Effects of proline on mitochondrial homeostasis and energy metabolism in the razor clam (Sinonovacula constricta) under high salinity stress

Extreme weather events are increasingly exacerbating salinity fluctuations in estuarine and intertidal zones, posing a serious threat to the habitat stability of benthic bivalves. These rapid and unpredictable salinity changes disrupt osmotic balance and impair critical physiological functions, often resulting in high mortality rates in natural populations and substantial economic losses in aquaculture. The razor clam Sinonovacula constricta, as an economically important bivalve species, is particularly susceptible to severe damage under sudden salinity shifts. Previous studies have shown that exogenous proline, acting as an osmolyte, can alleviate tissue damage and oxidative stress induced by high salinity, thereby enhancing the hyperosmotic tolerance of S. constricta. To investigate the protective role of exogenous proline in mitochondrial homeostasis and energy metabolism of S. constricta under high salinity stress, three experimental groups were established that a control group, a high-salinity group, and exogenous proline group. The study evaluated the ultrastructure of gill tissue, expression levels of key mitochondrial dynamics-related genes (pink, parkin, sirt, opa, mnf2, ampk, fis), and activities of energy metabolism enzymes (PK, MDH, SDH, LDH). Results revealed that high salinity induced severe mitochondrial damage, including membrane dissolution, cristae fragmentation, and vacuolization. Following proline supplementation, the mitochondrial area and vacuolization rate at both 48 h and 72 h of stress were significantly lower than those in the control group. Gene expression analysis showed that under high salinity stress, pink and parkin expression initially increased and then decreased, peaking at 48 h and 24 h, respectively. In contrast, the exogenous proline group sustained higher expression levels, significantly exceeding those of the high salinity group at 96 h. The sirt gene exhibited upregulation starting at 48 h under high salinity, peaking at 72 h, while proline treatment triggered a significant and continuous increase from 12 h onward. ampk expression showed a transient increase under high salinity, with an earlier and higher peak at 12 h in the proline group. opa and mnf2 expression peaked at 48 h under high salinity, but continued to rise in the exogenous proline group, ending significantly higher. fis expression was also upregulated initially under salinity stress, peaking at 24 h, but remained significantly lower in the exogenous proline group. Enzymatically, high salinity led to an initial increase followed by a decline in PK, MDH, and SDH activities, while LDH activity showed the opposite trend. Proline supplementation enhanced PK, MDH, and SDH activities and suppressed LDH activity, indicating a shift toward improved aerobic metabolism. In conclusion, proline stabilizes mitochondrial structure, regulates mitochondrial dynamics and quality control, promotes efficient energy production, and enhances high salinity tolerance in S. constricta.