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Parasitology Research

, Volume 107, Issue 4, pp 909–914 | Cite as

Ethanol-fixed material used for both classical and molecular identification purposes: Eudiplozoon nipponicum (Monogenea: Diplozoidae) as a case parasite species

  • Eva KoškováEmail author
  • Iveta Matějusová
  • Kristína Civáňová
  • Božena Koubková
Original Paper

Abstract

This study is focused on the feasibility of two treatments of alcohol-fixed monogenean parasites which are intended to be use for the combined morphological and molecular characterizations. The monogenean parasite, Eudiplozoon nipponicum, was selected as a model parasite species; however it is expected that these techniques will be suitable for other monogeneans and other parasitic families. The haptor of diplozoid parasites is equipped with sclerotized attachment clamps and central hooks which are utilized for morphological identification. As parasite tissue become very tough and rigid when preserved in ethanol, using these structures for species identification without additional treatment is difficult. We investigated two different techniques to digest the surrounding tissues, the first was treatment with 10% sodium dodecyl sulphate (SDS) and the second treatment was proteinase K. Tissue was successfully digested in both treatments and all clamps, central hook and even individual sclerites of the clamps were clearly visible and well defined. After treatment, the digest was used to extract genomic DNA, and the second internal transcribed spacer of the ribosomal DNA genes (rDNA) was amplified. Nucleic acid sequence was obtained from 90% of parasite specimens processed by both treatments. Treatment of haptors with SDS was proven to be more successful with no visible changes or damage observed to sclerites even after a month. This method represents a useful tool for the combined morphological and molecular studies as the correct sequence can be assigned to the same individual worm from which haptoral parts have been obtained.

Keywords

Sodium Dodecyl Sulphate Nucleic Acid Sequence ITS2 rDNA Parasite Tissue Monogenean Parasite 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

Acknowledgements

This research was supported by the Czech Science Foundation Grant No. 524/07/1610 and No. 526/09/H025, Long-Term Research Plan MSM 0021622416 and Ichthyoparasitology Research Centre of the Ministry of Education, Youth and Sports of the Czech Republic (LC 522).

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Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Eva Košková
    • 1
    Email author
  • Iveta Matějusová
    • 2
  • Kristína Civáňová
    • 1
  • Božena Koubková
    • 1
  1. 1.Department of Botany and Zoology, Faculty of ScienceMasaryk UniversityBrnoCzech Republic
  2. 2.Molecular Genetics, Marine LaboratoryAberdeenUK

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