Lack of involvement of reactive oxygen species in the bactericidal activity of Crassostrea virginica haemocytes in contrast to Morone saxatilis phagocytes

As part of ongoing efforts to determine the role of reactive oxygen species (ROS) in eastern oyster (Crassostrea virginica) immune reactions, the contribution of ROS to bactericidal activity was evaluated. By using striped bass (Morone saxatilis) macrophages as a positive comparative model, it was shown previously that the ROS-generating capability of eastern oyster haemocytes is comparatively low and may be unable to exceed bacterial antioxidant capability. The current study tested the hypothesis that eastern oyster haemocytes do not rely on ROS-dependent bactericidal mechanisms by evaluating the ability of haemocytes to kill the non-pathogenic bacteria Bacillus megaterium and Pseudomonas fluorescens in the presence and absence of an NADPH oxidase inhibitor. Striped bass macrophages again served as a positive comparative model. The inhibition of zymosan-stimulated ROS production by eastern oyster haemocytes and striped bass macrophages by the inhibitor diphenyleneiodonium (DPI) suggests that an NADPH oxidase-like activity is associated with these phagocytes. The eastern oyster haemocytes were considerably more sensitive to the effect of DPI on ROS generation than were the striped bass macrophages: elimination of superoxide production by the oyster and fish phagocytes occurred at 0.5 mu M and 5.0 mu M DPI, respectively. DPI at these respective concentrations did not affect phagocytic capability. A DPI concentration of 5.0 mu M resulted in an approximate 50% reduction in the ability of striped bass macrophages to kill B. megaterium and completely abolished bactericidal activity towards P. fluorescens. A contribution of ROS to the bactericidal activity of oyster haemocytes was not evident, as 0.5 mu M DPI did not affect the killing of B. megaterium and reduced bactericidal activity towards P. fluorescens by only 4%. Although significant stimulation of striped bass macrophage ROS production occurred in response to the test bacteria, exposure of oyster haemocytes to the bacteria did not result in enhanced ROS production. These data suggest that, in contrast to striped bass macrophages, eastern oyster haemocytes do not rely on ROS-dependent bacterial killing mechanisms. This conclusion does not preclude the possibility of alternative (e.g., regulatory) functions of phagocyte-generated ROS in eastern oyster and striped bass immunology. (C) 1999 Academic Press.