Effects of pesticide exposure and thermal stress in a model tropical reef fish, the damselfish Acanthochromis polyacanthus

Coastal ecosystems including tropical coral reefs are increasingly threatened by land based pollution which is now occurring simultaneously with an increase in ocean temperatures. Despite the high ecological and economical importance of these ecosystems, the impacts of pollutants and thermal stress on tropical marine organisms have only recently been considered and are usually studied in isolation to each other. In South-East Asia and North-Eastern Australia bordering the Great Barrier Reef (GBR), one of the most widely used insecticides is the organophosphate (OP) chlorpyrifos (CPF). In both regions, the wet season produces high rainfall and significant flood plumes transporting CPF onto inshore reefs at a time when reefs also experience high summer water temperatures. A few studies have shown that the damselfish Acanthochromis polyacanthus from the Indo-Pacific is sensitive to stress and this fish is becoming a model species in ecophysiology. Still, the underlying mechanisms leading to such sensitivity are largely unidentified. More importantly, very little is known about the effects of simultaneous stressors on reef fish generally. Consequently, this thesis aims to provide new insights into the neurophysiology, oxidative stress response and molecular stress response of a model tropical reef fish from the Indo-Pacific region, the damselfish Acanthochromis polyacanthus, after exposure to CPF and/or thermal stress under laboratory-controlled conditions.\ud \ud Juveniles of A. polyacanthus bred at James Cook University were exposed to a) CPF alone, b) temperature stress and c) simultaneous CPF and temperature stresses. Stress markers were used to examine different types of stress response. Activity of the neural enzyme Cholinesterase (ChE) was determined in muscle, as it is recognized as a sensitive indicator for OP exposure. Characterization of ChE was also undertaken to determine the predominant form of the enzyme in muscle tissue in this model reef fish species. To explore potential oxidative stress responses two key antioxidant molecules were examined. This included measurement of the ratio of the Coenzyme Q antioxidant form (CoQH₂) over total CoQ, as well as measurement of the activity of the detoxifying enzyme glutathione-S-transferase (GST) in liver tissue. Finally, expression of candidate stress-responsive genes (heat-shock protein 90, GST, Catalase, Elongation Factor 1α) was examined to explore the molecular response to CPF exposure and thermal stress in A. polyacanthus.\ud \ud Prior to experimental CPF exposure, ChE characterization revealed the presence of a complex mixture of ChEs in A. polyacanthus muscle tissue. This mixture was comprised of an atypical vertebrate acetylcholinesterase (AChE) form, as well as a typical vertebrate AChE and an atypical vertebrate butyrylcholinesterase (BChE). Reports in the literature of two other Perciforme species possessing similar ChEs and inhabiting coral reefs suggests evolutionary implications of atypical ChEs and raises the questions of their functional significance in coral reef fish.\ud \ud Laboratory-controlled CPF exposure revealed that fish exposed to 1 μg/L, 10 μg/L and 100 μg/L of CPF for 96 h (4 days) exhibited a 26%, 49% and 53% decrease in cholinesterase activity, respectively, compared to solvent control fish. This demonstrates that ChE inhibition is a sensitive marker for CPF exposure in A. polyacanthus. Furthermore, an increase in the antioxidant CoQ form was found after just 6 h of exposure to 10 g/L CPF, suggesting an early oxidative stress effect and potentially the involvement of CoQH2 to combat CPF-induced oxidative stress.\ud A thermal stress experiment using a slow increase in temperature revealed that exposure of the fish to 32°C or 34°C compared to 28°C induced a 50% reduction in ChE activity after 96 h of exposure. Most importantly, after 7 days of recovery at 28°C, ChE activity still remained 39% lower in thermally stressed fish, compared to fish only exposed to acclimation temperature (28°C) during the entire experiment. In vitro measurements of ChE activity indicated that temperature does not directly impact the activity of the enzyme itself between 26°C and 34°C, which clearly indicates that the in vivo decline in A. polyacanthus ChE activity associated with temperature increase is the result of a biological stress response in the fish. In addition, CoQ showed a decrease in antioxidant form in the early time points of the experiment in fish exposed to 34°C compared to 28°C, which suggests that this molecule is used to combat temperature-stress induced oxidative stress in liver cells. The results of this chapter demonstrate for the first time a link between thermal stress and a dysfunction of a neural enzyme in a tropical reef fish, which might have important implications for fish fitness and behaviour in the context of changing climates.\ud \ud CPF exposure and thermal stress were also applied simultaneously on juvenile A. polyacanthus. Combined effects were tested with 1 and 10 μg/L CPF at 28°C or 31°C. No interaction between the two factors was found for ChE activity, but ChE activity was decreased by CPF exposure after 7 days of exposure. In addition, the temperature increase of 3°C provided a small measure of protection against CPF at 31°C, since fish exposed to both CPF and 31°C exhibited higher ChE activity than fish exposed to CPF at 28°C. The results obtained for ChE activity across the three experiments indicated a thermal threshold for ChE activity between 31°C and 32°C in A. polyacanthus, which corroborates whole organism responses in the recent literature on this species. In addition, GST activity also decreased in fish exposed to 31°C compared to 28°C, further supporting the hypothesis of a fine thermal threshold for this species. Finally, four target genes exhibited lower expression in fish exposed to CPF compared to control fish, after 4 days of treatment. This may indicate that a general decrease in gene expression occurred in response to CPF exposure in A. polyacanthus. Such results indicate that CPF affects many cellular functions in A. polyacanthus over even this short time-frame.\ud \ud This thesis presents novel information on the response to stress in A. polyacanthus, a model species for the Indo-Pacific region. It identifies a strong and prolonged effect of temperature on ChE, a central enzyme in the nervous system of the fish, provides evidence for the use of antioxidant molecule during both CPF exposure and thermal stress, determines a general decline in gene expression as a result of CPF exposure and most importantly suggests that this species might possess a fine thermal threshold at between 31°C and 32°C. Together, these results indicate that A. polyacanthus is sensitive to both CPF exposure and thermal stress and that numerous functions are negatively affected by both stressors. While combined pressures of pollution and thermal stress are generally considered to be worse than individual pressures, this study also revealed that fish may in fact cope better with some pollutants at temperatures just below their thermal threshold. However, increases in runoff-associated pollution are likely to negatively affect A. polyacanthus and other tropical fish as climate change increases sea surface temperatures beyond their narrow thermal tolerance.\u