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

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 -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 (pH6 and pH9), low salinity (5ppt), or high carbonate alkalinity (5 and 10mmol/L) after 72h of exposure. However, mortalities increased up to 70% under extreme saline-alkaline stress (salinity 5ppt, carbonate alkalinity 10mmol/L, and pH9) after 14days 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.