Molecular characterization and DNA methylation analysis of carbonic anhydrase (Sp-CA) in the mud crab Scylla paramamosain: Its potential osmoregulation role under carbonate alkalinity stress

Abstract

Saline–alkali water represents a huge underdeveloped resource due to its harsh properties of high pH, high salinity, and complex ionic composition. In our previous research, the mud crab, Scylla paramamosain could adapt to the saline–alkali environment, which may bring dual benefits to high-quality aquaculture and the utilization of saline–alkali water. Yet, no systematic study related to S. paramamosain's ability to carbonate alkali, the key gene responsible for adaptation to the harsh environment, has been reported. In this study, we investigated the mud crab's response to a wide range of carbonate–alkali waters, verifying the specific gill tissue with the highest carbonic anhydrase (CA) expression. Afterward, we cloned the CA gene in S. paramamosain (Sp-CA) for the first time. The cDNA sequence is 1085 bp, with a 55-bp 5′-untranslated region (UTR) and 214-bp 3′-UTR. The open reading frame (ORF) of 816-bp nucleotides encodes a polypeptide of 271 amino acids (AAs). In silico analysis and phylogenetic analysis of crustacea's CAs were performed, elucidating the unique function and evolutionary relation of Sp-CA. We also conducted short-term and long-term stress experiments with mud crabs in a saline–alkali environment, with the relative mRNA expression and DNA methylation profile of Sp-CA under such stress having been clarified, bringing new insight into the understanding of mud crabs' adaptation and tolerance to a saline–alkali environment.