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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 (HO), superoxide (O), and singlet oxygen (O), 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 O. Under oxidative stress, when 8 mmol l HO 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 O (2×) and O (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.

the temperate A-type showing an especially high resistance to both stressors. In conclusion, a range of Symbiodinium types were shown to differ in their susceptibilities to both thermal and oxidative stress, though in contrast to our original hypothesis, sensitivity to oxidative stress did not necessarily predict thermal sensitivity (or vice versa).

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<p>Coral bleaching, the loss of symbiotic dinoflagellate algae (genus Symbiodinium) and/or photosynthetic algal pigments from their coral host has become a regular occurrence in the last few decades due to increasing seawater temperatures. A key consideration in bleaching susceptibility is the symbiotic alga‘s physiology and its capacity to deal with abiotic stress; oxidative stress is of particular interest given that this can arise from thermally induced photosynthetic dysfunction. The aim of this study was to compare the effects of thermal and oxidative stress on the photosynthetic performance of a range of Symbiodinium clades and types (i.e. sub-clades) in different states of symbiosis (in hospite, freshly isolated and in culture). Whether the responses to these two stressors are related was investigated; in particular, it was hypothesised that more thermally sensitive types would be more sensitive to oxidative stress. Furthermore, the study aimed to elucidate the role of antioxidants in the observed stress responses. The specific objectives were 1) to establish whether different types of cultured Symbiodinium have dissimilar sensitivities to oxidative stress, induced by hydrogen peroxide (H₂O₂), and whether these are related to their thermal sensitivities; 2) measure the activity and relative amounts of specific reactive oxygen species (ROS) in different types of cultured Symbiodinium in response to thermal and oxidative stress induced by H₂O₂; 3) measure total antioxidant activity in different cultured Symbiodinium types when under oxidative stress; and 4) compare and contrast the responses of different Symbiodinium types to thermal and oxidative stress when in hospite (i.e. in corals) and freshly isolated. In this study, I showed that different Symbiodinium clades and types can differ widely in their responses to both thermal and oxidative stress. This was indicated by photosynthetic performance measured by chlorophyll fluorescence, and differences in the quantity of specific ROS measured via fluorescent probes and flow cytometry. For instance, when adding H₂O₂ to Symbiodinium F1, originally from Hawaii, a decrease of > 99% in maximum quantum yield (Fv/Fm) was displayed, while there was no change in Fv/Fm in the temperate Symbiodinium A1, freshly isolated from the anemone Anthopleura aureoradiata from New Zealand. When comparing the difference in ROS production between the control (26 °C) and a thermal stress treatment (35 °C), type E1 from Okinawa showed no difference in any of the measured ROS. In contrast, a different A1 type from the Gulf of Aqaba displayed an increase in the overall production of ROS, and more specifically in the production of superoxide. Symbiodinium types also displayed differential oxidative stress resistance, which was apparent from their antioxidant activities; in particular, total antioxidant capacity was measured by the ferric reducing antioxidant potential (FRAP) and cellular antioxidant activity (CAA) assays. For example, the aforementioned Symbiodinium types, A1 from the Gulf of Aqaba and F1, increased their antioxidant activities with increasing H₂O₂ concentrations. Meanwhile, type E1 displayed higher baseline levels of antioxidants in comparison to the other two types (A1, F1), which then decreased with increasing H₂O₂. Specific activities of superoxide dismutase and ascorbate peroxidase were also measured. Stress susceptibility appears to be related both to Symbiodinium type and geographic origin, but greater sensitivity to thermal stress did not necessarily correlate with greater susceptibility to oxidative stress. The exact relationship between thermal and oxidative sensitivities in Symbiodinium spp. remains elusive, but it is suggested that different types might follow different strategies for dealing with stress. I propose that some Symbiodinium types rely more on photo-protection when exposed to thermal stress (and hence cope less with oxidative stress), while other types depend more on antioxidants and oxidative stress resistance. The latter might be the better strategy for types from more variable environments, such as higher latitude reefs or intertidal regions, where potentially stressful conditions may be encountered more frequently. This study gives new insights into the variability of stress responses in the genus Symbiodinium, and the complex relationship between thermal and oxidative stress. The implications of these findings for coral bleaching susceptibility and the biogeographic distribution of different Symbiodinium types are discussed.</p>