Flow cytometric analysis of haemocytes from eastern oysters, Crassostrea virginica, subjected to a sudden temperature elevation: II. Haemocyte functions: aggregation, viability, phagocytosis, and respiratory burst

The capability of an oyster to respond to environmental stresses, such as periodically high summer temperatures, as well as disease or parasite infections, depends, in large measure, upon the viability and functional capability of haemocytes. Eastern oysters (Crassostrea virginica) were subjected to a sudden increase in temperature from 20 to 28 °C for 1 week, and several haemocyte functions were determined before and after the temperature elevation using the flow cytometer. Previously, we described the characterization of different haemocyte types using new and modified flow cytometric methods. In this report, we provide detailed protocols for flow cytometric methods to: (1) determine haemocyte aggregation using paired samples with or without an antiaggregant solution; (2) assess haemocyte viability using propidium iodide (PI); (3) quantify haemocyte phagocytosis with fluorescent microbeads; and (4) measure the respiratory burst response of individual haemocytes using 2′,7′-dichlorofluorescein diacetate (DCFH-DA) and zymosan to activate the release of reactive oxygen species (ROS).The temperature increase caused no significant change in haemocyte aggregation, although there was a trend of increasing aggregation in granulocytes and small granulocytes, but a slight decrease in hyalinocyte aggregation. Phagocytosis of all haemocyte types decreased after the temperature increase. Significantly higher percentages of dead haemocytes in all haemocyte types (attributable to a large increase in mortality of hyalinocytes, the most numerous cells) were found after the temperature increase, suggesting generally less capable immune function. Numbers of dead small granulocytes and granulocytes tended to decrease, but this was not statistically significant. Effects of temperature elevation upon respiratory burst were not statistically significant; however, a trend of increased ROS production after temperature elevation was consistent for all haemocyte types. Granulocytes, hyalinocytes, and small granulocytes showed increased production of ROS in the presence of zymosan; granulocytes showed the highest induced fluorescence.