Flow-cytometric analysis of haemocytes from eastern oysters, Crassostrea virginica, subjected to a sudden temperature elevation : I. Haemocyte types and morphology

Abstract

In this report, we provide detailed protocols for flow-cytometric characterization of haemocytes from the eastern oyster, Crassostrea virginica, and report effects of sudden temperature elevation upon haemocyte characteristics. Haemocytes were differentiated from other particles in the haemolymph using a combination of the DNA-binding fluorochrome SYBR Green and internal cell complexity. In formalin-fixed haemolymph, four distinct subpopulations of haemocytes were found: small hyalinocytes, large hyalinocytes, small granulocytes, and granulocytes. Hyalinocytes were numerically dominant, small and large together accounting for 60% of the haemocytes; granulocytes accounted for 30–35%, and small granulocytes were least numerous at <5%. These percentages were the same for haemolymph analysed immediately after it was withdrawn from living oysters (without fixation) as for haemolymph fixed with formalin, although small and large hyalinocytes were not always clearly differentiated into two distinct populations. There was a general trend for the fixed cells to appear in the flow cytometer to be larger and more complex than the fresh ones (except for granulocyte complexity). Oysters subjected to a sudden increase in temperature from 20 to 28 °C for 1 week showed significant changes in haemocyte morphology. Sudden temperature elevation was associated with a general decrease in size of all haemocyte types. The ability to detect changes in individual haemocyte types for many oysters by these flow-cytometric methods should improve progress in understanding oyster mortality associated with combined effects of environmental stresses, disease, and parasites.