Protein Disulfide Isomerase A6 (PDIA6) Restrains Heat-Induced Oxidative Damage in Haemocytes of the Pacific Oyster (Crassostrea gigas)

Heat stress causes severe oxidative damage and immune cell death in marine bivalves, but its upstream regulators remain unclear. This study identified regulators linking heat stress to oxidative damage in Pacific oyster haemocytes. Under 30 °C exposure, the apoptosis rate of oyster haemocytes increased from ~4.54% to 17.40% at 24 h, accompanied by elevated ROS, malondialdehyde, and lipid hydroperoxide and reduced SOD activity. GSEA and protein interaction analysis of the haemocyte transcriptome pinpointed protein disulfide isomerase A6 (CgPDIA6) as the hub gene linking endoplasmic reticulum stress, apoptosis, and oxidative stress. Single-cell in silico knockout placed CgPDIA6 at the head of a coupled SOD–peroxiredoxin relay (CgSOD1, CgSOD2, CgPRDX6) and shifted haemocytes toward a stress-activated state. This prediction was confirmed by RNAi knockdown, in which silencing CgPDIA6 aggravated heat-induced oxidative injury, reduced the expression of antioxidant-related genes (CgSOD1, CgSOD2, and CgPRDX6), and further suppressed SOD activity. Molecular dynamics simulations showed that heat destabilized its catalytic thioredoxin domains, compromising its protective function. These results demonstrate that CgPDIA6 protects haemocytes against heat-induced oxidative damage by sustaining antioxidant enzyme activity, providing insights into redox regulation and heat adaptation in mollusks.