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.

Department of Genetics Medirex Laboratories a s Magnezitárska 2 C 04013 Košice Slovakia

Department of Zoology and Fisheries Faculty of Agrobiology Food and Natural Resources Czech University of Life Sciences Prague Kamýcká 129 16500 Praha Suchdol Czech Republic

Institute of Parasitology Slovak Academy of Sciences Hlinkova 3 04001 Košice Slovakia

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Jones AW, Mackiewicz JS. Naturally occurring triploidy and parthenogenesis in Atractolytocestus huronensis Anthony (Cestoidea: Caryophyllidea) from Cyprinus carpio L. in North America. J Parasitol. 1969;55:1105–1118. doi: 10.2307/3277242. DOI

Mackiewicz JS. Caryophyllidea (Cestoidea): evolution and classification. Adv Parasitol. 1982;19:139–206. doi: 10.1016/S0065-308X(08)60267-5. DOI

Olson PD, Poddubnaya LG, Littlewood DTJ, Scholz T. On the position of Archigetes and its bearing on the early evolution of the tapeworms. J Parasitol. 2008;94:898–904. doi: 10.1645/GE-1456.1. PubMed DOI

Li WX, Zhang D, Boyce K, Xi BW, Zou H, Wu SG, et al. The complete mitochondrial DNA of three monozoic tapeworms in the Caryophyllidea: a mitogenomic perspective on the phylogeny of eucestodes. Parasit Vectors. 2017;10:314. doi: 10.1186/s13071-017-2245-y. PubMed DOI PMC

Li WX, Fu PP, Zhang D, Boyce K, Xi BW, Zou H, et al. Comparative mitogenomics supports synonymy of the genera Ligula and Digramma (Cestoda: Diphyllobothriidae) Parasit Vectors. 2018;11:324. doi: 10.1186/s13071-018-2910-9. PubMed DOI PMC

Anthony JD. Atractolytocestus huronensis n. gen., n. sp. (Cestoda: Lytocestidae) with notes on its morphology. Trans Am Microsc Soc. 1958;77:383–390. doi: 10.2307/3223860. DOI

Králová-Hromadová I, Štefka J, Bazsalovicsová E, Boroková S, Oros M. The tapeworm Atractolytocestus tenuicollis (Cestoda: Caryophyllidea)—a sister species or ancestor of an invasive A. huronensis? Parasitol Res. 2013;112:3379–3388. doi: 10.1007/s00436-013-3516-y. PubMed DOI

Scholz T, Tavakol S, Halajian A, Luus-Powell WJ. The invasive fish tapeworm Atractolytocestus huronensis (Cestoda), a parasite of carp, colonises Africa. Parasitol Res. 2015;114:3521–3524. doi: 10.1007/s00436-015-4573-1. PubMed DOI

Bazsalovicsová E, Králová-Hromadová I, Xi B-W, Štefka J. Tour around the globe: the case of invasive tapeworm Atractolytocestus huronensis (Cestoda: Caryophyllidea), a parasite of common carp. Parasitol Int. 2018;67:366–374. doi: 10.1016/j.parint.2018.02.004. PubMed DOI

Králová-Hromadová I, Štefka J, Špakulová M, Orosová M, Bombarová M, Hanzelová V, et al. Intraindividual ITS1 and ITS2 ribosomal sequence variation linked with multiple rDNA loci: a case of triploid Atractolytocestus huronensis, the monozoic cestode of common carp. Int J Parasitol. 2010;40:175–181. doi: 10.1016/j.ijpara.2009.07.002. PubMed DOI

Oros M, Králová-Hromadová I, Hanzelová V, Bruňanská M, Orosová M. Atractolytocestus huronensis (Cestoda): a new invasive parasite of common carp in Europe. In: Sanders JD, Peterson SB, editors. Carp: habitat, management and diseases. New York: Nova Science Publishers, Inc; 2011. pp. 63–94.

Bruňanská M, Nebesářová J, Oros M. Ultrastructural aspects of spermatogenesis, testes, and vas deferens in the parthenogenetic tapeworm Atractolytocestus huronensis Anthony, 1958 (Cestoda: Caryophyllidea), a carp parasite from Slovakia. Parasitol Res. 2011;108:61–68. doi: 10.1007/s00436-010-2038-0. PubMed DOI

Oros M, Hanzelová V, Scholz T. The cestode Atractolytocestus huronensis (Caryophyllidea) continues to spread in Europe: new data on the helminth parasite of the common carp. Dis Aquat Organ. 2004;62:115–119. doi: 10.3354/dao062115. PubMed DOI

Frydrychová M, Marec F. Repeated losses of TTAGG telomere repeats in evolution of beetles (Coleoptera) Genetica. 2002;115:179–187. doi: 10.1023/A:1020175912128. PubMed DOI

Guerra Dos Santos M. Reviewing the chromosome nomenclature of Levan et al. Rev Brasil Genet. 1986;9:741–743.

Fernández R, Barragán MJL, Bullejos M, Marchal JA, Guardia R, Sánchez A. New C-band protocol by heat denaturation in the presence of formamide. Hereditas. 2002;137:145–148. doi: 10.1034/j.1601-5223.2002.01672.x. PubMed DOI

Ráb P, Roth P. Cold-blooded vertebrates. In: Balíček P, Forejt J, Rupeš J, editors. Methods of chromosome analysis. Brno: Czech Biological Society Publishing; 1988. pp. 115–124.

Frydrychová R, Grossmann P, Trubac P, Vítková M, Marec F. Phylogenetic distribution of TTAGG telomeric repeats in insects. Genome. 2004;47:163–178. doi: 10.1139/g03-100. PubMed DOI

Fuková I, Nguyen P, Marec F. Codling moth cytogenetics: karyotype, chromosomal location of rDNA, and molecular differentiation of sex chromosomes. Genome. 2005;48:1083–1092. doi: 10.1139/g05-063. PubMed DOI

Miller OL. The nucleolus, chromosomes, and visualization of genetic activity. J Cell Biol. 1981;91:15–27. doi: 10.1083/jcb.91.3.15s. PubMed DOI PMC

Bombarová M, Vítková M, Špakulová M, Koubková B. Telomere analysis of platyhelminths and acanthocephalans by FISH and Southern hybridization. Genome. 2009;52:897–903. doi: 10.1139/G09-063. PubMed DOI

Aksenova AY, Greenwell PW, Dominska M, Shishkin AA, Kim JC, Petes TD, Mirkin SM. Genome rearrangements caused by interstitial telomeric sequences in yeast. Proc Natl Acad Sci USA. 2013;110:19866–19871. doi: 10.1073/pnas.1319313110. PubMed DOI PMC

Aksenova AY, Han G, Shishkin AA, Volkov KV, Mirkin SM. Expansion of interstitial telomeric sequences in yeast. Cell Rep. 2015;13:1545–1551. doi: 10.1016/j.celrep.2015.10.023. PubMed DOI PMC

Rovatsos M, Kratochvíl L, Altmanová M, Johnson Pokorná M. Interstitial telomeric motifs in squamate reptiles: when the exceptions outnumber the rule. PLoS ONE. 2015;10:e0134985. doi: 10.1371/journal.pone.0134985. PubMed DOI PMC

Hastie ND, Allshire RC. Human telomeres: fusion and interstitial sites. Trends Genet. 1989;5:326–331. doi: 10.1016/0168-9525(89)90137-6. PubMed DOI

Hastie ND, Dempster M, Dunlop MG, Thompson AM, Green DK, Allshire RC. Telomere reduction in human colorectal carcinoma and with ageing. Nature. 1990;346:866–868. doi: 10.1038/346866a0. PubMed DOI

van Herwerden L, Blair D, Agatsuma T. Intra- and interindividual variation in ITS1 of Paragonimus westermani (Trematoda: Digenea) and related species: implications for phylogenetic studies. Mol Phylogen Evol. 1999;12:67–73. doi: 10.1006/mpev.1998.0572. PubMed DOI

Blair D, Nawa Y, Mitreva M, Doanh PN. Gene diversity and genetic variation in lung flukes (genus Paragonimus) Trans R Soc Trop Med Hyg. 2016;110:6–12. doi: 10.1093/trstmh/trv101. PubMed DOI

Špakulová M, Orosová M, Mackievicz JS. Cytogenetics and chromosomes of tapeworms (Platyhelminthes, Cestoda) Adv Parasitol. 2011;74:177–230. doi: 10.1016/B978-0-12-385897-9.00003-3. PubMed DOI

Vogt G, Falckenhayn C, Schrimpf A, Schmid K, Hanna K, Panteleit J, et al. The marbled crayfish as a paradigm for saltational speciation by autopolyploidy and parthenogenesis in animals. Biol Open. 2015;4:1583–1594. doi: 10.1242/bio.014241. PubMed DOI PMC

Martin P, Thonagel S, Scholtz G. The parthenogenetic Marmorkrebs (Malacostraca: Decapoda: Cambaridae) is a triploid organism. J Zool Syst Evol Res. 2016;54:13–21. doi: 10.1111/jzs.12114. DOI

Gutekunst J, Andriantsoa R, Falckenhayn C, Hanna K, Stein W, Rasamy J, et al. Clonal genome evolution and rapid invasive spread of the marbled crayfish. Nat Ecol Evol. 2018;2:567–573. doi: 10.1038/s41559-018-0467-9. PubMed DOI

Simon J-C, Delmotte F, Rispe C, Crease T. Phylogenetic relationships between parthenogens and their sexual relatives: the possible routes to parthenogenesis in animals. Biol J Linn Soc. 2003;79:151–163. doi: 10.1046/j.1095-8312.2003.00175.x. DOI

Xi BW, Wang GT, Wu SG, Nie P. New record of genus Atractolytocestus in China with redescription of A. sagittatus (Cestoda, Caryophyllidea) from Cyprinus carpio. Acta Zootax Sin. 2009;34:407–410.

Hossain MS, Patoka J, Kouba A, Buřič M. Clonal crayfish as biological model: a review on marbled crayfish. Biologia. 2018;73:841–855. doi: 10.2478/s11756-018-0098-2. DOI

Scholz T, Shimazu T, Olson PD, Nagasawa K. Caryophyllidean tapeworms (Platyhelminthes: Eucestoda) from freshwater fishes in Japan. Folia Parasitol (Praha). 2001;48:275–288. doi: 10.14411/fp.2001.046. PubMed DOI

Cuellar O. Animal parthenogenesis. Science. 1977;197:837–843. doi: 10.1126/science.887925. PubMed DOI

Pongratz N, Storhas M, Carranza S, Michielz NK. Phylogeography of competing sexual and parthenogenetic forms of a freshwater flatworm: patterns and explanations. BMC Evol Biol. 2003;3:23. doi: 10.1186/1471-2148-3-23. PubMed DOI PMC

Xu P, Zhang X, Wang X, Li J, Liu G, Kuang Y, et al. Genome sequence and genetic diversity of the common carp, Cyprinus carpio. Nat Genet. 2014;46:1212–1219. doi: 10.1038/ng.3098. PubMed DOI

Balon EK. Origin and domestication of the wild carp, Cyprinus carpio: from Roman gourmets to the swimming flowers. Aquaculture. 1995;129:3–48. doi: 10.1016/0044-8486(94)00227-F. DOI

Zhou JF, Wu QJ, Ye YZ, Tong J. Genetic divergence between Cyprinus carpio carpio and Cyprinus carpio haematopterus as assessed by mitochondrial DNA analysis, with emphasis on origin of European domestic carp. Genetica. 2003;119:93–97. doi: 10.1023/A:1024421001015. PubMed DOI

Bazsalovicsová E, Králová-Hromadová I, Štefka J, Scholz T. Molecular characterization of Atractolytocestus sagittatus (Cestoda: Caryophyllidea), monozoic parasite of common carp, and its differentiation from the invasive species Atractolytocestus huronensis. Parasitol Res. 2012;110:1621–1629. doi: 10.1007/s00436-011-2673-0. PubMed DOI

New cytogenetic data on Caryophyllaeus laticeps and Paracaryophyllaeus gotoi, parasites of evolutionary interest

Molecular cytogenetic analysis of a triploid population of the human broad tapeworm, Dibothriocephalus latus (Diphyllobothriidea)

Chromosomal study of Khawia abbottinae (Cestoda: Caryophyllidea): karyotype and localization of telomeric and ribosomal sequences after fluorescence in situ hybridization (FISH)