Oxidative stress inside cells occurs when the production of reactive oxygen species (ROS) is no longer efficiently counterbalanced by the generation of antioxidants. In this study, we measured the intracellular production of ROS, including hydrogen peroxide (H2O2), superoxide (O2-), and singlet oxygen (1O2), in cultured dinoflagellates of the genus Symbiodinium under thermal and oxidative stress. ROS tagged with fluorescent probes were measured by flow cytometry. Dissimilar Symbiodinium internal transcribed spacer 2 (ITS2) clades or phylotypes (A1, B2, E, F1) produced ROS in different quantities in response to stress. For example, when comparing the control (26 °C) to the high-temperature treatment (35 °C), Symbiodinium E showed no change in the intracellular concentrations of any of the ROS; but phylotype A1 displayed a 10-fold increase in the overall ROS concentration and a 4-fold increase in O2-. Under oxidative stress, when 8 mmol l-1 H2O2 was added to the cells, these same two Symbiodinium phylotypes increased their overall concentrations of ROS, but only Symbiodinium E showed an increase in the concentrations of O2- (2×) and 1O2 (3×). Therefore, not only were the stress responses of the various Symbiodinium phylotypes different but also the responses of individual phylotypes to thermal and oxidative stress were different in terms of ROS production. Variation in the quality and quantity of ROS generation and its implications for subsequent antioxidant production suggest that different stress mechanisms are at play. While our experiments were done under laboratory conditions that did not necessarily mirror ecological ones, these results provide new insight into processes inside Symbiodinium cells during stress events and add new explanations for a phylotype's susceptibility to stress.
Keywords: 1O2, singlet oxygen; APX, ascorbate peroxidase; CAT, catalase; DMSO, dimethyl sulfoxide; H2DCF-DA, 2′,7′-dichlorodihydrofluorescein diacetate; ITS2, internal transcribed spacer 2; NPQ, non-photochemical quenching; O2−, superoxide; OEC, oxygen-evolving complex; OH•, hydroxyl radical; PSI/II, photosystem I/II; ROS, reactive oxygen species; SOD, superoxide dismutase; SOG, singlet oxygen sensor green.