Abstract

Infectious diseases are a significant problem in oyster aquaculture, causing immense production losses. The protozoan parasites, Perkinsus marinus and Haplosporidium nelsoni, have generated losses estimated in the hundreds of millions of dollars over the last 35-45 years on the east coast of the United States. The relationship between parasites and defense mechanisms of oysters is unclear. A better understanding of the immunopathologic association may reduce these economic losses. We have designed assays to quantify the defense mechanisms of the Eastern Oyster (Crassostrea virginica) at the single cell level utilizing flow cytometry, which allows the discrimination of subpopulations of hemocytes. Phagocytosis of fluorescent microspheres was used to assess phagocytosis, the first line of defense that allows the engulfment of foreign microorganisms. The respiratory burst, the ability of cells to generate oxygen free radicals in order to kill and destroy the phagocytized microorganisms, was measured as the increase in dichlorofluorescein-associated fluorescence upon stimulation. Three sub-populations of hemocytes (granulocytes, hyalinocytes, and intermediate cells) were identified with unique functional characteristics. Granulocytes were the most active at phagocytosis and respiratory burst, while hyalinocytes were relatively inactive. Natural Killer (NK) cell-like activity was detected in oyster hemocytes, and was stimulated by human recombinant IL-2. TUNEL and Annexin V assays allowed quantification of hemocyte apoptosis, an important mechanism to induce cell death, which was detected at a higher frequency in dividing cells. We also demonstrated the influence of environmental conditions, such as temperature and salinity, on the immune functions of oyster hemocytes, suggesting that they could influence disease susceptibility. Similarly, we demonstrated that experimental infection with P. marinus influenced the induction of apoptosis in oyster hemocytes. Overall, flow cytometry can rapidly, accurately, and directly quantify the morphology and function of a large number of individual cells, and will lead to a better understanding of the bivalve immune system and susceptibility to disease under different environmental conditions.