Diverse responses of Symbiodinium types to menthol and DCMU treatment

Abstract

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 (IC₅₀) 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.

Keywords: Aposymbiotic coral; PSII system; ROS activity; Symbiodinium; Symbiodinium cell depletion.

Figure 1. Dose effects of menthol and dichlorophenyl dimethylurea (DCMU) on inactivation of photosystem II (PSII) function in freshly isolated Symbiodinium.

The means of PSII inhibition (n = 3) after 4 h of incubation in menthol (A) or DCMU (B) are plotted in the presence of various reagent concentrations.

Figure 2. Diverse reactive oxygen species (ROS) generation levels among Symbiodinium types when treated with artificial seawater (ASW) containing menthol.

Freshly isolated Symbiodinium (FIS) was incubated in menthol-supplemented ASW for 4 h, followed by 2′, 7′-dichlorofluorescin diacetate labeling and microscopic examination of fluorescence. Menthol concentrations used in FIS incubation were 1.73 mM for types C1 (S. goreaui), C3, C15, and D1a (S. trenchii), and 2.43 mM for B1 (S. minutum), which would cause complete breakdown of PSII activity in the algae. ROS signals in type C3 treated with the ASW control (A) and menthol-treated Symbiodinium types B1, D1a, C15, C3, and C1 (B∼F) are presented with a representative photo; n = 3, scale bar in the photo represents 50 µm.

Figure 3. The time course of photosystem II (PSII) activity decline and reactive oxygen species (ROS) generation when treating the Symbiodinium C3 with menthol and artificial seawater (ASW).

Changes in PSII activity (A) and ROS levels (B) in type C3 treated with 1.73 mM menthol were determined and plotted with incubation time. ASW-incubated congeneric Symbiodinium was used as a control.