UREA ON SHELL DAMAGE-REPAIR PROCESS OF MANTLE UNDER OCEAN ACIDIFICATION

Ocean acidification is widely recognized as a significant threat to marine bivalves, affecting their calcification, survival, and behavior. Mytilus coruscus, a crucial marine bivalve species with considerable economic and ecological importance, relies on the formation of calcareous shells for defense against predation. However, increasing evidence suggests that this crucial process might be sensitive to environmental stressors, including ocean acidification. Inhabiting the Yangtze River estuary sea area, M. coruscus experiences seasonal fluctuations in natural pH due to freshwater influx, with seawater pH decreasing rapidly in summer. This phenomenon implies potential impacts on M. coruscus if they fail to recover from short-term exposure to ocean acidification. Previous studies have revealed the effects of ocean acidification on Mytilus species, indicating a degree of tolerance. In addition, urea has been identified as potentially aiding in biomineralization in certain bacteria, operating through a mechanism involving urea-mediated carbanion provision. We speculate that urea may assist Mytilus in maintaining their shell biomineralization process under ocean acidification—a hypothesis partially supported by our previous work. However, the specific roles of urea in Mytilus shell biomineralization remain unclear. In this study, we constructed a Mytilus model with shell damage under acidification conditions and analyzed the mantle transcriptome of mussels with or without urea injection. In addition, we conducted microscopic observations and analyzed the free amino acid composition and intracellular calcium levels of mussel mantle under these conditions. Illumina sequencing yielded 42.3 Gb data, producing a total of 276334654 clean reads. Assembly of the sequencing data using the M. coruscus genome as reference generated 86924 transcripts, with 71437 (82.18%) transcripts annotated in various databases including GO, KEGG, COG, NR, SWISS-PROT, and InterPro. We identified 1798 differentially expressed genes, with 981 up-regulated and 817 down-regulated in urea-injected mussels compared to controls. KEGG enrichment analysis revealed 28 pathways enriched by upregulated genes and 47 by downregulated genes. Upregulated genes were predominantly associated with lysosome, phagosome, and apoptosis pathways, while downregulated genes were primarily linked to cancer, neuroactive ligand-receptor interaction, and focal adhesion pathways. The results showed that urea injection benefits mantle cell homeostasis, enhances energy metabolism, regulates immune balance, and promotes calcium recruitment in mussel mantles under acidification stress. Furthermore, urea injection upregulates free ammino acids involved in the tricarboxylic acid cycle, such as glutamate and aspartic acid, as well as urea-cycle related amino acids, such as arginine and ornithine. These suggests that urea injection facilitates energy capture during mantle shell damage-repair process under ocean acidification, with elevated arginine concentration potentially providing more precursor for urea biosynthesis in the mantle. Microscopic observations revealed increased secretion of mucinous ciliated substances from the epidermal layer of the mantle under ocean acidification, compared to urea-injected mussel. Furthermore, downregulation of mucin genes in urea-injected mussel mantles suggests that urea injection inhibits mucous secretion under ocean acidification. In conclusion, our findings shed light on the potential roles of urea in shell mineralization process of Mytilus under ocean acidification and provide insights for the development of mussel aquaculture amidst ocean acidification challenges.