Journal of fish pathology (한국어병학회지)

The Korean Society of Fish Pathology (한국어병학회)
권호 표지

Development of DNA probe for a protistan parasite of tunicate Halocynthia roretzi

  • Choi, Dong-Lim (Pathology Division, National Fisheries Research and Development Institute) ;
  • Hwang, Jee-Youn (Pathology Division, National Fisheries Research and Development Institute) ;
  • Choi, Hee-Jung (Pathology Division, National Fisheries Research and Development Institute) ;
  • Hur, Young-Baek (Aquaculture environment institute, National Fisheries Research and Development Institute)
  • Received : 2010.11.04
  • Accepted : 2010.12.08
  • Published : 2010.12.31
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Abstract

Edible tunicate Halocynthia roretzi, one of the most commercially important aquatic organisms in Korea, has been killed by tunic softness syndrome since last decade. The intracellular protistan parasite observed by the transmission electron microscope in hemocytes of the tunicate was considered to be the causative agent of the mass mortality. The goal of the present work is to examine the characteristic features of the parasite by identifying the 18S rDNA sequences of the parasite. The experiments conducted include amplification of presumptive 18S rDNA from diseased tunicate tissues with UNonMet-PCR and sequencing the product. A preliminary phylogenetic analysis was performed on the presumptive parasite rDNA. A digoxigenin labeled DNA probe was designed on the basis of the sequences of rDNA. Dig-ISH assay was conducted to diagnose the protistan parasite. A PCR using UNonMet-PCR primer generated 595 bp SSU rDNA fragment. Subsequently, PCRs with primer pair expended this sequence to 1542 bp. This is the first partial sequences of SSU rDNA gene to be published on the protistan parasite that has presumed causing the mass mortality of tunicate. Since the Dig-ISH technique demonstrated the presence of infection in hemocytes on the all host tissues, the fragment was confirmed to be the intracellular protistan parasite SSU rDNA. A phylogenetic analysis suggested that the protistan parasite may be a unique eukaryote that is closely related to Apicomplexa.

Keywords

References

  1. Adlard, R.D., and Lester, R.J.G.: Development of a diagnostic test for Mikrocytos roughleyi, the aetiological agent of Australian winter mortality of the commercial rock oyster, Saccostrea commercialis (Iredale & Roughley). J. Fish. Dis., 18:609-614, 1995. https://doi.org/10.1111/j.1365-2761.1995.tb00365.x
  2. Anderson, T.J., Adlard R.D., and Lester, R.J.G.: Molecular diagnosis of Marteilia sydneyi (Paramyxea) in Sydney rock oyster, Saccostrea commercialis (Angas). J. Fish. Dis., 18:507-510, 1995. https://doi.org/10.1111/j.1365-2761.1995.tb00354.x
  3. Bower, S.M., Carnegie, R.B., Goh, B., Jones, S.R., Lowe, G.J., and Mak, M.E.S.: Preferential PCR amplification of parasitic protistan small subunit rDNA from metazoan tissues. J. Eukaryot. Microbiol., 51(3):325-332, 2004. https://doi.org/10.1111/j.1550-7408.2004.tb00574.x
  4. Carnegie, R.B., Barber, B.J., Distel, D.L., and Culloty, S.C.: Development of PCR and in situ hybridization assays for detection of Bonamia ostrea in flat oysters, Ostrea edulis. J. Shellfish Res., 18(2):711-712, 1999.
  5. Carnegie, R.B., Meyer G.R., Blackbourn, J., Cochennec-Laureau, N., Berthe, F.C.J., and Bower, S.M.: Molecular detection of the oyster parasite Mikrocytos mackini, and a preliminary phylogenetic analysis. Dis. Aquat. Org., 54:219-227, 2003. https://doi.org/10.3354/dao054219
  6. Choi, D.L., Jee, B.Y., Choi, H.J., Hwang, J.Y., Kim, J.W., and Berthe, F.C.J.: First report on histology and ultrastructure of an intrahemocytic paramyxean parasite (IPP) from tunicate Halocynthia roretzi in Korea. Dis. Aquatic Org., 72:65-69, 2006. https://doi.org/10.3354/dao072065
  7. Ciancio, A., Scippa, S., Finetti-sialer, M., De Candia, A., Avallone, B., and De Vincentiis, M.: Redescription of Cardiosporidium cionae(Van Gaver and Stephan, 1907)(Apicomplexa: Piroplasmida), a plasmodial parasite of ascidian haemocytes. Eur.J.Protistol., 44:181-196, 2008 https://doi.org/10.1016/j.ejop.2007.11.005
  8. Felsenstein, J.: PHYLIP (phylogeny infernece package). Department of genetics, University of Washington, Seattle. 1996.
  9. Fong, D., Chan, M.M.Y., Rodriguez, R., Chen, C.C., Liang, Y., Littlewood, and D.T.J., Ford, S.E.: Small subunit ribosomal RNA gene sequence of the parasitic protozoan Haplosporidium nelsoni provides a molecular probe for the oyster MSX disease. Mol. Biochem. Parasitol., 62:139-142, 1993. https://doi.org/10.1016/0166-6851(93)90190-9
  10. Hillis, D.M., and Dixon, M.T.: Ribosomal DNA: molecular evolution and phylogenetic inference. Quart. Rev. Biol., 66:411-453, 1991. https://doi.org/10.1086/417338
  11. Howard, D.W., and Smith, C.S.: Histological techniques for bivalve mollusks. NOAA Tech Memo NMFSF/NEC-25:1-97. 1983.
  12. Le Roux, F., Audemard, C., Barnaud, A., and Berthe, F.: DNA probes as potential tools for the detection of Marteilia refingens. Mar. Biotechnol., 1:588-597, 1999. https://doi.org/10.1007/PL00011814
  13. Meyer, G.R., Bower, S.M., and Carnegie, R.B.: Sensitivity of a digoxigenin-labelled DNA probe in detecting Mikrocytos mackini, causative agent of Denman Island disease (mikrocytosis), in oysters. J. Invertebr. Pathol., 88:89-94, 2005. https://doi.org/10.1016/j.jip.2004.11.002
  14. Stokes, N.A., and Burreson, E.M.: A sensitive and specific DNA probe for the oyster pathogen Haplosporidium nelsoni. J. Eukaryot. Microbiol., 42(4):350-357, 1995. https://doi.org/10.1111/j.1550-7408.1995.tb01593.x
  15. Stokes, N.A., Siddall, M.E., and Burreson, E.M.: Detection of Haplosporidium nelsoni (Haplosporidia: Haplosporidiidae) in oysters by PCR amplification. Dis. Aquatic Org., 23:145-152, 1995a https://doi.org/10.3354/dao023145
  16. Stokes, N.A., Siddall, M.E., and Burreson, E.M.: Small subunit ribosomal RNA gene sequence of Minchinia teredinis (Haplosporidia: Haplosporidiidae) and a specific DNA probe and PCR primers for its detection. J. Invertebr. Pathol., 65:300-308, 1995b. https://doi.org/10.1006/jipa.1995.1046
  17. Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F., and Higgins, D. G.: The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res., 25:4876, 1997. https://doi.org/10.1093/nar/25.24.4876