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

[摘要] 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 fragmentsfollowing 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 establishcultures 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 everyculture. 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 arecharacteristics 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-2Athat 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.

[学科分类] 生物科学（综合）

[关键词] Perkinsus atlanticus;Tapes decussatus;In vitro culture;Clam parasite;Ribosomal RNA gene complex;ITS;Ultrastructure