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 to 45 years along the United States East Coast. The relationship between parasites and oyster defense mechanisms is unclear. A better understanding of the immunopathologic association may reduce these economic losses.

Defense mechanisms of the eastern oyster (Crassostrea virginica) were quantified at the single cell level utilizing flow cytometry. Phagocytosis was measured using fluorescent beads. Respiratory burst activity was quantified as the increase in dichlorofluorescein-associated fluorescence upon stimulation. Apoptosis was evaluated with TUNEL assay and Annexin V. Three sub-populations of hemocytes (granulocytes, hyalinocytes and intermediate cells) were identified with unique functional characteristics. Granulocytes were most active at phagocytosis and peroxide production, while hyalinocytes were relatively inactive. TUNEL and Annexin V assays allowed quantification of hemocyte apoptosis, which was detected at a higher frequency in dividing cells. 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.