4.6 Metabolism and salinity acclimation
Our analyses showed changes in the expression and methylation of genes involved in lipid, protein and carbohydrate metabolism as well as mitochondrial functions. The transition from a seawater- to a freshwater physiology requires gill epithelium turnover and active ion transport and is thus an energy consuming process. This energy is provided by metabolites related to carbohydrates, proteins/amino acids and lipids, transported from liver stores to the blood, towards the gill. In addition to energy sources provided by the blood, there is also a local energy supply at the gill level necessary for the modulation and stimulation of gill epithelium reorganization and ion transport mechanisms (Hwang and Lee., 2007).
Lipid metabolism in fish gills has been widely overlooked, despite the growing number of studies in mammals indicating the importance of lipids in ion channel regulation notably through lipid-protein interactions (Rosenhouse-Dantsker et al., 2012). In both analyses (GO-term and KEGG enrichment analyses), the sphingolipid synthesis and signaling pathways were significantly enriched and are thus worth considering. Sphingolipids are involved in multiple functions, such as immune-related functions, cellular growth, differentiation, apoptosis and have been argued to have second-messenger functions (Shayman, 2000). Sphingolipids and mainly sphingomyelin, are also major components of membrane microdomains, called lipid rafts, where they are involved in the regulation of ion channels (Rosenhouse-Dantsker et al., 2012). Lipid raft abundance in fish gills can in fact change upon salinity transfer. They can be enriched in membrane transporters like Na+/K+-ATPase, as shown in tilapiaO. mossambicus and milkfish Chanos chanos gills (Lin et al., 2021). Hydrolysis of sphingomyelin due to a cell stress can lead to ceramide formation, which is implicated in numerous physiological functions. Several ion channels such as CFTR (Ramu et al., 2007) and voltage gated K+ channels (Fan et al., 1997) have been shown to be inhibited by ceramide. In this study, sphingomyelin synthase (sgms1 ) was upregulated and hypomethylated in FW. We also observed a downregulation of serine palmitoyltransferase(sptlc2 ) which is involved in the first step of sphingolipid biosynthesis (Hanada., 2003) and ceramide synthase 5(cers5 ). Altogether, these results indicate a potential change in the ceramide species profiles (Gault et al., 2010). However, the potential functional link between methylation changes and expression changes of genes involved in sphingolipid metabolism is not clear and needs further investigation.
Studies have pointed out that the enzymes involved in methylation and demethylation have substrates that are responsive to cellular metabolism (Reid et al., 2017). Mitochondria provide key metabolites for epigenetic processes (Shaughnessy et al., 2014). The availability of these metabolites change, when fish are energetically challenged by environmental stressors. For DNA methyltransferases, substrates and cofactors include methylthioadenosine (MTA), S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) for example whereas for DNA demethylases (TET-family), other metabolites are used as succinate, fumarate, … being intermediates of the TCA cycle (Reid et al., 2017). We observed changes in the expression of genes linked to methionine metabolism (ahcyl1, 2, mtap ) and an overall overexpression of genes involved in the TCA cycle (dlst ,sdha, sdhd, sdhb , cs ). This clearly displays changes in metabolic pathways and energetic status, which could directly affect the epigenome and DNA methylation dynamics. In gills of sea bass transferred to FW, ionocytes, previously called mitochondria-rich cells due to their high abundance of mitochondria, are present at higher densities with higher energy-consuming Na+/K+-ATPase activities (Masroor et al., 2018). We also measured higher expression of paralogs encoding for the V-H+-ATPase indicating a high energy demand for transepithelial ion transport in FW. In accordance with this, we observed higher mitochondrial activities in FW. Mitochondria have a central role in energy (ATP) production but also in metabolite production in the TCA cycle and mitochondria-nuclear signaling. These processes can be linked to epigenetic regulation. As chromatin-modifying enzymes use as substrates and cofactors metabolites derived from diverse metabolic pathways including notably the TCA cycle (Lopes, 2020), salinity-driven changes in transcription of these genes might also directly affect the availability of substrates for chromatin-modifying enzymes and affect DNA methylation dynamics. Relationships between genes, environment and epigenetic marks, and the variation of those marks still require more investigations to gather a full understanding of the determinism of salinity acclimation in fish.