Diverse responses of Symbiodinium types to menthol and DCMU treatment

To understand the mechanism of photosynthetic inhibition and generation of reactive oxygen species (ROS) in Symbiodinium types under stress, chemicals such as dichlorophenyl dimethylurea (DCMU) are widely used. Moreover, DCMU and recently menthol were used to generate aposymbiotic cnidarian hosts. While the effects of DCMU on Symbiodinium cells have been extensively studied, no studies have shown the mechanism behind menthol-induced coral bleaching. Moreover, no study has compared the effects of DCMU and menthol treatments on photosystem II (PSII) activity and generation of ROS in different Symbiodinium types. In this study, we utilized five freshly isolated Symbiodinium types (S. minutum (B1), S. goreaui (C1), C3, C15, and S. trenchii (D1a)) to compare the effects of DCMU and menthol treatments. Symbiodinium cells were exposed to DCMU and menthol at different concentrations for 4 h. Results showed that values of the 50% inhibitory concentration (IC(50)) for PSII inhibition were 0.72∼1.96 mM for menthol-treated cells compared to 29∼74 pM for DCMU-treated cells. Diverse responses of Symbiodinium types were displayed in terms of PSII tolerance to menthol (S. minutum > S. trenchii = C15 > C3 = S. goreaui), and also in the response curves. In contrast, responses were not so diverse when the different types were treated with DCMU. Three of five menthol-treated Symbiodinium types showed instant and significant ROS generation when PSII activity was inhibited, compared to no ROS being generated in DCMU-treated Symbiodinium types. Both results indicated that menthol inhibited Symbiodinium PSII activity through Symbiodinium type-dependent mechanisms, which were also distinct from those with DCMU treatment. This study further confirmed that photosynthetic functions Symbiodinium have diverse responses to stress even within the same clade.