Metabolomics and biochemical assays reveal the metabolic responses to hypo-salinity stress and osmoregulatory role of cAMP-PKA pathway in Mercenaria mercenaria

Abstract

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.

Keywords: Energy metabolism; Hypo-salinity tolerance; Metabolomics; Osmoregulation; Oxidative stress.

Graphical abstract

Fig. 1

Histological observation of M. mercenaria gills exposed to different durations of hypo-salinity stress. (A) S30; (B) S10_1d; (C) S10_5d. Intracellular and extracellular vacuoles are marked by red arrows. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Ion concentration and NKA enzyme activity variations in M. mercenaria gills during hypo-salinity stress. (A) Na⁺ concentration; (B) Cl^- concentration; (C) NKA enzyme activity.

Fig. 3

OPLS-DA of the metabolites in M. mercenaria gill samples. (A) S10_8h vs S30; (B) S10_1d vs S30; (C) S10_5d vs S30.

Fig. 4

Variations in the composition of differential metabolites in S10_8h vs S30, S10_1d vs S30, and S10_5d vs S30. The differential metabolites belonging to different classes are marked by different colors.

Fig. 5

Expression variations in seven critical genes in cAMP-PKA pathway during hypo-salinity stress. Expression levels of the selected genes were normalized to EF1α expression level. Data are means (±SE) of three replicates. Different letters above the bars indicate significant difference in gene expression between groups (P < 0.05).

Fig. 6

Schematic diagram of M. mercenaria cAMP-PKA pathway involved in osmoregulation. The four boxes in a row below each metabolite or gene represent different groups: S30, S10_8h, S10_1d, and S10_5d. Colors of each box indicate the relative content or expression level of each metabolite or gene. Red indicates a high level, and blue indicates low. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 7

Heatmap analysis depicts the content variations in dipeptides (A), and lysophosphatide (B) in hard clams during hypo-salinity stress.

Fig. 8

Schematic diagram of the metabolic responses of hard clams to hypo-salinity stress. The four boxes in a row near each metabolite represent different groups: S30, S10_8h, S10_1d, and S10_5d. Colors of each box indicate the relative content of each metabolite. Red indicates high content, and blue indicates low. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)