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Diseases of Aquatic Organisms

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DAO 52:217-231 (2002)  -  doi:10.3354/dao052217

Continuous in vitro culture of the carpet shell clam Tapes decussatus protozoan parasite Perkinsus atlanticus

Sandra M. Casas1, Jerome F. La Peyre2, Kimberly S. Reece3, Carlos Azevedo4, Antonio Villalba1,*

1Centro de Investigacións Mariñas, Consellería de Pesca e Asuntos Marítimos, Xunta de Galicia, Aptdo. 13, 36620 Vilanova de Arousa, Spain
2Cooperative Aquatic Animal Health Research Program, Department of Veterinary Science, Louisiana State University Agricultural Center, Baton Rouge, Louisiana 70803, USA
3School of Marine Science, Virginia Institute of Marine Science, The College of William and Mary, Gloucester Point, Virginia 23062, USA
4Department of Cellular Biology, Institute of Biomedical Sciences, University of Oporto, 4050 Porto, Portugal
*Corresponding author. Email:

ABSTRACT: Continuous in vitro cultures of the clam Tapes decussatus parasite Perkinsus atlanticus were established from infected gill fragments, infected haemolymph and parasite hypnospores isolated from infected gill fragments following incubation in Ray¹s fluid thioglycollate medium (RFTM). No continuous cultures could be initiated from P. atlanticus zoospores. Cultures initiated from hypnospores yielded the highest percentage of continuous cultures (100%, 6/6), followed by cultures initiated from gill fragments (93%, 43/46) and from haemolymph (30%, 3/10). Failures to establish continuous cultures were due to microbial contamination. The source of parasite influenced the success rate, the time taken to establish cultures and the size of cultured cells. In vitro proliferation of parasite cells was mainly by vegetative multiplication. Zoosporulation, yielding motile biflagellated zoospores, was observed at a low frequency (<1% of dividing cells) in every culture. Morphology of cultured cells examined with light and transmission electron microscopy corresponded to that of P. atlanticus found in clam tissues. Cultured cells enlarged in RFTM and stained blue-black with Lugol¹s solution, which are characteristics of the Perkinsus species cells. DNA sequences of the internal transcribed spacer (ITS) region of the ribosomal RNA gene complex matched those of P. atlanticus. All cultures were established in a medium designated JL-ODRP-2A that was similar in composition to the culture medium JL-ODRP-1 originally used to propagate Perkinsus marinus in vitro. Proliferation of P. atlanticus in vitro could be supported by the commercial culture medium (1:2 v/v) DME:Ham¹s F-12 with fetuin.

KEY WORDS: Perkinsus atlanticus · Tapes decussatus · In vitro culture · Clam parasite · Ribosomal RNA gene complex · ITS · Ultrastructure

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