Benefit or burden? Effects of repeated heat exposure on thermal tolerance, energetics, and immunity in the blue mussel Mytilus edulis.

Irregular heat spikes associated with marine heat waves may either induce thermal hardening and enhance resilience or exceed physiological limits and compromise future stress tolerance in marine ectotherms. Understanding the mechanisms underlying these contrasting responses is critical for predicting the impacts of increasing thermal variability. We investigated whether prior heat exposure induces protective responses in Baltic blue mussels (Mytilus spp.). Mussels were exposed to repeated heat stress (RHS; four cycles of 2.5 h at 27°C followed by 21.5 h recovery at 15°C) and subsequently allowed a 4-day recovery period. Cardiac performance during acute warming (15-35°C) was assessed using heart rate and Arrhenius breakpoint temperature (ABT). In addition, RHS-exposed and naïve mussels were subjected to prolonged heat exposure (27°C, 5 days), during which energy status, mitochondrial activity, and transcriptional responses associated with proteostasis, oxidative stress, immunity, and apoptosis were evaluated. RHS induced a transient proteostatic and immune response, characterized by increased expression of hsp70A and immune-related genes (MyD88a, MyD88b, MyD88c, C1, and TLRb), which was not maintained during recovery. During prolonged heat exposure, RHS-exposed mussels exhibited 100% mortality by day 5, indicating the absence of a beneficial hardening response. Prolonged heat exposure induced chaperone responses (hsp60 and hsp90) in both groups, reflecting activation of stress response pathways. In contrast, RHS-exposed mussels showed reduced expression of antioxidant genes, including glutathione peroxidase (GPx) and manganese superoxide dismutase (MnSOD), suggesting compromised antioxidant defence and increased vulnerability to cellular damage. Despite maintaining high ATP levels and stable mitochondrial electron transport system activity, RHS-exposed mussels appeared unable to effectively allocate energy towards repair and stress mitigation. Overall, repeated heat stress and recovery did not induce thermal hardening of Baltic blue mussels under the tested conditions but instead reduced tolerance to prolonged heat exposure. This outcome may reflect the combined effects of the high prior heat exposure temperature, low salinity, and population-specific characteristics influencing adaptive thermal responses.