Oxidative stress and antioxidant defenses in experimentally bleached and copper exposed sea anemones (Anemonia viridis).
Supervisors: Ibon Cancio, Oihane Diaz de Cerio (UPV/EHU)
Symbiotic relationship between anthozoans and their associated photosynthetic dinoflagellates (Symbiodinium) provides a highly valuable adaptive advantage for survival in the intertidal marine environment. However, photosynthesis also poses risks for the host animals especially under environmental stress, in some cases leading to overproduction of reactive oxygen species (ROS). Under prolonged stress, this can present a challenge for the antioxidant defenses and ultimately lead to bleaching and breakdown of the symbiotic relationship as it is occurring with coral reefs under climate change. In the present study, aposymbiotic sea anemones, Anemonia viridis, were produced by menthol treatment (0.38 mmol/L for three days followed by 0.19 mmol/L up to 30  days) to observe the cellular and molecular responses after bleaching. Additionally, the response of bleached and unbleached organisms was studied under exposure for 1 and 7 days to the prooxidant metal copper (10 μg/L of CuCl2.2H2O). A nearly complete loss of Symbiodinium was observed after menthol treatment, and Cu induced a more limited bleaching effect on individuals not treated with menthol. ROS generation was significantly induced only in tentacles of bleached anemones exposed to Cu, but not in bleached vs unbleached (unexposed) ones. To study the transcription levels of antioxidant enzymes first the partial CDS of ovothiol synthase A (ovoA) was sequenced and annotated in A. viridis. The transcription levels of ovoA, gpxa, gpxj and gpxde were then analyzed in the tentacles of anemones without observing any clear pattern of regulation in any of the four experimental groups. Menthol bleaching did not modify the transcription pattern of these genes. Targeted metabolomic analysis revealed low levels of all three forms of the H2O2 detoxifying ovothiol (OSH-A, -B and -C) in the tentacles of the studied animals, OSH-B being the prevailing form. Higher levels of OSH-B were quantified in the menthol bleached group (day-1) suggesting a response to the ROS generated during the bleaching process but not to Cu exposure. Glutathione appeared mainly in its reduced form (GSSG) in all groups of organisms indicating strong pro-oxidant state in general in the tentacles of A. viridis in the present experiment. In consequence, menthol induced bleaching cannot be demonstrated to result in increased oxidative stress in sea anemones, while Cu exposure really induces an excess production of ROS without a concomitant clear elevation of the antioxidant defense barriers. Sea anemones could constitute a useful model to study the mechanisms underlying bleaching effects in cnidarians under laboratory set-ups using menthol or temperature induced bleaching. However, and in the future, this process should be monitored during the bleaching process, and not once it has finished.