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The influence of increasing temperatures in thermal and oxidative stress responses were studied in the muscle of several estuarine fish species (Diplodus vulgaris, Diplodus sargus, Dicentrarchus labrax, Gobius niger and Liza ramada). Selected fish were collected in July at the Tagus estuary (24±0.9°C; salinity of 30±4‰; pH=8). Fish were subjected to a temperature increase of 1°C.h−1 until they reached their Critical Thermal Maximum (CTMax), starting at 24°C (control temperature). Muscle samples were collected during the trial and results showed that oxidative stress biomarkers are highly sensitive to temperature. Results from stress oxidative enzymes show alterations with increasing temperature in all tested species. Catalase (CAT; EC 1.11.1.6) activity significantly increased in L. ramada, D. labrax and decreased in D. vulgaris. Glutathione S-transferase (GST; EC 2.5.1.18) activity increased in L. ramada, D. sargus, D. vulgaris, and D. labrax. In G. niger it showed a cycle of increase-decrease. Lipid peroxidation (LPO) increased in L. ramada, D. sargus and D. labrax. With respect to correlation analysis (Pearson; Spearman r), the results showed that oxidation products and antioxidant defenses were correlated in L. ramada (LPO-CAT and LPO-GST, D. sargus (LPO-CAT), and D. labrax (LPO-CAT). Oxidative biomarkers were correlated with thermal stress biomarker (Hsp70) in L. ramada (CAT-Hsp70), D. vulgaris (LPO-Hsp70), D. labrax (GST-Hsp70) and G. niger (LPO-Hsp70). In conclusion, oxidative stress does occur with increasing temperatures and there seems to be a relation between thermal stress response and oxidative stress response. The results suggest that oxidative stress biomarkers should be applied with caution, particularly in field multi-species/multi-environment studies. [ABSTRACT FROM AUTHOR]

• Mussels exhibited intraspecific differences depending on each environmental scenario. • Harbour mussels exhibited differences in lethal and sublethal responses. • Female mussels showed more sensitivity after chemical and warming insults. • Lethal responses could determine the non-reversibility imprint of biomarker responses. The intertidal mussel B. rodriguezii is a representative species from hard bottom substrates where both anthropogenic and natural stressors are present. Pre-exposure to these different stressors can modify the tolerance to additional stressors such as warming. Moreover, this tolerance can vary depending on intraspecific variables such as the organism's sex. The effects of warming and its intraspecific variability in representative coastal species are crucial to understanding the tolerance to future environmental scenarios. The mussels were collected in different environmental scenarios, including low (Control), chemical (Harbour) and natural stressed (Estuary) sites, and then exposed to different water temperatures (10–30 °C) for 14 days. Lethal and sublethal responses were evaluated in different mussel populations. Thus, cumulative death rate, air survival time, heat shock proteins (HSC70/HSP70), total ubiquitin, catalase (CAT), glutathione-s-transferase (GST) and lipid peroxidation (TBARS) were assessed in mussels from different areas and different sexes. The results revealed diminished air survival time and high cumulative mortality rate in mussels collected at the harbour and those exposed to higher temperatures, respectively. The sublethal responses of the field animals showed different patterns according to the different areas investigated. Besides, the results revealed that these differences were also observed between sexes. Regarding the sublethal responses in mussels exposed to warming, the interactive effects of temperature and sites showed a strong influence on all biochemical parameters analyzed (p < 0.001). Therefore, harbour mussels showed a distinct pattern compared to other locations and reflecting the most damaging effects of warming. The influence of sex and its interactions with warming were also crucial in most of the sublethal responses (p < 0.05). Multivariate analysis was performed with all sublethal responses, and the different warming scenarios showed different groups according to the sites. In the predicted warming scenarios, males showed no differences between sites. In contrast to males, females showed differences between sites in the predicted and the worse-case warming scenarios. Our results highlight the importance of compensatory mechanisms in the mussel warming tolerance like HSP70. The influence of sex is also crucial in understanding warming tolerance in mussels chronically exposed to pollutants in their natural environment. Also, lethal endpoints are essential for understanding the non-reversibility signature of the observed biochemical responses. [ABSTRACT FROM AUTHOR]