Integrated behavioural and physiological responses of sand smelt larvae to the effects of warming and hypoxia as combined stressors.

Forecasts indicate that rising temperatures towards the future and the expansion of dead zones will change environmental suitability for fish early stages. Therefore, we assessed the chronic effects of warming (26 °C), hypoxia (<2-2.5 mg L-1) or their combination on mortality rate, growth, behaviour, energy metabolism and oxidative stress using Atherina presbyter larvae as a model species. There were no differences between the treatments in terms of mortality rate. The combination of warming and hypoxia induced faster loss of body mass (+22.7%). Warming, hypoxia or their combination enhanced boldness (+14.7-25.4%), but decreased exploration (-95%-121%), increased the time in frozen state (+60.6-80.5%) and depleted swimming speed (-45.6-50.5%). Moreover, routine metabolic rate was depleted under hypoxia or under the combination of warming and hypoxia (-56.6 and 57.2%, respectively). Under hypoxia, increased catalase activity (+56.3%) indicates some level of antioxidant defence capacity, although increased DNA damage (+25.2%) has also been observed. Larvae also exhibited a great capacity to maintain the anaerobic metabolism stable in all situations, but the aerobic metabolism is enhanced (+19.3%) when exposed to the combination of both stressors. The integrative approach showed that changes in most target responses can be explained physiologically by oxidative stress responses. Increased oxidative damages (lipid peroxidation and DNA damage) and increased interaction between antioxidant enzymes (superoxide dismutase and catalase) are associated to increased time in frozen state and decreased swimming activity, growth rates and boldness. Under all stressful situations, larvae reduced energy-consuming behaviours (e.g. depleted exploration and swimming activity) likely to stabilize or compensate for the aerobic and anaerobic metabolisms. Despite being an active small pelagic fish, we concluded that the sensitive larval phase exhibited complex coping strategies to physiologically acclimate under thermal and hypoxic stress via behavioural responses.

Early life stage mechanisms of an active fish species to cope with ocean warming and hypoxia as interacting stressors.

Ocean's characteristics are rapidly changing, modifying environmental suitability for early life stages of fish. We assessed whether the chronic effects of warming (24 °C) and hypoxia (<2-2.5 mg L-1) will be amplified by the combination of these stressors on mortality, growth, behaviour, metabolism and oxidative stress of early stages of the white seabream Diplodus sargus. Combined warming and hypoxia synergistically increased larval mortality by >51%. Warming induced faster growth in length and slower gains in weight when compared to other treatments. Boldness and exploration were not directly affected, but swimming activity increased under all test treatments. Under the combination of warming and hypoxia, routine metabolic rate (RMR) significantly decreases when compared to other treatments and shows a negative thermal dependence. Superoxide dismutase and catalase activities increased under warming and were maintained similar to control levels under hypoxia or under combined stressors. Under hypoxia, the enzymatic activities were not enough to prevent oxidative damages as lipid peroxidation and DNA damage increased above control levels. Hypoxia reduced electron transport system activity (cellular respiration) and isocitrate dehydrogenase activity (aerobic metabolism) below control levels. However, lactate dehydrogenase activity (anaerobic metabolism) did not differ among treatments. A Redundancy Analysis showed that ∼99% of the variability in mortality, growth, behaviour and RMR among treatments can be explained by molecular responses. Mortality and growth are highly influenced by oxidative stress and energy metabolism, exhibiting a positive relationship with reactive oxygen species and a negative relationship with aerobic metabolism, regardless of treatment. Under hypoxic condition, RMR, boldness and swimming activity have a positive relationship with anaerobic metabolism regardless of temperature. Thus, seabreams may use anaerobic reliance to counterbalance the effects of the stressors on RMR, activity and growth. The outcomes suggests that early life stages of white seabream overcame the single and combined effects of hypoxia and warming.