Parasitology Research

, Volume 114, Issue 4, pp 1473–1483 | Cite as

Cytogenetics of Aspidogaster limacoides (Trematoda, Aspidogastrea): karyotype, spermatocyte division, and genome size

  • Marta Bombarová
  • Marta Špakulová
  • Martin Kello
  • Petr Nguyen
  • Eva Bazsalovicsová
  • Ivica Králová-Hromadová
Original Paper


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.


Fluke Chromosome Meiosis Fluorescent in situ hybridization C value Evolution 



We wish to thank to František Marec (the Institute of Entomology, Biology Centre ASCR, České Budějovice, CR) for kind help and providing facilities during research stays of M.B. We are indebted to M. Oros and V. Hanzelová (the Institute of Parasitology SAS, Košice, SR) for their help with collecting the material, as well as to M. Farský from the Slovak Fishing Association Štúrovo, SR, for a kind provision of fish. The work was supported by the Slovak Grant Agency VEGA under contract no. 2/0168/13. P.N. was supported by Grant 14-35819P of the Czech Science Foundation. M.K. was supported (50 %) by the project “Medicínsky univerzitný park v Košiciach” (MediPark, Košice) ITMS: 26220220185 (95 %) based on the support of the Operational Programme Research and Development (OP VaV-2012/2.2/08-RO) (contract no. OPVaV/12/2013). The publication has also been created within realization of the project Centre of Excellence for Parasitology (Code ITMS: 26220120022), based on the support of the Operational Programme “Research and Development” funded from the European Fund of Regional Development (rate 0.2).


  1. Baršienė J (1993) The karyotypes of trematodes. Academia, Vilnius, 370 pp. (In Russian)Google Scholar
  2. Bazsalovicsová E, Králová-Hromadová I, Špakulová M, Reblánová M, Oberhauserová K (2010) Determination of ribosomal internal transcribed spacer 2 (ITS2) interspecific markers in Fasciola hepatica, Fascioloides magna, Dicrocoelium dendriticum and Paramphistomum cervi (Trematoda), parasites of wild and domestic ruminants. Helminthologia 47:76–82. doi: 10.2478/s11687-010-0011-1 CrossRefGoogle Scholar
  3. 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 113:4111–4116. doi: 10.1007/s00436-014-4082-7 CrossRefPubMedGoogle Scholar
  4. Birstein VJ, Mikhailova NA (1990) On the karyology of trematodes of the genus Microphallus and their intermediate gastropod host, Littorina saxatilis I. Chromosome analysis of three Microphallus species. Genetica 80:159–165. doi: 10.1007/BF00137320 CrossRefGoogle Scholar
  5. Chen MX, Zhang LQ, Wen CG, Sun J, Gao Q (2010) Phylogenetic relationship of species in the genus Aspidogaster (Aspidogastridae, Aspidogastrinae) in China as inferred from its rDNA sequences. Acta Hydrobiol Sin 34:312–316. doi: 10.3724/sp.j.1035.2010.00312 CrossRefGoogle Scholar
  6. Comai L (2005) The advantages and disadvantages of being polyploidy. Nat Rev Genet 6:836–846. doi: 10.1038/nrg1711 CrossRefPubMedGoogle Scholar
  7. Dobigny G, Ducroz JF, Robinson TJ, Volobouev V (2004) Cytogenetics and cladistics. Syst Biol 53:470–484. doi: 10.1080/10635150490445698 CrossRefPubMedGoogle Scholar
  8. Doležel J, Bartoš J, Voglmayr H, Greihuber J (2003) Nuclear DNA content and genome size of trout and human. Cytom Part A 51A:127–128. doi: 10.1002/cyto.a.10013 CrossRefGoogle Scholar
  9. Doussau De Bazignan M, Ozouf-Costaz C (1985) Une technique rapide d’analyse chromosomique appliquée à sept espèces de poissons antarctiques. Cybium 9:57–74Google Scholar
  10. Fernández R, Barragán MJL, Bullejos M, Marchal JA, Díaz de la Guardia R, Sánchez A (2002) New C-band protocol by heat denaturation in the presence of formamide. Hereditas 137:145–148CrossRefPubMedGoogle Scholar
  11. Fletcher HL, Hoey EM, Orr EM, Trudgett A, Fairweather I, Robinson MV (2004) The occurrence and significance of triploidy in the liver fluke, Fasciola hepatica. Parasitology 128:69–72. doi: 10.1017/S003118200300427X CrossRefPubMedGoogle Scholar
  12. Frydrychová R, Marec F (2002) Repeated losses of TTAGG telomere repeats in evolution of beetles (Coleoptera). Genetica 115:179–187. doi: 10.1023/A:1020175912128 CrossRefPubMedGoogle Scholar
  13. Fuková I, Nguyen P, Marec F (2005) Codling moth cytogenetics: karyotype, chromosomal location of rDNA, and molecular differentiation of sex chromosomes. Genome 48:1083–1092. doi: 10.1139/g05-063 CrossRefPubMedGoogle Scholar
  14. Gibson DI (1987) Questions in digenean systematics and evolution. Parasitology 95:429–460. doi: 10.1017/S0031182000057851 CrossRefPubMedGoogle Scholar
  15. Grzywacz B, Chobanov DP, Maryańska-Nadachowska A, Karamysheva TV, Heller K-G, Warchałowska-Śliwa E (2014) A comparative study of genome organization and inferences for the systematics of two large bushcricket genera of the tribe Barbitistini (Orthoptera: Tettigoniidae: Phaneropterinae). BMC Evol Biol 14:48. doi: 10.1186/1471-2148-14-48 CrossRefPubMedCentralPubMedGoogle Scholar
  16. Hirai H, Spotila LD, LoVerde PT (1989) Schistosoma mansoni: chromosomal localization of DNA repeat elements by in situ hybridization using biotinylated DNA probes. Exp Parasitol 69:175–188. doi: 10.1016/0014-4894(89)90186-0 CrossRefPubMedGoogle Scholar
  17. Johnston DA (2006) Genomes and genomics of parasitic flatworms. In: Maule AG and Marks NJ (eds) Parasitic flatworms: molecular biology, biochemistry, immunology and physiology. CAB International, pp 38–80Google Scholar
  18. Králová-Hromadová I, Špakulová M, Horáčková E, Turčeková Ľ, Novobilský A, Beck R, Koudela B, Marinculić A, Rajský D, Pybus M (2008) Sequence analysis of ribosomal and mitochondrial genes of the giant liver fluke Fascioloides magna (Trematoda: Fascioloidae): intraspecific variation and differentiation from Fasciola hepatica. J Parasitol 94:58–67. doi: 10.1645/GE-1324.1 CrossRefPubMedGoogle Scholar
  19. Králová-Hromadová I, Bazsalovicsová E, Boroková S, Hanzelová V (2013) Ribosomal ITS2 structure in Caryophyllaeus laticeps and Caryophyllaeus brachycollis (Cestoda: Caryophyllidea), parasites of cyprinid fish. Helminthologia 50:235–273. doi: 10.2478/s11687-013-0135-1 CrossRefGoogle Scholar
  20. Koroleva YI (1969) Karyology of some species of Diplozoon. Parazitologyia 3:411–414 (In Russian)Google Scholar
  21. Košková E, Špakulová M, Koubková B, Reblánová M, Orosová M (2011) Comparative karyological analysis of four diplozoid species (Monogenea, Diplozoidae), gill parasites of cyprinid fishes. Parasitol Res 108:935–941. doi: 10.1007/s00436-010-2135-0 CrossRefPubMedGoogle Scholar
  22. Leitch IJ, Bennett MD (2004) Genome downsizing in polyploid plants. Biol J Linn Soc 82:651–663. doi: 10.1111/j.1095-8312.2004.00349.x CrossRefGoogle Scholar
  23. Levron C, Poddubnaya L, Oros M, Scholz T (2010) Vitellogenesis of basal trematode Aspidogaster limacoides (Aspidogastrea: Aspidogastridae). Parasitol Int 59:532–538. doi: 10.1016/j.parint.2010.06.011 CrossRefPubMedGoogle Scholar
  24. LoVerde PT, Fredericksen DW (1978) The chromosomes of Cotylogaster occidentalis and Cotylaspis insignis (Trematoda: Aspidogastrea) with evolutionary considerations. Proc Helminthol Soc Wash 45:158–161Google Scholar
  25. Macgregor HC (1993) Introduction to animal cytogenetics. Chapman and Hall, London, 238 pp. ISBN 0-412-54600-0Google Scholar
  26. Martens PM, Curini-Galletti MC, Van Oostveldt P (1989) Polyploidy in Proseriata (Plathyhelminthes) and its phylogenetical implications. Evolution 43:900–907CrossRefGoogle Scholar
  27. Nguyen P, Sahara K, Yoshido A, Marec F (2010) Evolutionary dynamics of rDNA clusters on chromosomes of moths and butterflies (Lepidoptera). Genetica 138:343–354. doi: 10.1007/s10709-009-9424-5 CrossRefPubMedGoogle Scholar
  28. Olson PD, Cribb TH, Tkach VV, Bray RA, Littlewood DTJ (2003) Phylogeny and classification of the Digenea (Platyhelminthes: Trematoda). Int J Parasitol 33:733–755. doi: 10.1016/S0020-7519(03)00049-3 CrossRefPubMedGoogle Scholar
  29. Orosová M, Oros M (2012) Classical and molecular cytogenetics of Khawia sinensis (Cestoda: Caryophyllidea), invasive parasite of carp, Cyprinus carpio. Parasitol Res 110:1937–1944. doi: 10.1007/s00436-011-2720-x CrossRefPubMedGoogle Scholar
  30. Palacios-Gimenez OM, Castillo ER, Martí DA, Cabral-de-Mello DC (2013) Tracking the evolution of sex chromosome systems in Melanoplinae grasshoppers through chromosomal mapping of repetitive DNA sequences. BMC Evol Biol 13:167. doi: 10.1186/1471-2148-13-167 CrossRefPubMedCentralPubMedGoogle Scholar
  31. Park GM, Im K, Huh S, Yong TS (2000) Chromosomes of the liver fluke, Clonorchis sinensis. Korean J Parasitol 38:201–206. doi: 10.3347/kjp.2000.38.3.201 CrossRefPubMedCentralPubMedGoogle Scholar
  32. Petkevičiūtė R (2001) Chromosomes of Aspidogaster conchicola. J Helminthol 75:295–297. doi: 10.1079/JOH200050 PubMedGoogle Scholar
  33. Petkevičiūtė R, Stužėnas V, Stanevičiūtė G (2014) Differentiation of European freshwater bucephalids (Digenea: Bucephalidae) based on karyotypes and DNA sequences. Syst Parasitol 87:199–212. doi: 10.1007/s11230-013-9465-0 CrossRefPubMedGoogle Scholar
  34. Poddubnaya L, Levron C, Gibson DI (2011) Ultrastructural characteristics of the uterine epithelium of aspidogastrean and digenean trematodes. Acta Parasitol 56:131–139. doi: 10.2478/s11686-011-0016-z CrossRefGoogle Scholar
  35. Ráb P, Roth P (1988) Cold-blooded vertebrates. In: Balíček P, Forejt J, Rupeš J (eds) Methods of chromosome analysis. Czech Biological Society Publishing, Brno, pp 115–124Google Scholar
  36. Reblánová M, Špakulová M, Orosová M, Králová-Hromadová I, Bazsalovicsová I, Rajský D (2011) A comparative study of karyotypes and chromosomal location of rDNA genes in important liver flukes Fasciola hepatica and Fascioloides magna (Trematoda: Fasciolidae). Parasitol Res 109:1021–1028. doi: 10.1007/s00436-011-2339-y CrossRefPubMedGoogle Scholar
  37. Rohde K (1973) Structure and development of Lobatostoma manteri sp.nov. (Trematoda: Aspidogastrea) from the Great Barrier Reef, Australia. Parasitology 66:63–83. doi: 10.1017/S0031182000044450 CrossRefPubMedGoogle Scholar
  38. Rohde K (1994) The minor groups of parasitic Platyhelminthes. Adv Parasitol 33:145–234, 0-12-031733-8CrossRefPubMedGoogle Scholar
  39. Rohde K (2001) The Aspidogastrea, an archaic group of Platyhelminthes. In: Littlewood DTJ, Bray RA (eds) Interrelationships of the Platyhelminthes. Taylor and Francis, London, pp 159–167. ISBN 0-7484-0903-3Google Scholar
  40. Rohde K (2002) Subclass Aspidogastrea Faust & Tang, 1936. In: Gibson DI, Jones A, Bray RA (eds) Keys to the Trematoda, vol I. CABI Publishing and the Natural History Museum, Wallingford, pp 5–14. ISBN 0-85199-547-0Google Scholar
  41. Roussel P, Hernandez-Verdun D (1994) Identification of Ag-NOR proteins, markers of proliferation related to ribosomal gene activity. Exp Cell Res 214:465–472. doi: 10.1006/excr.1994.1283 CrossRefPubMedGoogle Scholar
  42. Snyder SD, Tkach VV (2007) Neosychnocotyle maggiae n. gen, n. sp. (Platyhelminthes: Aspidogastrea) from freshwater turtles in northern Australia. J Parasitol 93:399–403. doi: 10.1645/GE-1001R.1 CrossRefPubMedGoogle Scholar
  43. Swiderski Z, Poddubnaya LG, Gibson DI, Młocicki D (2012) Advanced stages of embryonic development and cotylocidial morphogenesis in the intrauterine eggs of Aspidogaster limacoides Diesing, 1835 (Aspidogastrea), with comments on their phylogenetic implications. Acta Parasitol 57:131–148. doi: 10.2478/s11686-012-0025-6 CrossRefPubMedGoogle Scholar
  44. Špakulová M, Orosová M, Mackiewicz JS (2011) Cytogenetics and chromosomes of tapeworms (Platyhelminthes, Cestoda). Adv Parasitol 74:177–230, 978-0-12-385897-9CrossRefPubMedGoogle Scholar
  45. Tiersch TR, Chandler RW, Wachtel SS, Elias S (1989) Reference standards for flow cytometry and application in comparative studies of nuclear DNA content. Cytometry 10:706–710CrossRefPubMedGoogle Scholar
  46. Wachtler F, Stahl A (1993) The nucleolus: a structural and functional interpretation. Micron 24:473–505. doi: 10.1016/0968-4328(93)90026-W CrossRefGoogle Scholar
  47. 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 61:84–86. doi: 10.1016/j.parint.2011.06.027 CrossRefPubMedGoogle Scholar
  48. Zamparo D, Brooks DR (2003) Phylogenetic systematic assessment of the Aspidobothrea (Platyhelminthes, Neodermata, Trematoda). Zool Scr 32:83–93. doi: 10.1046/j.1463-6409.2003.00088.x CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Marta Bombarová
    • 1
  • Marta Špakulová
    • 1
  • Martin Kello
    • 2
  • Petr Nguyen
    • 3
    • 4
  • Eva Bazsalovicsová
    • 1
  • Ivica Králová-Hromadová
    • 1
  1. 1.Institute of ParasitologySlovak Academy of SciencesKošiceSlovakia
  2. 2.Department of Pharmacology, Faculty of MedicinePavol Jozef Šafárik UniversityKošiceSlovakia
  3. 3.Institute of EntomologyBiology Centre AS CR, v.v.iČeské BudějoviceCzech Republic
  4. 4.Faculty of ScienceUniversity of South Bohemia in České BudějoviceČeské BudějoviceCzech Republic

Personalised recommendations