THE POTENTIAL IMPACT OF MACROALGAL GENERATED HYDROGEN PEROXIDE LEVELS ON CO-OCCURRING ORGANISM IN ROCK POOLS by AGAPERY YOANE PATTINASARANY Abstract Macroalgae have been identified as a potential source of ROS (e.g., hydrogen peroxide) in some rock pools and enclosed habitats; the accumulation of ROS may be harmful to other organisms. Although some studies have found vulnerability to these ROS, there has been little research on the potential impact of photosynthesis-induced oxidative stress on organisms living in rock pools. The aims of this study were to investigate the levels of hydrogen peroxide produced by macroalgae and to assess the effect of similar levels of H2O2 on a rock-pool resident, the snakelocks anemone. The objective of the first experimental chapter was to assess the physicochemical parameters in rock pools during a tidal cycle, including hydrogen peroxide levels. During this work, two methods for measuring H2O2 levels were used. A fluorescence method, which is commonly used in laboratories, was developed further by examining whether the reaction product was sufficiently stable to carry out the fluorogenic reaction in the field and then measure the fluorescence back in the lab, whereas the luminescence method involved a novel application of a low-cost portable luminometer designed for measuring environmental ATP using firefly bioluminescence. According to this study, the ‘weediest’ pools had the highest H2O2 levels. Temperature, pH, PAR, and salinity were also measured at low tide to see how much the conditions changed hourly depending on the presence or absence of macroalgae (weediness). Photosynthetic activity was detected in the pools as evidenced by an increase in pH and dissolved oxygen, which was greater in weedy pools than in non-weedy pools. Furthermore, the influence of abiotic stress, i.e., different physicochemical circumstances, on the steady state hydrogen peroxide production (Chapter 2) of three species of intertidal macroalgae, a green alga (Ulva lactuca), a brown alga (Fucus serratus), and a red alga (Chondrus crispus), was investigated in the laboratory. All three species reached steady-state levels of hydrogen peroxide after 4 h of exposure at various temperatures, pH levels, salinities, and light intensities. The highest level of H2O2 was found with F. serratus (4.6 µM), while the lowest was with C. crispus (1 µM). H2O2 production was doubled when macroalgae were desiccated, but it was reduced when C. crispus was present. The fluorescence of chlorophyll a was also measured to investigate the effect of abiotic stress on photosynthesis in the macroalgae. The raw fluorescence of the macroalgae showed the typical polyphasic O-J-I-P increase. Even under stress, photosynthetic units are active, and there was an effect on the downregulation of the photochemical machinery, which manifested as dynamic photoinhibition; however, it was only an increase in light intensity that had a substantial impact on photosynthesis. The goal of the final experimental chapter was to investigate the effect of ambient H2O2 on two morphs of the snakelocks anemone (Anemonia viridis). Following tissue extraction, total glutathione and TBARS levels were measured, as well as superoxide dismutase and catalase activity, measured using spectrophotometry. Furthermore, zooxanthellae counts were performed. Notably, no other physiological indicators were altered in the anemone, including total glutathione, implying that the pool of reduced glutathione (GSH) remained stable. Furthermore, despite the fact that superoxide dismutase (SOD) activity increased as H2O2 concentration increased, there was no significant difference between treatments. Catalase (CAT) activity, on the other hand, increased dramatically at the highest H2O2 concentration used. This suggests that H2O2 alone was unlikely to have changed zooxanthellae density in this study.

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