Regulatory mechanisms in Anadara granosa with different hemoglobin concentrations in response to heat stress

Climate change impacts the physiological processes of species, with rising seawater temperatures posing severe existential threats to marine mollusks. Therefore, studying the responses of marine organisms to heat stress is crucial. The blood clam, Anadara granosa, is a marine bivalve species that contains hemoglobin, and individuals with higher hemoglobin concentrations (HCs) exhibit better physiological health. In this study, we analyzed the regulatory mechanisms in the blood clam with different HCs in response to heat stress. Enzyme activity indicates that blood clams with different HC levels respond to heat stress by regulating oxidative stress and altering metabolic pathways, with clams with higher HCs showing greater adaptability and stress resistance. Transcriptome analysis of the control group (C), the high-HC heat-stressed group (TH), and the low-HC heat-stressed group (TL) revealed that endoplasmic reticulum processing and cytochrome P450 metabolism are crucial for the heat stress response. Additionally, the IP3R/Ca²⁺ signaling pathway regulates the response in clams with higher HCs. Combined miRNA-mRNA analysis showed that clams with higher HCs have more heat-responsive genes, forming a more complex regulatory network. Dual-luciferase reporter assays revealed that agr-miR-936 negatively regulates Calr, involved in the heat stress response, while agr-miR-1935-Cenph may affect HC differences by regulating total hemocyte counts. Our findings deepen the understanding of the molecular regulatory mechanisms that allow blood clams to cope with heat stress, offering new insights for aquaculture to manage extreme temperatures amid global warming.