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The winter syndrome in the gilthead sea bream Sparus aurata indicates that the species is exposed to critically low temperatures in Mediterranean aquaculture in winter. The present study of metabolic patterns and molecular stress responses during cold exposure was carried out to investigate this "disease", in light of the recent concept of oxygen and capacity limited thermal tolerance. The metabolic profile of fuel oxidation was examined by determining the activities of the enzymes hexokinase (HK), aldolase (Ald), pyruvate kinase (PK), L-lactate dehydrogenase (L-LDH), citrate synthase (CS), malate dehydrogenase (MDH) and 3-hydroxyacyl CoA dehydrogenase (HOAD) in heart, red and white muscle after exposure to temperatures of 10, 14 and 18°C. Especially, the increase in LDH activity combined with the accumulation of L-lactate in tissues indicates that temperatures below 14°C are critical for Sparus aurata and stimulate the anaerobic component of metabolism. Increase in the activity of HOAD suggests that oxidation of free fatty acids might contribute to ATP turnover at low temperatures. The expression of Hsp70 and Hsp90 in all tissues examined revealed a cellular stress response during cooling below 18°C. In the light of winter temperatures in S. aurata cultures around 10°C, our data suggest that the fish are exposed to stressful conditions at the low end of their thermal tolerance window. These conditions likely impair the aerobic capacity of the fish, compromise the rates of growth and reproduction and may contribute to elicit pathological conditions.

The present study assesses the resilience of the Mediterranean gilthead seabream (Sparus aurata) to acute warming and water acidification, using cellular indicators of systemic to molecular responses to various temperatures and CO2 concentrations. Tissue metabolic capacity derived from enzyme measurements, citrate synthase, 3-hydroxyacyl CoA dehydrogenase (HOAD), as well as lactate dehydrogenase. Cellular stress and signaling responses were identified from expression patterns of Hsp70 and Hsp90, the phosphorylation of p38 MAPK, JNKs and ERKs, from protein ubiquitylation and finally from the levels of transcription factor Hif-1α as an indicator of systemic hypoxemia. Exposure to elevated CO2 levels at temperatures higher than 24 °C generally caused an increase in fish mortality above the rate caused by warming alone, indicating effects of the two factors and a failure of acclimation and thus the limits of phenotypic plasticity to be reached. As a potential reason, tissue-dependent induction and stabilization of Hif-1α indicate hypoxemic conditions. Their exacerbation by enhanced CO2 levels is linked to the persistent expression of Hsp70 and Hsp90, oxidative stress and activation of MAPK and ubiquitin pathways. Antioxidant defence is enhanced by expression of catalase and glutathione reductase, however, leaving superoxide dismutase suppressed by elevated CO2 levels. On longer timescales in specimens surviving warming and CO2 exposures, various metabolic adjustments initiate a preference to oxidize lipid via HOAD for energy supply. These processes indicate significant acclimation up to a limit and a time-limited capacity to survive extreme conditions passively by exploiting mechanisms of cellular resilience.

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Long-term exposure of Mytilus galloprovincialis to temperatures beyond 26oC triggers mussels' mortality. The present study aimed to integratively illustrate the correlation between intermediary metabolism, Hsp gene expression and oxidative stress related proteins in long-term thermally stressed Mytilus galloprovincialis and whether they are affected by thermal stress' magnitude and duration. We accordingly evaluated the gene expression profiles, in the posterior adductor muscle (PAM) and the mantle, concerning hsp70 and hsp90, and the antioxidant defense indicators Mn-SOD, Cu/Zn-SOD, catalase, glutathione S-transferase, and the metallothioneins mt-10 and mt-20. Moreover, we determined antioxidant enzymes' activities, oxidative stress through lipid peroxidation and activities of intermediary metabolism enzymes. The pattern of changes in relative mRNA expression levels indicate that mussels are able to sense thermal stress even when exposed to 22oC and before mussels' mortality is initiated. Data indicates a close correlation between the magnitude and duration of thermal stress with lipid peroxidation levels, changes in the activity of antioxidant enzymes and the enzymes of intermediary metabolism. The gene expression and increase in the activities of antioxidant enzymes support a scenario, according to which exposure to 24oC might trigger ROS production, which is closely correlated with anaerobic metabolism under hypometabolic conditions. Increase and maintenance of oxidative stress in conjunction with energy balance disturbance seems to trigger mussels' mortality after long term exposure at temperatures beyond 26oC. Eventually, in the context of preparation for oxidative stress, certain hypotheses and models are suggested, integrating the several steps of cellular stress response.

The present study aimed to examine the expression and activation of MAPKs (p38 MAPK, ERK1/2, and JNKs) in red blood cells (RBCs) of the gilthead sea bream, Sparus aurata, during thermal stress and investigate their involvement in the expression of heat shock proteins. The data showed that only p38 MAPK is detected in RBCs of Sparus aurata and it is phosphorylated and activated during exposure to increased temperature. Induction of Hsp70 in thermally stressed RBCs was abolished in the presence of the p38 MAPK inhibitor, SB203580, suggesting the involvement of the kinase in this response. This mechanism might play a cytoprotective role in the RBCs of the gilthead sea bream.

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