Hypo-salinity events frequently occur in marine ecosystem due to persistent rainfall and freshwater inflow, reducing the cytosol osmolarity and triggering cellular stress responses in aquatic organisms. Euryhaline bivalves have developed sophisticated regulatory mechanisms to adapt to salinity fluctuations over a long period of evolution. In this study, we performed multiple biochemical assays, widely targeted metabolomics, and gene expression analysis to investigate the comprehensive metabolic responses to hypo-salinity stress and osmoregulation mechanisms in hard clam Mercenaria mercenaria, which is a euryhaline bivalve species widely cultured in China. During hypo-salinity stress, increased vacuoles appeared in gill filaments. The Na⁺ and Cl^- concentrations in gills significantly decreased because of the up-regulation of Na⁺/K⁺-ATPase (NKA) activity. The cAMP content dramatically decreased at 5 d post hypo-salinity stress. Meanwhile, the gene expression levels of adenylate cyclase, proteinkinase A, and sodium and calcium channel proteins were evidently down-regulated, suggesting that cAMP-PKA pathway was inhibited to prevent ambient inorganic ions from entering the gill cells. Antioxidant metabolites, such as serine and Tyr-containing dipeptides, were significantly up-regulated to resist oxidative stress. Glycerolipid metabolism was strengthened to stabilize membrane structure when hypo-salinity stress was prolonged to 5 days. At 1 d post hypo-salinity stress, an increase in alanine and lactate contents marked the initiation of anaerobic metabolism. Acylcarnitines accumulation indicated that fatty acids β-oxidation was promoted to provide energy for osmoregulation. The potential biomarkers of hypo-salinity stress were identified in hard clams. This study provides novel insights into the metabolic regulatory mechanisms to hypo-salinity stress in euryhaline bivalves.

由于持续降雨和淡水流入，海洋生态系统中经常发生低盐度事件，降低细胞质渗透压并引发水生生物的细胞应激反应。Euryhaline 双壳贝类已经发展出复杂的调节机制，以适应长期进化过程中的盐度波动。在这项研究中，我们进行了多项生化分析、广泛靶向代谢组学和基因表达分析，以研究硬蛤Mercenaria mercenaria对低盐胁迫和渗透压调节机制的综合代谢反应，这是一种在中国广泛养殖的广盐双壳类物种。在低盐胁迫期间，鳃丝中出现增加的液泡。Na ⁺和 Cl ^{-由于 Na +} /K ⁺的上调，鳃中的浓度显着降低-ATP酶（NKA）活性。cAMP 含量在低盐胁迫后 5 d 显着下降。同时，腺苷酸环化酶、蛋白激酶A、钠和钙通道蛋白的基因表达水平明显下调，表明cAMP-PKA通路受到抑制，阻止环境无机离子进入鳃细胞。抗氧化代谢物，如丝氨酸和含酪氨酸二肽，显着上调以抵抗氧化应激。当低盐胁迫延长至5天时，甘油脂代谢增强，膜结构稳定。在低盐胁迫后 1 d，丙氨酸和乳酸含量的增加标志着厌氧代谢的开始。酰基肉碱的积累表明促进了脂肪酸的β-氧化，为渗透压调节提供能量。在硬蛤中发现了潜在的低盐胁迫生物标志物。这项研究为广盐双壳贝类低盐胁迫的代谢调节机制提供了新的见解。