RESPONSE OF CRASSOSTREA-VIRGINICA TO IN-VITRO CULTURED PERKINSUS-MARINUS - PRELIMINARY COMPARISONS OF 3 INOCULATION METHODS

The recent development of in vitro culture methods for the oyster path ogen Perkinsus marinus (Mackin, Owen & Collier) provides a bountiful s upply of axenic parasites for biological investigation. Understanding how this parasite interacts with its host, Crassostrea virginica (Gmel in), is of paramount importance. Here we report and discuss the result s of several preliminary experiments on the response of C. virginica t o in vitro-cultured P. marinus and the early fate of these cultured ce lls. In three separate experiments, doses of 10(2)-10(7) parasites per oyster of in vitro-cultured parasites were used to challenge healthy oysters (mean wet tissue weight = 13.8 g) via feeding, shell cavity in jection, or adductor muscle injection. After 7 wk, no oysters from the feeding trial were infected. Most oysters in the shell cavity and add uctor muscle injection trials had detectable infections, but variabili ty was high. Mean infection intensity increased with dosage, but the e ffect of dosage was not significant in all trials, probably because of low sample size. The cultured parasites produced infection intensitie s that appeared to be markedly lower than those reported for natural c ells under similar experimental dosing conditions. Sixty-two percent o f shell cavity and adductor muscle injections, excluding controls, pro duced infections with fewer than 100 parasites per oyster, the lowest dosage used in this study. In another experiment, the early fate of cu ltured cells was investigated for each inoculation method by sampling rejecta over 4 days after dosing. On average, 4% of fed cells, 7% of c ells injected into the shell cavity, and 13% of cells injected into th e adductor muscle were recovered. Eighty-two percent of discarded para sites, many in phagocytes; were found on Day 1 postinoculation. Oyster s were sacrificed on Day 4 to determine total body parasite burdens. R egardless of delivery method, total recovery of parasites (discarded a nd total parasite burden) was low compared with the dose administered: 4% from feedings, 12% from shell cavity injections, and 21% from addu ctor muscle injection. Finally, transmission electron microscopy of he mocytes removed at 2, 6, and 18 h postinoculation appeared to indicate that hemocytes can digest in vitro-cultured parasites, possibly expla ining the low recovery rates and indicating a mechanism for the appare ntly low pathogenicity of cultured P. marinus.