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

  • Martina OrosováEmail author
  • Irena Provazníková
  • Bing Wen Xi
  • Mikuláš Oros
Fish Parasitology - Original Paper


An original cytogenetic study combining classical karyotype analysis and modern fluorescence in situ hybridization using telomeric (TTAGGG)n and ribosomal sequences (18S rDNA) was performed in Khawia abbottinae (Cestoda, Caryophyllidea), a parasite of Chinese false gudgeon (Abbottina rivularis) from China. Analyses based on conventional Giemsa staining, DAPI, YOYO-1 dye, and silver (Ag) staining were also carried out. The karyotype is composed of eight pairs of metacentric and telocentric chromosomes (2n = 16, n=5m + 3t). Constitutive heterochromatin was mainly positioned at pericentromeric regions, and telomeric sequences (TTAGGG)n were restricted to the end of all chromosomes. In mitotic preparations stained with Giemsa, both homologues of chromosome pair 4 showed a distinct secondary constriction. FISH with rDNA probe confirmed that this secondary constriction contains a nucleolar organizer region (NOR). The process of spermatocyte meiosis and the dynamics of nucleolus degradation in dividing cell were scrutinized. The present study and its results enhance the limited knowledge on basic karyotype characteristics and 18S rDNA clusters location in caryophyllidean tapeworms.


Cestoda Karyotype FISH 18S rDNA mapping Telomeres 



This work was supported by the Slovak Research and Development Agency under contract No. SK-CN-2017-0007 and Bilateral Mobility Project SAV-18-20 and by the Research & Development Operational Program funded by the ERDF: Environmental protection against parasitozoonoses under the influence of global climate and social changes (code ITMS: 26220220116; rate 0.2).

Compliance with ethical standards

Conflict of interest

On behalf of all authors, the corresponding author states that there is no conflict of interest.


  1. Barčák D, Oros M, Hanzelová V, Scholz T (2017) A synoptic review of Caryophyllaeus Gmelin, 1790 (Cestoda: Caryophyllidea), parasites of cyprinid fishes. Folia Parasitol 64:027Google Scholar
  2. Birstein VJ (1991) On the karyotypes of the Neorhabdocoela species and karyological evolution of Turbellaria. Genetica 83:107–120Google Scholar
  3. Bombarová M, Špakulová M (2015) New chromosome characteristics of the monozoic tapeworm Caryophyllaeus laticeps (Cestoda, Caryophyllidea). Helminthologia 52:336–340Google Scholar
  4. Bombarová M, Špakulová M, Koubková B (2014) New data on the karyotype and chromosomal rDNA location in Paradiplozoon megan (Monogenea, Diplozoidae), gill parasite of chubs. Parasitol Res 113:4111–4116Google Scholar
  5. Bombarová M, Marec F, Nguyen P, Špakulová M (2007) Divergent location of ribosomal genes in chromosomes of fish thorny-headed worms, Pomphorhynchus laevis and Pomphorhynchus tereticollis (Acanthocephala). Genetica 131:141–149Google Scholar
  6. Bombarová M, Špakulová M, Kello M, Nguyen P, Bazsalovicsová E, Kráľová-Hromadová I (2015) Cytogenetics of Aspidogaster limacoides (Trematoda, Aspidogastrea): karyotype, spermatocyte division, and genome size. Parasitol Res 114:1473–1483Google Scholar
  7. Bombarová M, Vítková M, Špakulová M, Koubková B (2009) Telomere analysis of platyhelminths and acanthocephalans by FISH and southern hybridization. Genome 52:897–903Google Scholar
  8. Brabec J, Scholz T, Kráľová-Hromadová I, Bazsalovicsová E, Olson PD (2012) Substitution saturation and multiple copies of standard nuclear and mitochondrial phylogenetic markers in an unusual group of unsegmented tapeworms (Platyhelminthes). Int J Parasitol 42:259–267Google Scholar
  9. Cabral-de-Mello D, Moura RC, Souza A, Martinus C (2011) Evolutionary dynamics of heterochromatin in the genome of Dichotomius beetles based on chromosomal analysis. Genetica 139:921–931Google Scholar
  10. Dobigny G, Oyouf-Costaz C, Bonillo C, Volobuev V (2002) “Ag-NORs” are not always true NORs: new evidence in mammals. Cytogenet Genome Res 98:75–77Google Scholar
  11. Grelon M (2016) Meiotic recombination mechanisms. CR Biol 339:247–251Google Scholar
  12. Grey AJ (1979) A comparative study of the chromosomes of twenty species of caryophyllidean tapeworms. Dissertation, College of Arts and Sciences, Department of Biology, State University of New York at Albany, USA. 214 pp. (data used with written permission of Dr. Anthony J. Grey, PhD)Google Scholar
  13. Hanzelová V, Oros M, Barčák D, Miklisová D, Kirin D, Scholz T (2015) Morphological polymorphism in tapeworms: redescription of Caryophyllaeus laticeps (Pallas, 1781) (Cestoda:Caryophyllidea) and characterisation of its morphotypes from different fish hosts. Syst Parasitol 90:177–190Google Scholar
  14. Hirai H, Taguchi T, Saitoh Y, Kawanaka M, Sugiyama H, Habe S, Okamoto M, Hirata M, Shimada M, Tiu WU, Lai K, Upatham ES, Agatsuma T (2000) Chromosomal differentiation of the Schistosoma japonicum complex. Int J Parasitol 30:441–452Google Scholar
  15. Howell WM, Black DA (1980) Controlled silver staining of nucleolus organizer regions with a protective colloidal developer: a 1-step method. Experientia 36:1014–1015Google Scholar
  16. Hsü HF (1935) Contribution à ľétude des cestodes de Chine. Rev Suisse Zool 42:485–492Google Scholar
  17. Kato A, Albert PS, Vega JM, Bichler JA (2006) Sensitive fluorescence in situ hybridization signal detection in maize using directly labeled probes produced by high concentration DNA polymerase nick translation. Biotech Histochem 81:71–78Google Scholar
  18. John B (1990) Meiosis. Cambridge: Cambridge University Press.
  19. Kráľová-Hromadová I, Štefka J, Špakulová M, Orosová M, Bombarová M, Hanzelová V, Bazsalovicsová E, Scholz T (2010) Intraindividual ITS1 and ITS2 ribosomal sequence variation linked with multiple rDNA loci: a case of triploid Atractolytocestu huronensis, the monozoic cestode of common carp. Int J Parasitol 40:175–181Google Scholar
  20. Kráľová-Hromadová I, Bazsalovicsová E, Oros M, Scholz T (2012) Sequence structure and intragenomic variability of ribosomal ITS2 in monozoic tapeworms of the genus Khawia (Cestoda: Caryophyllidea), parasites of cyprinid fish. Parasitol Res 111:1621–1627Google Scholar
  21. Littlewood DTJ, Olson PD (2001) Small subunit rDNA and the Platyhelminthes: signal, noise, conflict and compromise. In: Littlewood DTJ, Bray RA (eds) Interrelationships of the Platyhelminthes. Taylor and Francis, London, pp 262–278Google Scholar
  22. Levan A, Fredga K, Sandberg A (1964) Nomenclature for centromere position on chromosomes. Hereditas 52:201–220Google Scholar
  23. Mackiewicz JS (1994) Order Caryophyllidea van Beneden in Carus, 1863. In: Khalil A, Jones A, Bray RA (eds) Keys to the Cestode parasites of vertebrates. CAB International, Wallingford, pp 21–43Google Scholar
  24. Meyne J, Ratliff RL, Moyzis RK (1989) Conservation of the human telomere sequence (TTAGGG)n among vertebrates. PNAS 86:7049–7053Google Scholar
  25. Meyne J, Baker RJ, Hobart HH, Hsu TC, Ryder OA, Ward OG, Wiley JE, Wurster-Hill DH, Yates TL, Moyzis RK (1990) Distribution of non-telomeric sites of the (TTAGGG)n telomeric sequence in vertebrate chromosomes. Chromosoma 99:3–10Google Scholar
  26. Mutafova T, Gergova S (1994) Cytological studies on three hymenolepidid species. J Helminthol 68:323–325Google Scholar
  27. Mutafova T, Nedeva I (1999) Karyological study of Khawia sinensis Hsü, 1935 (Cestoda, Lytocestidae). Acta Parasitol 44:206–208Google Scholar
  28. Nguyen P, Sahara K, Yoshido A, Marec F (2010) Evolutionary dynamics of rDNA clusters on chromosomes of moths and butterflies (Lepidoptera). Genetica 138:343–354Google Scholar
  29. Orosová M, Oros M (2012) Classical and molecular cytogenetics of Khawia sinensis (Cestoda: Caryophyllidea), invasive parasite of carp, Cyprinus carpio. Parasitol Res 110:1937–1944Google Scholar
  30. Orosová M, Špakulová M (2018) Tapeworm chromosomes: their value in systematics with instructions for cytogenetic study. Folia Parasitol 65:001Google Scholar
  31. Orosová M, Marec F, Oros M, Xi BW, Scholz T (2010a) A chromosome study and 18S rDNA in situ hybridization of Khawia saurogobii (Cestoda: Caryophyllidea). Parasitol Res 106:587–593Google Scholar
  32. Orosová M, Kráľová-Hromadová I, Bazsalovicsová E, Špakulová M (2010b) Karyotype, chromosomal characteristics of multiple rDNA clusters and intragenomic variability of ribosomal ITS2 in Caryophyllaeides fennica (Cestoda). Parasitol Int 59:351–357Google Scholar
  33. Pawlovski WP, Cande WZ (2005) Coordinating the events of the meiotic prophase. Trends Cell Biol 15:674–981Google Scholar
  34. Petkevičiūtė R (1998) A chromosome study of Khawia sinensis. Acta Zool Lithuanica 8:35–39Google Scholar
  35. Petkevičiūtė R (2001) Chromosomes of Aspidogaster conchicola. J Helminthol 75:295–297Google Scholar
  36. Petkevičiūtė R (2003) Comparative karyological analysis of three species of Bothriocephalus Rudolphi, 1808 (Cestoda: Pseudophyllidea). Parasitol Res 5:358–363Google Scholar
  37. Petkevičiūtė R, Bondarenko SK (2001) Comparative karyological studies on the species of Eubothrium Nybelin, 1922 (Cestoda: Pseudophyllidea). Syst Parasitol 50:127–134Google Scholar
  38. Petkevičiūtė R, Stunžėnas V, Stanevičiūtė G (2018) Comments on species divergence in the genus Sphaerium (Bivalvia) and phylogenetic affinities of Sphaerium nucleus and S. corneum var. mamillanum based on karyotypes and sequences of 16S and ITS1 rDNA. PLoS ONE 13(1):e0191427Google Scholar
  39. Rausch RL, Rausch VR (1990) Reproductive anatomy and gametogenesis in Shipleya inermis (Cestoda: Dioecocestidae). Ann Parasitol Hum Comp 65:229–237Google Scholar
  40. Sahara K, Marec F, Traut W (1999) TTAGG telomeric repeats in chromosomes of some insects and other arthropods. Chromosom Res 76:449–460Google Scholar
  41. Sessions SK (1996) Chromosomes: molecular cytogenetics. In: Hills DM, Moritz C, Mable BK (eds) Molecular systematics, Second edn. MA Press, Sinauer Asociates, Sunderland, pp 121–168Google Scholar
  42. Sheng MY, Wang LJ (2010) Chromosomal localization of 45S and 5S rDNA in 14 species and the implications for genome evolution of genus Epimedium. Plant Syst Evol 290:65–73Google Scholar
  43. Scholz T, Oros M (2017) Caryophyllidea van Beneden in Carus, 1863. In: Caira JN, Jensen K (eds) Planetary Biodiversity Inventory (2008–2016): tapeworms from vertebrate bowels of the earth, University of Kansas, Natural History Museum, Lawrence, special publication no, vol 25, pp 47–64Google Scholar
  44. Scholz T, Brabec J, Kráľová-Hromadová I, Oros M, Bazsalovicsová E, Ermolenko A, Hanzelová V (2011) Revision of Khawia spp. (Cestoda: Caryophyllidea), parasites of cyprinid fish, including a key to their identification and molecular phylogeny. Folia Parasitol 58:197–223Google Scholar
  45. Scholz T, Oros M, Bazsalovicsová E, Brabec J, Waeschenbach A, Xi BW, Aydoğdu A, Besprozvannykh V, Shimazu T, Králová-Hromadová I, Littlewood DTJ (2014) Molecular evidence of cryptic diversity in Paracaryophyllaeus (Cestoda: Caryophyllidea), parasites of loaches (Cobitidae) in Eurasia, including description of P. vladkae n. sp. Parasitol Int 63:841–850Google Scholar
  46. Stanevičiūtė G, Stunžėnas V, Petkevičiūtė R (2015) Phylogenetic relationships of some species of the family Echinostomatidae Odner, 1910 (Trematoda), inferred from nuclear rDNA sequences and karyological analysis. Comp Cytogen 9:257–270Google Scholar
  47. Schubert I (2007) Chromosome evolution. Curr Opin Plant Biol 10:109–115Google Scholar
  48. Silva-Neto LC, Bernardino ACS, Loreto V, Moura RC (2015) Physical mapping of 18S and 5S rDNA loci and histone H3 gene in grasshopper species of the subfamily Gomphocerinae (Acrididae). Gen Mol Res 14:15008–15015Google Scholar
  49. Singh M, Kumar R, Nagpure NS, Kushwaha B, Gond I, Lakra WS (2009) Chromosomal localization of 18S and 5S rDNA using FISH in the genus Tor (Pisces, Cyprinidae). Genetica 137:245–252Google Scholar
  50. Solovyeva AI, Stefanova VN, Podgornaya OI, Demin S (2016) Karyotype features of trematode Himasthla elongate. Mol Cytogenet 9:34Google Scholar
  51. Supiwong W, Pinthong K, Seetapan K, Saenjundaeng P, Bertollo LAC, Oliveira EA, Yano CF, Liehr T, Phimphan S, Tanomtong A, Cioffi MB (2019) Karyotype diversity and evolutionary trends in the Asian swamp eel Monopterus albus (Synbranchiformes, Synbranchidae): a case of chromosomal speciation? BMC Evol Bio 19:73Google Scholar
  52. Špakulová M, Scholz T (1999) A chromosome study of the tapeworm Bathybothrium rectangulum (Bloch, 1782) (Cestoda: Pseudophyllidea). Parasitol Res 85:270–273Google Scholar
  53. Špakulová M, Orosová M, Mackiewicz JS (2011) Cytogenetics and chromosomes of the tapeworms (Platyhelmithes, Cestoda). Adv Parasit 74:177–230Google Scholar
  54. Špakulová M, Bombarová M, Miklisová D, Nechybová S, Langrová I (2019) 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). Parasit Vectors 12(1):161. Google Scholar
  55. Teixeira GA, Barros LAC, Anguiar HJAC, Pompolo SG (2017) Comparative physical mapping of 18S rDNA in the karyotypes of six leafcutter ant species of the genera Atta and Acromyrmex (Formicidae: Myrmicinae). Genetica 145:351–357Google Scholar
  56. Traut W, Sahara K, Otto TD, Marec F (1999) Molecular differentiation of sex chromosomes probed by comparative genomic hybridization. Chromosoma 108:173–180Google Scholar
  57. Traut W, Szczepanowski M, Vitková M, Opitz C, Marec F, Zrzavy J (2007) The telomere repeat motif of basal Metazoa. Chromosom Res 15:371–382Google Scholar
  58. White MJD (1973) Modes of speciation. WH Freeman and Co., San FranciscoGoogle Scholar
  59. Xi BW, Oros M, Chen K, Scholz T, Xie J (2018) A new monozoic tapeworm, Parabreviscolex niepini n. g., n. sp. (Cestoda: Caryophyllidea), from schizothoracine fishes (Cyprinidae: Schizothoracinae) in Tibet, China. Parasitol Res 117:347–354Google Scholar
  60. Xi BW, Oros M, Wang GT, Scholz T, Xie J (2013) Khawia abbottinae sp. n. (Cestoda: Caryophyllidea) from the Chinese false gudgeon Abbottina rivularis (Cyprinidae: Gobioninae) in China: morphological and molecular data. Folia Parasitol 60:141–148Google Scholar
  61. Zadesenets KS, Karamysheva TV, Katokhin AV, Mordvinov VA, Rubtsov NB (2012) Distribution of repetitive DNA sequences in chromosomes of five opisthorchid species (Trematoda, Opisthorchiidae). Parasitol Int 61:84–86Google Scholar
  62. Zrzavá M, Hladová I, Dalíková M, Šíchová J, Ounap E, Kubíčková S, Marec F (2018) Sex chromosomes of the iconic moth Abraxas grossulariata (Lepidoptera, Geometridae) and its congener A. sylvata. Genes 9:279 doi:

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Institute of ParasitologySlovak Academy of SciencesKošiceSlovakia
  2. 2.Institute of EntomologyBiology Centre CASČeské BudějoviceCzech Republic
  3. 3.Faculty of ScienceUniversity of South Bohemia in České BudějoviceČeské BudějoviceCzech Republic
  4. 4.Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture, Freshwater Fisheries Research CenterChinese Academy of Fishery SciencesWuxiChina

Personalised recommendations