The purpose of our study is to provide new cytogenetic data on A. huronensis and to improve previous karyological analyses of A. huronensis from the USA [1] and Europe [10, 11]. All previous studies clearly proved that the triploid tapeworm karyotype comprises 8 chromosome triplets (3n = 24). The slight discrepancy in classification of triplets 2, 3 and 7 (acrocentric versus subtelocentric) reported in geographically distant parasite populations (USA, Europe) might be based on either nomenclature differences or different karyological techniques used [1, 10]. Another view might relate to the above-mentioned affiliation of the American and European tapeworms to phylogenetically differentiated intraspecific clusters [9]. However, this last distinction was really insignificant as the total intrapopulation diversity was extremely low. In any case, chromosome characteristics should be re-examined in most world locations using standardized techniques.
Nevertheless, previous cytogenetic studies have not analysed morphological differences among homologue chromosomes within individual triplets, although photos from Králová-Hromadová et al. [10] suggest their possible existence. The analysis in the present study deals with newly collected worms of A. huronensis. These specimens were taken from a relatively isolated pond in eastern Slovakia, which is regularly stocked with carp. The cytogenetic analysis not only confirms the triploid nature of the A. huronensis karyotype but also reveals a much more intimate chromosome structure. Indeed, nearly all triplets were created from statistically distinct elements differing in macromorphology and/or banding patterns. As shown in Fig. 2, each of triplets 1, 4, 5 and 6 included a pair of slightly similar homologues (1BC, 4BC, 5BC, 6BC); however, even these similar elements within pairs might be more or less unequal in length. The third element of each mentioned triplet was visibly different, deviating significantly in length (1A), morphology (4A, 5A) or in the presence of AT-rich bands and/or interstitial telomeric signals (4A, 5A, 6A). The inner heterogeneity within triplets 2, 3 and 7 was even greater: each triplet possessed morphologically and structurally diverse chromosomes which differed in nearly all of the above-mentioned features (see Figs. 1, 2).
As with the previous analysis of another European population [10], two tandemly arranged clusters of ribosomal DNA (i.e. nucleolar organizer regions, NORs) were located on the long arm of chromosomes 2A, 2B and 2C. One cluster was found in the in the post-centromeric and the other in the intercalary position. However, silver-staining analysis, which highlights only active NORs [20], showed that not all the ribosomal gene loci were regularly functional, and the number of positive signals varied from cell to cell. This fact was reflected in the numbers of nucleoli (from one to five) apparent in interphase cells (not shown).
Successful application of the telomeric (TTAGGG)n probe in A. huronensis has proved the existence of this ancestral sequence repeat in additional cestode species, which has been detected so far in two caryophyllidean and one nippotaeniidean tapeworms [21]. However, extremely weak terminal telomeres were accompanied by the irregular presence of interstitial telomeric sequences (ITSs) detected in five individual chromosomes, each belonging to various triplets. These interstitial signals, positioned close to the centromeres, had more intense colours in comparison to repeats on the chromosome ends. In general, ITSs, known in many eukaryotic genomes (from yeast to human), are thought to be linked to the disruption of genome integrity, but the detailed molecular mechanisms responsible for ITSs-mediated genome instability remain unclear [22, 23]. However, it has been proposed that ITSs are usually derived from ancestral telomere fusion events during karyotype evolution [24, 25]. They could act as hotspots for breakage and induce high rates of chromosome rearrangements; the breakage and fragility might facilitate chromosome remodelling and cell transformation [26]. In A. huronensis, ITSs occur only in one of three elements of triplets 2, 4, 6, 7 and 8. However, minimization of telomeres at the chromosome ends was evident in all elements.
The extreme diversification of chromosomes within individual triplets was evident in the course of the meiotic prophase of spermatocytes. Similarly to the previous analysis by Jones & Mackiewicz [1], a different frequency of univalents, bivalents and trivalents was detected depending on the type of individual chromosome. Our study clearly confirmed that meiotic division is abnormal and that spermatogenesis fails to produce functional sperm. While the course of spermatogonial mitoses seemed regular, all stages of spermatocyte divisions were aberrant. During the prophase stages, irregular multivalent associations or unpaired univalents were common. In the segregation of chromosomes to opposite poles during the early anaphase, univalents predated the paired multivalents which dwelled longer in the cell centre; as a result, this process was once again considered uneven. Homeotypical division results in abnormal sperm containing an irregular number of chromosomes. Using section methodology, Jones & Mackiewicz [1] described additional meiotic disorders such as chromosome non-disjunctions accompanied by spindle irregularities, as well as bridges between spermatid nuclei in telophase II (due to lagging chromosomes), etc. One ultramicroscopic study [12] showed a fragmentation of nuclei in A. huronensis spermatocytes, which is clearly a feature of cell degeneration and can be a consequence of the aberrant first meiotic division. That study did not detect any mature, functional spermatozoon [12]. In summary, all existing studies show that A. huronensis is a triploid parthenogen. It is, moreover, the only cestode species in which triploidy, parthenogenesis, multiple loci for ribosomal DNA (NORs) and intragenomic ITS paralogues are mutually linked [9, 10]. Such a combination of original genetic phenomena was also observed in a lung fluke [Paragonimus westermani (Kerbert, 1878), Digenea: Paragonimidae], a parasite that belongs to a species complex and has triploid populations occurring in sympatry with diploid and occasionally with tetraploid specimens in eastern Asia [27, 28].
Among tapeworms, the tendency for triploid individuals, or even triploid populations, to occur within a common diploid species is obvious in three basal groups of Eucestoda, namely the orders Spathebothriidea, Diphyllobothriidea and Caryophyllidea [29]. However, A. huronensis is the only stable, exclusively triploid parthenogenetic tapeworm species. Such cases are frequent among plants but extremely rare in animals. An example of a well-researched triploid invertebrate is the marbled crayfish [Procambarus virginalis (Lyko, 2017), Decapoda, Procambaridae] [30,31,32] which is an all-female species reproduced by cloning (apomictic parthenogenesis). Atractolytocestus huronensis and P. virginalis are the only obligatory parthenogens in their groups (Cestoda and Decapoda), and their type of reproduction is consistent with the extremely reduced diversity or even genetic identity of mitochondrial DNA [9, 30]. Both are successful invasive organisms with the potential to colonize new territories [33,34,35]. The crayfish has been distributed worldwide via the pet trade and has demonstrated its viability once released into the wild. The specific carp endoparasite A. huronensis is widespread together with its fish host throughout four continents [9]. However, through a series of phylogenetic approaches, it has been proven that the marbled crayfish is an evolutionarily young autopolyploid descendant of the sexually reproducing diploid congener, the slough crayfish (P. fallax Hagen, 1870) [32]. The origin of the polyploid nature of A. huronensis, however, is still uncertain.
Jones & Mackiewicz [1] took into account both possible variants of the hypothetic origin of the A. huronensis triploidy (autopolyploidization or interspecific hybridization). The authors expressed the view that if triploidy arose by genetic but not interspecific hybridization, “then the immediate ancestor of the triploid line may still exist in the carp” (p. 1117 in [1]). Two possible living candidates for ancestors may be the only two congeners Atractolytocestus tenuicollis (Li, 1964) and A. sagittatus (Kulakovskaya & Akhmerov, 1965), both of which parasitize the common carp exclusively [7, 36]. Both species appear to reproduce sexually, and their distribution is restricted to several regions in East Asia [7, 11]; A. huronensis is, on the contrary, a successful invasive species, widespread nearly worldwide, including the lower Chinese Yangtze River basin [9]. These data are consistent with the hypothesis that a non-overlapping spatial distribution pattern of sexuals and parthenogens is frequently found; typically, the sexual populations are located in the distribution centre while parthenogens are present at the margin of the distribution [37, 38]. Since all three Atractolytocestus congeners are specific carp parasites, it is likely that all of them were distributed worldwide; however, only the parthenogenetic A. huronensis possessed the ability to survive and complete its life-cycle out of Asia. In fact, parthenogenesis, together with polyploidy, of asexual lineages in new habitats are thought to be an ecological advantage when compared to their sexual diploid counterparts [33].
Recently, new light has been shed on certain problems concerning the molecular phylogeny of both the parasite A. huronensis [9] and its fish host C. carpio [39]. An analysis of various samples of A. huronensis from continental Europe, UK, USA, China and South Africa, along with the haplotype network based on ribosomal ITS2 variants (paralogues), reveals that there is a relatively increased level of diversity in tapeworm populations from China. This fact indicates that the region of East Asia likely played a role in the origin of A. huronensis and might serve as a source location for global expansion of this tapeworm. Molecular analysis of world carp populations [39] revealed that the latest round of genome duplication (allotetraploidization, 2n = 100) occurred approximately 8.2 million years ago, and a single origin of C. carpio from the Caspian Sea was confirmed. The previously designed [40, 41] historic distribution of carp into Europe and the eastern mainland of Asia is accepted as a very likely scenario. Moreover, carp domestication was confirmed through the Middle Ages; two independent regions were involved, namely the Roman empire and East Asia [39]. Since then, the long-lasting, intense worldwide carp trade was illustrated by extensive genetic admixtures occurring in both the feral and farmed C. carpio populations. The European lineage of mirror carp carried admixtures from Asian populations, whilst North American carp showed evidence of multiple introductions from both Europe and Asia [39].
Historically, the carp became a host of several sexual caryophyllidae parasites in Asia; however, the origin of triploidy in A. huronensis remains unknown. The current discrepancies within chromosome triplets suggest that a more likely explanation may be of hybrid origin, which is a major route to parthenogenesis in animals [33]. The triploid karyotype of A. huronensis comprises two more or less similar chromosome sets while the third set is significantly different in many characters. It is most likely that the mutational load was caused by long-lasting asexual reproduction, manifested by the accumulation of deleterious aberrations in two original genomes at different intensities. Seemingly, A. tenuicollis might be the ideal candidate for an ancestral sparring partner for A. huronensis concerning interspecific hybridization [7]. The sexually reproducing diploid A. tenuicollis forms a genetically closest cluster with A. huronensis [9]. These two species are morphologically similar and occur in China [7, 11, 34]. On the other hand, the last congener A. sagittatus originated from other parts of Asia (the Amur River basin, Caspian Sea Drainage, and Japan), and its genetic affinity with A. huronensis is a bit weaker [9, 36, 42]. Hopefully, detailed cytogenetic research of sexual Atractolytocestus spp. as well as the Chinese A. huronensis population will solve the problem.