BEAULIEU, L.; ALLEN, S.; BURNETT, K.; BURNETT, L.*: Low pH Effects on the Killing Activity of Oyster Hemocytes

An assay was used to measure the ability of hemocytes of the Eastern oyster, Crassostrea virginica to kill the bacterium Vibrio parahaemolyticus. Experiments were carried out at pH 7.6 and 6.6 mimicking the in vivo hemolymph environment within the oyster under well aerated conditions (7.6) and during air exposure (6.6). Assays were carried out using 96-well plates to expose hemocytes to Vibrio (1:20 ratio) for 3 hours at room temperature during which time the hemocytes phagocytized the Vibrio. The phagocytosis stage of the assay was terminated by adding 0.2% Tween to the wells, killing the hemocytes but not the Vibrio. A culture medium was then added to each well and the remaining Vibrio allowed to grow for 2 hours. The number of live Vibrio in each well after the growout period was measured using a colorimetric cellproliferation assay. A killing index was calculated based on the number of Vibrio in wells with hemocytes compared to their control wells (i.e., with hemocytes killed at time zero). A killing index of Vibrio was expressed as a percentage of the control, e.g., where an index of 50% indicates that 50% of the bacteria were killed by hemocytes. The average killing index at pH 7.7 was 49.2 (1.86 SEM; n=4) and lower, but not significantly different at pH 6.6, 38.8 (1.80 SEM; n=4). Under these assay conditions hemocyte viability, as judged by trypan blue exclusion, was high at the beginning of the assay (90.4-98% viable at pH 7.6; 87.4-95% at pH 6.6) and remained high for three hours (83.2-92% viable at pH 7.6; 86-92% at pH 6.6). These results suggest that low pH does not cause a significant reduction in the killing activity of oyster hemocytes. Supported by NSF-REU DBI-9876926 & ODRP NA96RG0488.