For a comprehensive understanding of fish responses to increasing thermal stress in marine environments, we investigated tissue energetics, antioxidant levels, inflammatory and cell death responses in Sparus aurata (gilthead seabream) red muscle during exposure to elevated temperatures (24 °C, 26 °C, 30 °C) compared to the control temperature of 18 °C. Energetic aspects were assessed by determining lactate, glucose and lipids levels in blood plasma, ATP, ADP and AMP levels, and AMPK phosphorylation as an indicator of regulatory changes in energy metabolism, in tissue extracts. Oxidative defence was assessed by determining superoxide dismutase, catalase and glutathione reductase maximum activities. Moreover, xanthine levels were determined as an indicator of purine conversion to xanthine and associated ROS production. In the context of inflammatory response and cell death due to oxidative stress, pro-inflammatory cytokines (IkBα phosphorylation, IL-6 and TNFα) levels, and LC3 II/I ratio and SQSTM1/p62 as indicators of autophagic-lysosomal pathway were also determined. A recovery in the efficacy of ATP production after a marked decrease during the 1st day of exposure to 24 °C is observed. This biphasic pattern is paralleled by antioxidant enzymes' activities and inflammatory and autophagy responses, indicating a close correlation between ATP turnover and stress responses, which may benefit tissue function and survival. However, exposure beyond 24 °C caused tissue's antioxidant capacity loss, triggering the inflammatory and cell death response, leading to increased fish mortality. The results of the present study set the thermal limits of the gilthead seabream at 22-24 °C and establish the used cellular and metabolic indicators as tools for the definition of the extreme thermal limits in marine organisms.
Keywords: Antioxidant responses; Cell death; Fish; Inflammation; Metabolic responses; Temperature.