Characterization, functional analysis, and expression levels of three carbonic anhydrases in response to pH and saline–alkaline stresses in the ridgetail white prawn Exopalaemon carinicauda

Abstract

Carbonate alkalinity, salinity, and pH are three important stress factors for aquatic animals in saline–alkaline water. Carbonic anhydrases (CAs) catalyze the reversible reaction of CO₂ reported to play an important role in the acid–base regulation in vertebrates. To explore the molecular mechanism of CAs efficacy in shrimp after their transfer into saline–alkaline water, the cDNAs of three CAs (EcCAc, EcCAg, and EcCAb) were cloned from Exopalaemon carinicauda. Sequence analysis showed that EcCAc and EcCAg both possessed a conserved α-CA domain and a proton acceptor site, and EcCAb contained a Pro-CA domain. Tissue expression analysis demonstrated that EcCAc and EcCAg were most abundantly in gills, and EcCAb was highly expressed in muscle. The cumulative mortalities remained below 25% under exposure to pH (pH 6 and pH 9), low salinity (5 ppt), or high carbonate alkalinity (5 and 10 mmol/L) after 72 h of exposure. However, mortalities increased up to 70% under extreme saline–alkaline stress (salinity 5 ppt, carbonate alkalinity 10 mmol/L, and pH 9) after 14 days of exposure. The EcCAc and EcCAg expressions in gills were significantly upregulated during the early period of pH and saline–alkaline stresses, while the EcCAb expressions showed no regular or large changes. The two-way ANOVA found significant interactions between salinity and carbonate alkalinity observed in EcCAc, EcCAg, and EcCAb expressions (p < 0.05). Furthermore, an RNA interference experiment resulted in increased mortality of EcCAc- and EcCAg-silenced prawns under saline–alkaline stress. EcCAc knockdown reduced expressions of Na⁺/H⁺ exchanger (EcNHE) and sodium bicarbonate cotransporter (EcNBC), and EcCAg knockdown reduced EcCAc, EcNHE, EcNBC, and V-type H⁺-ATPase (EcVTP) expressions. These results suggest EcCAc and EcCAg as important modulators in response to pH and saline–alkaline stresses in E. carinicauda.