Examination of the regulation of energy metabolism, antioxidant response, and ammonia detoxification in hard clam, Mercenaria mercenaria, under hypersalinity stress

With global warming on the rise, hypersalinity events are occurring with increasing frequency due to accelerated evaporation rates of seawater. Hypersalinity affects the distribution, growth, and physiological processes of aquatic organisms, especially for osmoconforming bivalves that lack complete means of osmoregulation. In this way, knowledge concerning comprehensive metabolic regulation is limited in hypersalinity-stressed bivalves. The hard clam, Mercenaria mercenaria, is an important pond-farmed bivalve in China, with an annual output value of 150 million USD. This species can be farmed in salt pans because it is highly resistant to hypersalinity. In this study, we investigated the comprehensive pattern of metabolic regulation in hard clams under hypersalinity stress. We identified 979 metabolites in the gills of hard clams, 295 of which were differentially accumulated after different periods of exposure to hypersalinity (8 h, 1 day, and 5 days), compared with the control. Critical hypersalinity-responsive metabolites, including acyl carnitine, oxidized lipids, and several antioxidants were identified using K-means analysis. During hypersalinity exposure, the activities of hexokinase and pyruvate kinase, and the pyruvic acid content significantly increased, suggesting that glycolysis was enhanced. Alanine and glycine were identified as key free amino acids involved in osmoregulaion. After 5 days of hypersalinity exposure, the accumulation of alanine and lactic acid marked the initiation of anaerobic glycolysis. The up-regulation of amino acid catabolism and inhibition of ammonia excretion led to ammonia accumulation in the gills. Glutamine synthetase and glutamate dehydrogenase activities significantly increased to reduce ammonia toxicity. Many antioxidant metabolites, such as allantoin, cinnamic acid, and trigonelline, were dramatically up-regulated to relieve hypersalinity-induced oxidative stress. The accumulation of lysophosphatides suggested that cell membranes damaged by prolonged oxidative stress and/or hyperosmosis were dissolved by phospholipase A1. This process helped to maintain overall cellular homeostasis. Potential biomarkers of hypersalinity stress in hard clams were identified. These results will facilitate the assessment of the physiological status of hard clam in aquaculture and provide novel insights into the metabolic regulatory mechanisms of bivalves under hypersalinity stress.