Oxidative burst in hard clam ( Mercenaria mercenaria) haemocytes

Haemocytes of bivalve molluscs are known to be responsible for many immunological functions, including recognition, phagocytosis, and killing or elimination of invading microorgansisms, such as potentially infective bacteria and parasites. In many bivalves, killing of microorganisms engulfed by haemocytes is accomplished by a sudden release of reactive oxygen species (ROS) within the haemocytes; this response is referred to as an oxidative burst. Previous studies have failed to detect oxidative burst in haemocytes of the hard clam (northern quahog), Mercenaria mercenaria. In the present study, we applied a widely used chemical probe for ROS detection in haemocytes, dichlorofluorescin-diacetate (DCFH-DA), to haemocytes from this clam species and used flow cytometry to quantify fluorescence in individual haemocytes. Oxidation of DCFH-DA to the fluorescent product, DCF, within unstimulated haemocytes indicated that ROS were clearly produced in these cells. Two activators of oxidative burst, zymosan and bacterial extracellular products, which have been applied successfully to haemocytes in other species, stimulated large increases in ROS production in hard clam haemocytes. Furthermore, two inhibitors of ROS production, W-13 and diphenylene iodinium (DPI), significantly suppressed ROS production by haemocytes. Nitric oxide synthase inhibitors, NMMA and L-NIO, did not suppress ROS production, indicating that the observed oxidation of DCFH-DA is not mediated by nitric oxide. These results show unequivocally that haemocyte oxidative burst is active in M. mercenaria and, therefore, is a likely mechanism in host response to pathogens and parasites.