Presence of the distinct systems responsible for superoxide anion and hydrogen peroxide generation in red tide phytoplankton Chattonella marina and Chattonella ovata

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.