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 q Ivica Králová-Hromadová a, * , Jan Štefka b,c , Marta Špakulová a , Martina Orosová b , Marta Bombarová a , Vladimíra Hanzelová a , Eva Bazsalovicsová a , Tomáš Scholz b a Parasitological Institute, Slovak Academy of Sciences, Hlinkova 3, 040 01 Košice, Slovakia b Biology Centre ASCR, Institute of Parasitology, Branišovská 31, 370 05 C ˇ eské Bude ˇjovice, Czech Republic c Faculty of Science, University of South Bohemia, C ˇ eské Bude ˇjovice, Czech Republic article info Article history: Received 24 June 2009 Accepted 6 July 2009 Keywords: Fish parasites Caryophyllidea Ribosomal divergence Karyotype FISH abstract Complete sequences of the ribosomal internal transcribed spacers (ITS1 and ITS2) and karyological char- acters of the monozoic (unsegmented) tapeworm Atractolytocestus huronensis Anthony, 1958 (Cestoda: Caryophyllidea) from Slovakia were analysed, revealing considerable intra-genomic variability and trip- loidy 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 sub- stitutions 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 + 1 minute 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. Ó 2009 Australian Society for Parasitology Inc. All rights reserved. 1. Introduction Caryophyllidean cestodes are common parasites of freshwater siluriform and cypriniform fishes widely distributed in most zoo- geographical regions, except for the Neotropics (Mackiewicz, 1982). Caryophyllideans, as tapeworms with a very specific mor- phology (monopleuroid body lacking proglottisation and segmen- tation) and life cycle (exclusively with tubificid annelids as intermediate hosts) occupy a key position in the evolution and in- ter-relationships of cestodes (Mackiewicz, 2003). The caryophyllidean species Atractolytocestus huronensis An- thony, 1958 was originally described from the common carp Cyp- rinus carpio carpio L. from the Huron River, Michigan, USA (Anthony, 1958). The tapeworm has been imported from North America to Europe recently, most probably due to insufficient vet- erinary control of transported fish. Its occurrence in Europe was reported for the first time in England (Chubb, J.C., Kirk, R., Wellby, I., 1996. Caryophyllaeid tapeworm A. huronensis Anthony, 1958 (= Markevitschia sagittata Kulakovskaya et Akhmerov, 1965) in carp Cyprinus carpio L. in British Isles – another translocation? In: Ab- stracts of the Spring Meeting of the British Society of Parasitology, University of Wales, Bangor, p. 66; Kirk, R.S., Veltkamp, J.C., Chubb, J.C. 2003. Identification of A. huronensis (Caryophyllidea: Lytocesti- dae) from carp (Cyprinus carpio) using histological and ashing tech- niques. In: Abstract of the Spring Meeting of the British Society of Parasitology, Manchester, pp. 45–46.). Subsequently, the parasite was found in continental Europe, in four neighbouring countries (Hungary, Slovakia, Czech Republic, Germany), in feral as well as farmed carp (Majoros et al., 2003; Oros et al., 2004; Kappe et al., 2006). There is a high probability that this tapeworm could soon invade other territories via the human import and trade activities, and potentially via the natural migration of carp. 0020-7519/$36.00 Ó 2009 Australian Society for Parasitology Inc. All rights reserved. doi:10.1016/j.ijpara.2009.07.002 q Nucleotide sequence data reported in this paper are available in the GenBank TM , EMBL and DDBJ databases under Accession Nos. FJ475069–FJ475108. * Corresponding author. Tel.: +421 55 6334455; fax: +421 55 6331414. E-mail address: hromadova@saske.sk (I. Králová-Hromadová). International Journal for Parasitology 40 (2010) 175–181 Contents lists available at ScienceDirect International Journal for Parasitology journal homepage: www.elsevier.com/locate/ijpara