Integrated analysis of whole transcriptomes and free amino acid (FAA) profiles identified potential ncRNA-mRNA regulatory networks governing FAA metabolism in the gill of Crassostrea hongkongensis under osmotic stress

Oyster aquaculture in South China is persistently challenged by seasonal salinity increases, primarily due to the limited high-salinity tolerance of the dominant species, Crassostrea hongkongensis . Osmotic stress, a major consequence of salinity fluctuation, is often mitigated by marine bivalves through employing free amino acids (FAA) as compatible intracellular osmolytes. To elucidate the regulatory mechanisms underlying FAA-mediated osmotic response in C. hongkongensis , we conducted an integrative analysis of whole-transcriptome sequencing data and FAA profiles from gill tissues of oysters under different salinity stresses. Weighted gene co-expression network analysis identified the turquoise mRNA module, which not only exhibited a strong negative correlation with FAA levels but was also significantly enriched in amino acid metabolic pathways. Subsequent ncRNA-mRNA interaction analysis identified several miRNA-mediated regulatory relationships targeting genes involved in amino acid metabolism. Notably, three miRNAs (miR-425-x, novel-m0160–5p and novel-m0161–3p) were predicted to downregulate Mho_042430, which encodes a functionally characterized SHMT1 homolog critical for serine-glycine interconversion during hyperosmotic stress in C. hongkongensis . This study reveals potential ncRNA-mRNA interactions that regulate amino acid metabolism during osmotic stress, providing novel mechanistic insights into FAA-mediated osmoregulation in C. hongkongensis . • WGCNA identified mRNA and lncRNA modules associated with FAA levels. • Interaction analysis identified miRNA-mediated regulation targeting SHMT1 . • CeRNA networks involved a novel FAA-associated hub gene MSTRG.72154.