ROS-induced moderate autophagy of haemocytes confers resistance of Mercenaria mercenaria to air exposure stress.

Air exposure (AE) is a significant environmental stressor that can lead to desiccation, hypoxia, starvation, and disruption of cellular homeostasis in marine bivalves. Autophagy is a highly conserved catabolic pathway that facilitates the degradation of damaged macromolecules and organelles, thereby supporting cellular stress responses. To date, autophagy-mediated resistance mechanisms to AE stress remain largely elusive in bivalves. In this study, we performed a multi-tool approach to investigate the autophagy-related physiological regulation in hard clams (Mercenaria mercenaria) under different duration of AE (T = 0, 1, 5, 10, 20, 30 days). We observed that autophagy of haemocytes was significantly activated on day 5. However, autophagy activity began to significantly decline from day 10 to day 30. Autophagy was significantly inhibited after antioxidant treatment, indicating that reactive oxygen species (ROS) was an endogenous inducer of autophagy. A significant decline in the survival rate of hard clams was observed after injection of ammonium chloride or carbamazepine during AE stress, suggesting that moderate autophagy was conducive for clam survival under AE stress. We also observed DNA breaks and high levels of apoptosis in haemocytes on day 10. Activation of apoptosis lagged behind autophagy, and the relationship between autophagy and apoptosis might shift from antagonism to synergy with the duration of stress. This study provides novel insights into the stress resistance mechanisms in marine bivalves.