Anesthetizing solar-powered sea slugs for photobiological studies

Photosynthetic sea slugs have the ability to "steal" chloroplasts (kleptoplasts) from marine macroalgae and keep them structurally intact and physiologically functional. The photosynthetic activity of these symbioses has been assessed using pulse amplitude modulated (PAM) fluorometry. However, the movement of these sacoglossan slugs can impair specific photobiological studies on kleptoplasts. Thus, immobilizing sacoglossan slugs while not interfering with the photosynthetic activity would be a methodological advance for research in this field. We evaluated the effect of two anesthetics, eugenol and MS-222, on the photosynthetic activity of kleptoplasts and on the behavior of the kleptoplasts-bearing slug Elysia viridis. Anesthetics promoted relaxation of sea slug muscle with no touch reaction in about 6 min. Sea slugs immobilized for 120 min completely recovered after anesthetic removal. No significant differences were found on photosynthetic parameters measured immediately (0-1 min) after immobilization. The effective quantum yield of photosystem II of E. viridis after 120 min of immobilization was significantly decreased by 12% in the MS-222 treatment, while eugenol promoted no significant effect. Photosynthetic activity assessed by rapid light-response curves (RLC) of relative electron transport rates (rETR) revealed a significant decrease in both initial response to light (-34%) and maximum rETR (rETR(m)) (-60%), after 120 min of immobilization using MS-222. After 120 min of immobilization with eugenol, the initial response to light significantly decreased 15% and rETR(m) decreased 27%. We conclude that, whenever photobiological studies employing PAM fluorometry require immobilization of photosynthetic sea slugs, eugenol can be used as a powerful anesthetic with little impact on the photosynthetic activity of kleptoplasts.