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 cont ribution of ROS to bactericidal activity was evaluated. By using striped ba ss (Morone saxatilis) macrophages as a positive comparative model, it was s hown previously that the ROS-generating capability of eastern oyster haemoc ytes is comparatively low and may be unable to exceed bacterial antioxidant capability. The current study tested the hypothesis that eastern oyster ha emocytes do not rely on ROS-dependent bactericidal mechanisms by evaluating the ability of haemocytes to kill the non-pathogenic bacteria Bacillus meg aterium and Pseudomonas fluorescens in the presence and absence of an NADPH oxidase inhibitor. Striped bass macrophages again served as a positive com parative model. The inhibition of zymosan-stimulated ROS production by east ern oyster haemocytes and striped bass macrophages by the inhibitor dipheny leneiodonium (DPI) suggests that an NADPH oxidase-like activity is associat ed with these phagocytes. The eastern oyster haemocytes were considerably m ore sensitive to the effect of DPI on ROS generation than were the striped bass macrophages: elimination of superoxide production by the oyster and fi sh phagocytes occurred at 0.5 mu M and 5.0 mu M DPI, respectively. DPI at t hese respective concentrations did not affect phagocytic capability. A DPI concentration of 5.0 mu M resulted in an approximate 50% reduction in the a bility of striped bass macrophages to kill B. megaterium and completely abo lished bactericidal activity towards P. fluorescens. A contribution of ROS to the bactericidal activity of oyster haemocytes was not evident, as 0.5 m u M DPI did not affect the killing of B. megaterium and reduced bactericida l activity towards P. fluorescens by only 4%. Although significant stimulat ion of striped bass macrophage ROS production occurred in response to the t est 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 b acterial killing mechanisms. This conclusion does not preclude the possibil ity of alternative (e.g., regulatory) functions of phagocyte-generated ROS in eastern oyster and striped bass immunology. (C) 1999 Academic Press.