Synergy of environmental variables alters the thermal window and heat shock response: an experimental test with the crab Pachygrapsus marmoratus.

The intertidal zone is an extremely variable habitat, imposing stressful conditions on its inhabiting communities. Tolerance towards extremes of temperature, salinity and pH are crucial in these habitats. Despite the vast literature on stress tolerance, few studies have focused on the synergistic effects of several variables on thermal tolerance and HSP70 (heat shock protein 70 kDa) levels. In this work, the crabs were exposed to three experimental conditions 1) thermal ramp at standard pH (8) and saline conditions (35‰) (named T), 2) thermal ramp at standard pH (8) and hyposaline conditions (15‰) (named T plus HypoS), and 3) thermal ramp at lower pH (7) and standard saline conditions (35‰) (named T plus A). Two physiological parameters (Critical Thermal Maximum - CTMax, and osmolality) and a stress biomarker (HSP70) were chosen for this analysis. These parameters were measured in all of the aforementioned conditions. CTMax for each set of conditions was reached by exposing the organisms to a rate of temperature increase of 1 °C h(-1) until loss of equilibrium. Haemolymph samples were taken every 2 °C to quantify HSP70 and osmolality. Results showed that CTMax did not differ between crabs solely exposed to T stress and crabs exposed to T plus HypoS stress. However, HSP70 production was impaired in T plus HypoS stress. When crabs were exposed to T plus A stress, they showed a significantly higher CTMax, suggesting that short-term exposure to acidified conditions may alter the thermal window of this species. Nevertheless, in T plus A conditions HSP70 production was impaired as well. Regarding osmolality it decreased according to temperature increase in all tested stress conditions. This study showed that the heat stress response is altered by the synergistic effect of variables. Physiological end-points (i.e. CTMax) may vary and the expression of stress proteins such as HSP70 may be impaired.