Abstract:The global levels of soil and water salinization and alkalinization are increasing with the influence of climate and topography changes, as well as other natural and human factors. Saline-alkaline water (SAW) all over the world has specific characteristics such as high alkalinity, high pH, and complex water quality types, which inhibits the survival and culture of common aquatic animals. The ridgetail white prawn Exopalaemon carinicauda is an economically important marine shrimp with many advantages, such as widely environmental adaptability, rapid growth, and good reproductive capability. It is potentially suitable for large-scale culture in SAW; however, its adaptability to this environment remains unclear. Exploring the E. carinicauda adaptability mechanism to SAW will help to guide culture management for marine crustaceans. In this study, the DNA methylomes of the E. carinicauda gill tissue cultured in SAW and normal seawater (SW) were analyzed and the impact on gene regulation was investigated by MethylRAD sequencing. The results showed 2 347 003 and 416 176 methylations at the CG and CWG sites (W = A or T), respectively. Comparing the SAW and SW groups, the CG and CWG loci in the SAW group increased slightly, indicating that SAW induced more DNA methylation in the gill cells that activated or inhibited pathways and played a crucial role in the environmental changes adaption. Methylation was prevalent in the exon, intron, splice site, and upstream and downstream regions of the E. carinicauda gill genes, as well as in the intergenic regions. DNA methylation sites were mostly distributed in the Genebody. The DNA methylation distribution curve peaked in the downstream sequence of the transcription start site and upstream sequence of the transcription termination site. The methylation label frequency was significantly higher in these regions in relation to other sequences. A total of 8805 differential methylation sites (DMSs) were screened, including 8189 CG DMSs and 616 CWG DMSs. Obviously, the CG DMS was significantly higher than the CWG DMS. The intergenic and intron regions accounted for a large proportion of the DMS observed. Overall, the DMS showed a higher trend in the genic downstream regions of the gene relative to upstream regions. The Gene Ontology (GO) enrichment analysis of the differentially expressed genes (DEGs) based on the DMS showed enrichment of genes involved in the "development, heterochrony, and protein disables isomerase activity," which played a role in the CG level down-regulation. In addition, "incubation involved in sorocarp development" and "nucleus and double-stranded RNA binding" were molecular functions up-regulated by the CG methylations. The down-regulated genes with CWG methylation were enriched for the "regulation of transcription and DNA template" process, while the up-regulated genes were enriched for the "epithelial cell migration and open trail” system. These two processes were induced in E. carinicauda cultured in saline-alkali water. The key genes in the HIF-1 signal and spliceosome pathways, such as hif-p, hk, and sf3b1, play an important role in response to saline-alkaline stress; therefore, the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis based on DMS of the DEG was performed. In addition, 158 CG methylated genes (MGs) were detected in DEGs from shrimps exposed to SAW, among which 77 and 81 were up- and down-regulated, respectively. Moreover, 94 CWG MGs were differentially expressed, from which 33 and 61 were up- and down-regulated, respectively. GO enrichment analysis of the CG MGs showed significant enrichment of the "muscle organ development" process; CWG MGs were significantly enriched for the "vesicle-m transport" and "membrane mediated zinc binding plasma transport" processes. This indicated that E. carinicauda was damaged by the saline-alkaline environment, but the shrimp may adapt to this stress by adjusting the ion balance. The KEGG enrichment analysis indicated that lipid metabolism and signal transduction pathways may play crucial roles in the E. carinicauda gill tissue response to saline-alkaline stress. Moreover, changes in lipid peroxidation and physiological metabolism may be caused by long-term saline-alkaline stress. The energy metabolism pathway was significantly enriched and many different genes in the lipid metabolism pathway were expressed in the stressed E. carinicauda. Therefore, it was speculated that the changes in DNA methylation level might play an important role in response to saline-alkaline stress. Overall, the results showed that a series of E. carinicauda physiological activities related to environmental adaptation was activated by SAW. In addition, a small number of methylated loci were negatively correlated with gene expressions, which indicated a complex relationship between DNA methylation and gene regulation. Although ADAM17, FPC-l, and CYP450 showed hypomethylation, the corresponding differentially MGs showed a significantly increased expression level in a saline-alkaline environment. Therefore, genomic DNA methylation may promote gene expression under saline-alkaline stress, including the ADAM17, FPC-l, and CYP450 genes. This study analyzed the DNA methylation levels in gill tissue of E. carinicauda cultured with SAW and provided information that will further elucidate the molecular mechanisms involved in crustacean adaptation to saline-alkaline environment.
