Abstract
Marine heatwaves repeatedly trigger summer mortality of the farmed Pacific oyster (Crassostrea gigas), forcing its haemocytes to reprogramme metabolism under thermal stress. The metabolic nodes governing this adaptation remain poorly defined. Integrating publicly available transcriptomics, single-cell analysis, enzyme inhibition, and molecular dynamics simulation, we identify argininosuccinate synthetase (CgASS) as a key node of this response. Heat stress drove sustained metabolic remodelling of the haemocyte transcriptome, and network analysis placed CgASS at the centre, where it was preferentially expressed in haemocytes and developmentally regulated. Single-cell virtual knockout selectively reshaped minor haemocyte populations, markedly expanding a stress-activated effector population while contracting immunomodulatory and cytoskeletal populations. In vivo inhibition with α-methyl-DL-aspartate raised citrulline, lowered argininosuccinate, and further suppressed respiration and feeding under heat, confirming that CgASS activity sustains aerobic performance. CgASS inactivation constricts arginine supply, thereby depriving both the nNOS–NO pathway that sustains respiration and feeding and the AMD–polyamine pathway that supports cytoprotection. Molecular dynamics revealed that CgASS collapses into an abnormally compact, rigid conformation at 30 °C, constraining the flexibility required for catalysis. Together, these results suggest CgASS as a potential key node of a metabolic compensation–decompensation axis and a molecular target for breeding thermally resilient shellfish.