauna in the Tisa River system (Slovakia) after a catastrophic pollution event

An extensive survey of helminth parasites in 1,316 freshwater fish representing 31 species from two aquatic ecosystems in southeastern Slovakia characterized by different level of environmental pollution was carried out. The helminth species diversity was compared between the Tisa River, heavily polluted with cyanides and heavy metals after a series of ecological disasters in 2000, and the less anthropogenically influenced Latorica River. A parasitological survey found 31 gastrointestinal helminths: Trematoda (11 species), Cestoda (14), Acanthocephala (3) and Nematoda (3). As many as 70 host−parasite combinations have been found. Twenty of them (28.6%) represent new host−parasite finding records for the territory of Slovakia. The component communities were species-poor in both rivers, with high dominance of one to three helminth species. Even though the fish communities were qualitatively similar (ICS = 81%) and the number of fish examined was approximately the same (676 and 640) in both localities, the helminth species richness and diversity of host−parasite combinations were two times lower in the more polluted Tisa River. The helminth communities were also much less abundant in the Tisa River. Based on the Czekanowski−Sørensen similarity index (ICS = 48.8%) and the Percentage similarity index (PI = 19.5%), the helminth communities were qualitatively and quantitatively different in the two rivers. The remarkable lack of species diversity in the Tisa River can be explained by the negative impact of residual contamination of the Tisa river bottom on certain freshwater invertebrates (bivalves and prosobranch mollusks, copepods and amphipods) which serve as obligatory intermediate hosts for the helminths. Four species, the aspidogastrean Aspidogaster limacoides Diesing, 1835, the acanthocephalan Pomphorhynchus tereticollis (Rudolphi, 1809) tapeworms Atractolytocestus huronensis Anthony, 1958 and Khawia sinensis Hsü, 1935 are recorded in Slovakia for the first time.

Phylogenetic relationships of the monozoic tapeworms (Eucestoda: Caryophyllidea) inferred from morphological characters

Phylogenetic relationships of all genera of the order Caryophyllidea, possibly the earliest branching group of true tapeworms (Platyhelminthes: Eucestoda) and the only one that is monozoic, have been assessed for the first time. Results of this cladistic analysis, inferred from 30 unweighted morphological characters, are only partly congruent with the existing classification, which consists of four families based on the position of the inner longitudinal muscles in relation to the internal genital organs. Whereas all but five genera of the Caryophyllaeidae form a monophyletic clade, members of the Capingentidae are split, occurring within six unrelated groups. The Lytocestidae is also paraphyletic, as some genera appear in four unrelated clades. Archigetes appears in a derived clade, indicating that its direct (monoxenous) life-cycle involving only tubificid oligochaetes is secondarily derived and not plesiomorphic among the Eucestoda, as postulated by some authors.

Perch and Its Parasites as Heavy Metal Biomonitors in a Freshwater Environment: The Case Study of the Ružín Water Reservoir, Slovakia

Heavy metal concentrations were determined in 43 perches (Perca fluviatilis) and in two of its most common parasites, the acanthocephalan Acanthocephalus lucii and the cestode Proteocephalus percae, collected in the period 2009–2010 from Ružín, a seriously polluted water reservoir in Slovakia. Samples of muscle, liver, kidney, brain, male and female reproductive organs and adipose tissue of fish and both parasites were analyzed for As, Cd, Cr, Cu, Hg, Mn, Ni, Pb and Zn, by ICP-MS. Mean concentrations of individual heavy metals in all fish samples decreased in the order zinc > copper > manganese > mercury > arsenic > chromium > cadmium > nickel > lead. Zinc was found to be the dominant element and its antagonistic interaction with copper was confirmed. The kidney was a key target organ receiving the highest mean concentrations of all analyzed metals, but some metals showed specific affinity for particular tissues. In terms of human health, concentration of Hg in fish muscle, which exceeded more than two-times its maximum level admitted in foodstuffs in European countries, is of great importance and should be taken into account. Bioaccumulation factors (C[parasite]/C[fish tissue]) calculated for all elements indicated much higher detection skills of A. lucii and P. percae parasites than fish organs and hence, present results allow proposing both parasite models as useful tools to monitor aquatic environmental quality. Acanthocephalans, however, seem to be superior for heavy metal monitoring, also demonstrated under experimental conditions. Present results also indicate the decreasing heavy metal burden of the reservoir and its gradual recovery in the course of time.

Tapeworm Khawia sinensis: review of the introduction and subsequent decline of a pathogen of carp, Cyprinus carpio.

The Asian tapeworm Khawia sinensis Hsü, 1935 (Cestoda: Caryophyllidea) is a large-sized (body length up to 11.5 cm) monozoic (unsegmented) parasite of common carp (Cyprinus carpio L.) that may cause mortality of young fish (fry). Since the 1960s, this cestode successfully colonized a large part of Europe, including the British Isles, North America and Japan. However, a review of published records provides evidence that the tapeworm K. sinensis, invasive parasite of carp, has become less common during the last two decades. Decline of K. sinensis may have been related to the recent introduction of another invasive tapeworm, the caryophyllidean Atractolytocestus huronensis Anthony, 1958 to Europe. Other factors that may have caused that K. sinensis is much less common than previously are also briefly discussed. A comparison of K. sinensis from feral and cultured carp, published to date, with those recently found for the first time in wild populations of carp in Slovakia did not reveal any marked differences in their morphology or measurements.

A comparison of the internal transcribed spacer of the ribosomal DNA for Eubothrium crassum and Eubothrium salvelini (Cestoda : Pseudophyllidea), parasites of salmonid fish

The internal transcribed spacers (ITS-1 and ITS-2) and 5.8S subunit of the ribosomal RNA gene of Eubothrium crassum and Eubothrium salvelini, cestode parasites of salmonid fish, were sequenced and compared. The lengths and GC contents of the regions sequenced were 1119 bp and 50.1% for E. crassum, and 1117 bp and 50.5% for E. salvelini, respectively. Sequence alignment and comparison of both taxa showed 97.9% similarity. Within the ITS-1 and ITS-2 of both species, the restriction enzymes MspI and Bsh1236I possessed different cleavage sites and could be employed for rapid differentiation of both species of Eubothrium by PCR-RFLP.

Neuropeptides and serotonin in the cestode, Proteocephalus exiguus: an immunocytochemical study.

Neuropeptides and serotonin in the cestode, Proteocephalus exiguus: an immunocytochemical study. International Journal for Parasitology 25: 673-682. Neuropeptide F (NPF), RFamide and serotonin (5-HT) immunoreactivities have been detected in the nervous system of P. exiguus procercoids and adults, using an indirect immunocytochemical technique in conjunction with confocal scanning laser microscopy. The peptidergic nervous system of the procercoid is well developed, with two brain ganglia, three pairs of longitudinal nerve cords, transverse ring commissures and nerves in the suckers, all showing NPF-immunostaining. Strong NPF- and RF-immunostaining was observed in the CNS and PNS of the adult worm. The distribution patterns of the two neuropeptides were similar. Immunoreactivity for 5-HT was found only in the CNS.

The synonymy of Proteocephalus neglectus La Rue, 1911 with P. exiguus La Rue, 1911, two fish cestodes from the Holarctic Region.

Two cestode species of the genus Proteocephalus Weinland, 1858, P. neglectus La Rue, 1911, parasitic in salmonid fishes, and P. exiguus La Rue, 1911, occurring mostly in coregonids, were evaluated taxonomically. Comparison of the morphology and morphometry revealed that the taxa apparently represent morphological forms of one polymorphic species, for which the name Proteocephalus exiguus La Rue, 1911 is proposed. A redescription of this species, based on extensive comparative material from different fish hosts and geographical areas, is given. The reliability of criteria (size of the body and shape of segments, characteristics relating to the head morphology, presence/absence of microtriches on the scolex surface, relative size of the cirrus-sac, number of testes and testicular layers, shape of genital atrium, size of the egg) used previously for differentiating these species is also discussed.

Host specificity and geographical distribution of Eubothrium in European salmonid fish

The host specificity and distribution of Eubothrium crassum (Bloch, 1779) and Eubothrium salvelini (Schrank, 1790), morphologically fairly similar pseudophyllidean tapeworms parasitizing salmonid fish, were critically assessed on the basis of morphological and genetic evaluation of extensive material collected from different definitive hosts and geographical regions in Europe. Eubothrium crassum occurs in fish of the genera Salmo, i.e. salmon (S. salar - both freshwater and marine), sea trout (S. trutta trutta), brown trout (S. trutta fario), and lake trout (S. trutta lacustris), and also in Danubian salmon (Hucho hucho) and vendace (Coregonus albula). Eubothrium salvelini parasitizes Arctic char (Salvelinus alpinus) and brook trout (Salvelinus fontinalis) in Europe, and also whitefish (Coregonus wartmanni). Rainbow trout (Oncorhynchus mykiss), which is not a native European fish species, was found to be a suitable definitive host for both Eubothrium species, which may occur simultaneously in the same fish. Previous records of E. crassum in Arctic char and brook trout, and those of E. salvelini in fish of the genus Salmo were most probably misidentifications. Most studies of Eubothrium have involved salmonids from the northern part of Europe, with few records from southern and south-eastern Europe. This study also confirmed the reliability of the morphology of the apical disc for the discrimination of E. crassum and E. salvelini.