How to become a successful invasive tapeworm: a case study of abandoned sexuality and exceptional chromosome diversification in the triploid carp parasite Atractolytocestus huronensis Anthony, 1958 (Caryophyllidea: Lytocestidae).

BACKGROUND: A cytogenetic analysis of the new local triploid population of the caryophyllidean tapeworm Atractolytocestus huronensis, a unique parthenogenetic species with the ability to colonise new regions, was performed to understand the inner structure of its chromosome complement. METHODS: A karyotype analysis was carried out using classical Giemsa staining and C-banding combined with fluorescent DAPI staining. A hypothesis that triplets are composed from three homologue chromosomes of approximately the same length and same centromere position was tested statistically for multiple dependent variables using a non-parametric Friedman's ANOVA. The chromosomal location of ribosomal DNA clusters within the nucleolar organization region (NORs) and telomeric (TTAGGG)n sequences were detected by fluorescent in situ hybridization (FISH). Chromosomes were subjected to AgNO3 staining in order to determine whether the rDNA sites represent active NORs. RESULTS: The cytogenetic analysis confirmed the karyotype composed from eight chromosome triplets (3n = 24) as well as the existence of a pair of NORs located on each chromosome of the second triplet. Six NORs varied their activity from cell to cell, and it was reflected in the numbers of nucleoli (from 1 to 5). A huge morphological diversification of homologue chromosomes was originally detected in six out of eight triplets; the homologue elements differed significantly either in length and/or morphology, and some of them carried discernible interstitial telomeric sequences (ITSs), while the end telomeres were minute. The heterochromatin bands with high AT content varied irregularly, and the course of aberrant spermatogenesis was evident. CONCLUSIONS: Diversification of homologues is a unique phenomenon very likely caused by the long-term absence of a recombination and consequential accumulation of chromosome rearrangements in the genome of A. huronensis during species evolution. Unalterable asexual reproduction of the tapeworm, along with international trade in its host (carp), is facilitating its ongoing spread.

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.

New data on the karyotype and chromosomal rDNA location in Paradiplozoon megan (Monogenea, Diplozoidae), gill parasite of chubs.

New morphological data on chromosome complement of diplozoid parasite Paradiplozoon megan from the chub Squalius cephalus are shown herein. The karyotype of P. megan is characterized by seven pairs (2n = 14) of medium long (up to 11 µm) one-armed chromosomes which are nearly identical in number and morphological classification with chromosomes of other Paradiplozoon species described karyologically to date (Paradiplozoon bliccae, Paradiplozoon nagibinae, Paradiplozoon sapae, Paradiplozoon pavlovskii and Paradiplozoon homoion). A single locus for ribosomal RNA genes, visualized in the secondary constriction site by the fluorescent in situ hybridization method, is situated interstitially on a median part of a long arm of the smallest, 7th chromosome pair in P. megan. Phylogenetic interrelationships within the members of the family Diplozoidae and hypothesis for the ancestral karyotype are discussed here.

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.

Telomere analysis of platyhelminths and acanthocephalans by FISH and Southern hybridization.

We examined the composition of telomeres in chromosomes of parasitic worms, representatives of the flatworm groups Monogenea and Cestoda (Platyhelminthes), and thorny-headed worms (Syndermata: Acanthocephala) by fluorescence in situ hybridization (FISH) with different telomeric repeat probes. Our results show that the (TTAGGG)n sequence, supposed to be the ancestral telomeric repeat motif of Metazoa, is conserved in Monogenea (Paradiplozoon homoion) and Cestoda (Caryophyllaeus laticeps, Caryophyllaeides fennica, and Nippotaenia mogurndae) but not in Acanthocephala (Pomphorhynchus laevis and Pomphorhynchus tereticollis). In the Pomphorhynchus species, no hybridization signals were obtained with the "nematode" (TTAGGC)n, "arthropod" (TTAGG)n, and bdelloid (TGTGGG)n telomeric probes using FISH with their chromosomes and Southern hybridization with P. laevis DNA. Therefore, we suggest that parasitic Acanthocephala have evolved yet unknown telomeric repeat motifs or different mechanisms of telomere maintenance.

Divergent location of ribosomal genes in chromosomes of fish thorny-headed worms, Pomphorhynchus laevis and Pomphorhynchus tereticollis (Acanthocephala).

We studied distribution of ribosomal DNA (rDNA) sequences along with chromosomal location of the nucleolar organizer regions (NORs) in males of two fish parasites, Pomphorhynchus laevis and Pomphorhynchus tereticollis (Acanthocephala). Fluorescence in situ hybridization with 18S rDNA probe identified two clusters of rDNA in each species, but revealed a remarkable difference in their location on chromosomes. In P. laevis, the rDNA-FISH signals were found in long arms of the first chromosome pair and in short arms of the second pair. Whereas in P. tereticollis, rDNA clusters were located in long arms of both the first and second chromosome pairs. The divergent location of rDNA clusters in the chromosome No. 2 supports current classification of P. tereticollis, previously considered a synonym of P. laevis, as a separate species. A possible scenario of the second chromosome rearrangement during karyotype evolution of the two species involves two successive pericentric inversions. In both species, one or two prominent nucleoli were apparent within interphase nuclei stained with either silver nitrate or a fluorescent dye YOYO-1. However, a single large nucleolus was observed in early stages of mitosis and meiosis I regardless the number of rDNA clusters. Nevertheless, two bivalents with silver-stained NORs in diakinesis and two silver-stained sites in early prophase II nuclei indicated that all NORs are active. This means that each Pomphorhynchus NOR generates a nucleolus, but the resulting nucleoli have a strong tendency to associate in a large body.