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 p1gtoplankters; blooms of these phytoplankters often cause severe damage to Ph farming. Previous studies have demonstrated that C marina and C. ovata continuously produce reactive oxygen species (ROS) such as superoxide anion (O-2(-)) 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 O-2(-) generation. To investigate the localization of 02 and H2O2 generatioin C. marina and C. ovata, we employed 2-methyl-6(p-methoxyphenyl)-3,7-dihydroi lmidazo[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 0(2)(-) 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, 02 levels of C. marina and C. ovata decreased significantly, whereas a few times higher levels of H2O2 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 O-2(-) and H2O2 generation may be distinct and such systems are independently operating in the cells.