Raphidophycean flagellates, Chattonella marina and C. ovata, are harmful red tide phytoplankters; blooms of these phytoplankters often cause severe damage to fish farming. Previous studies have demonstrated that C. marina and C. ovata continuously produce reactive oxygen species (ROS) such as superoxide anion (O2-) hydrogen peroxide (H2O2) under normal growth conditions, and an ROS-mediated toxic mechanism against fish and other marine organisms has been proposed. Although the exact mechanism of ROS generation in these phytoplankters still remains to be clarified, our previous study suggested that NADPH oxidase-like enzyme located on the cell surface of C. marina may be involved in O2- generation. To investigate the localization of O2- and H 2O2 generation in C. marina and C. ovata, we employed 2-methyl-6(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazin-3-one and 5-(and-6)-carboxy-2′,7′-dichlorodihydrodihydrofluorescein dictate, acetyl ester, which are specific fluorescent probe for detecting O 2- and H2O2, respectively. Observation by fluorescence microscopy of live phytoplankters incubated with each probe revealed that O2- is mainly generated on the cell surface, whereas H2O2 is generated in the intracellular compartment in these phytoplankters. When the cells were ruptured by ultrasonic treatment, O2- levels of C. marina and C. ovata decreased significantly, whereas a few times higher levels of H 2O2 were detected in the ruptured cell suspensions when compared with the levels of the live cell suspension. In immunoblotting analysis, the protein recognized by anti-human gp91 phox was detected in both species. These results suggest that, in both phytoplankters, the underlying mechanisms of O2- and H2O2 generation may be distinct and such systems are independently operating in the cells.
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