Enzyme system responsible for the generation of superoxide anion in red tide plankton, Chattonella marina

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Chattonella marina, a raphidophycean flagellate, is one of the most noxious red tide phytoplankton and is highly toxic to fish, especially to yellowtail Seriola quinqueradiata. Although the precise mechanism of the toxic action of Chattonella sp. remains unclear, it has been demonstrated that a decrease in oxygen partial pressure of arterial blood is the earliest physiological disturbance observed in fish after exposure" to C. marina. Physiological and histological studies of fish exposed to C.\marina also'suggested that;the blockade of respiratory water flow through the gill lamellae caused by excessive mucusiinterferes with O2 transfer, resulting in asphyxia. C. marina is known to produce reactive oxygen species (ROS) such as superoxide anion (O2-), hydrogen peroxide (H2O2), and hydroxyl radical (-OH) under normal physiological conditions. Although several lines of evidence suggest that ROS are involved in the mortality of fish exposed to C. marina, the mechanism; of ROS generation in C. marina is remained to be clarified. In this study, we found that the cell-free supernatant prepared from C. marina cells showed NAD(P)H-dependent O2- generation; and this response was inhibited by diphenyleneiodonium (DPI), a specific inhibitor of mammalian NADPH oxidase. When the cell-free supernatant of C. marina was analyzed by immunoblotting using antibody raised against the human neutrophil cytochrome b558 large subunit (91 kDa), a main band with approximately 110 kDa was detected. Furthermore, the cell surface localization of the epitope recognized with this antibody was demonstrated in C. marina by indirect immunofluorescence. These results provide evidence for the involvement of enzymatic system analogous to the neutrophil NADPH oxidase as a source of O2- production in C. marina.