IN-VITRO CULTURE OF THE EASTERN OYSTER PARASITE PERKINSUS-MARINUS - OPTIMIZATION OF THE METHODOLOGY

We have succeeded in establishing an in vitro continuous culture of the eastern oyster (Crassostrea virginica) parasite Perkinsus marinus (Gauthier and Vasta, 1993) and have now characterized a number of variables, such as temperature, salinity, pH, seeding density, and selected nutritional requirements that affect parasite proliferation. Optimum temperature, salinity, and pH ranges were 28-32 degrees C, 25-30 ppt, and 6.6-6.8, respectively. Proliferation rates during the first 48 hr of culture were directly related to the size of the inoculum. The addition of Ham's F12 nutrient mixture (a serum replacement) to Dulbecco modified Eagle's (DME) base medium substantially enhanced proliferation, particularly at a DME:Ham's F12 ratio of 1:2. Five percent fetal bovine serum was required for optimal growth, but higher concentrations (10-20%) were inhibitory. The presence of oyster plasma enhanced growth only at low concentrations of fetal bovine serum (0-0.1%) and doubled addition of Ham's F12 (DME:Ham's 1:2). After approximately 30 passes over a 6-month period, the cultured parasite exhibited morphological and virulence features that are similar to those from the freshly collected specimens. The optimized medium apparently provides an environment comparable to the intracellular conditions of the host since the small (approximately 4 mu m) trophozoite stage frequently observed to proliferate within the oyster hemocyte is maintained. (C) 1995 Academic Press, Inc.