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.

Keywords: Carbonic anhydrase; Exopalaemon carinicauda; Expression analysis; RNA interference; Saline–alkaline stress; pH stress.

Fig. 1

Cumulative mortality rates of prawns under pH stress (a) and saline–alkaline stress (b). Each bar represents the mean value from three repetitions with standard error (SE)

Fig. 2

Phylogenetic analysis of CAs in various species based on neighbor-joining method. The species with the GenBank accession numbers are Aedes albopictus CAc (XP_019541540.1), Anopheles aquasalis CAc (JAA98551.1), Anopheles gambiae CAc (ABF66618.1), Bemisia tabaci CA13 (XP_018906696.1), Callinectes sapidus CAc (ABN51213.1), Cherax cainii CA (AJO70180.1), Cherax destructor CA (AJO69996.1), Cherax quadricarinatus CAc (AIW68600.1), Drosophila rhopaloa CA (XP_016989668.1), Homo sapiens CA1 (NP_001729.1), Homo sapiens CA2 (NP_000058.1), Limulus polyphemus CA2 (XP_013785730.1), Litopenaeus vannamei CAI (ADM16544.2), Penaeus monodon CAI ABV65904.1, Portunus trituberculatus CAc (AFV46144.1), Tribolium castaneum CA1 (XP_974322.1); Anolis carolinensis CA15 (XP_008115548.1), Branchiostoma belcheri CA (XP_019637467.1), Callinectes sapidus CAg (ABN51214.1), Carcinus maenas CAg (ABX71209.1), Cherax cainii CAg (AJO70181.1), Cherax destructor CAg (AJO69997.1), Crassostrea gigas CA (XP_019930779.1), Daphnia magna α-CA (KZS12866.1), Hadrurus spadix CA1 (JAV48261.1), Halocaridina rubra CAg (AIM43573.1), Hyalella azteca CA2 (XP_018027123.1), Limulus polyphemus CA1 (XP_013777310.1), Litopenaeus vannamei CAg (AGC70493.1), Monopterus albus CA4 (XP_020456274.1), Patella vulgata CA (CCJ09593.1), Portunus trituberculatus CAg (AFV46145.1); Camponotus floridanus CAb1 (XP_011261355.1), Cherax cainii CAb (AJO70182.1), Cherax destructor CAb (AJO69999.1), Cherax quadricarinatus CAb (AIW68602.1), Culex quinquefasciatus CA (XP_001849341.1), Daphnia magna CAb (JAN40855.1), Drosophila rhopaloa CAb1 (XP_016978326.1), Fopius arisanus CAb1 (XP_011300350.1), Hyalella Azteca CAb1 (XP_018006429.1), Nasonia vitripennis CAb1 (XP_001606972.1), Zootermopsis nevadensis CAb1 (KDR11644.1). The numbers at the forks indicate the bootstrap proportions

Fig. 3

Relative expression levels of EcCAc (a), EcCAg (b), and EcCAb (c) in different tissues of E. carinicauda. Vertical bars represent the mean ± SD from three prawns (n = 3). Different lowercase letters meant significant differences (p < 0.05)

Fig. 4

Relative expression levels of EcCAc (a), EcCAg (b), and EcCAb (c) in response to pH stress in E. carinicauda. At every time point of pH stress, different lowercase letters indicated significant differences (p < 0.05) among different pH treatments

Fig. 5

Relative expression levels of EcCAc (a), EcCAg (b), and EcCAb (c) in response to saline–alkaline stress in E. carinicauda. Different lowercase letters indicate significant differences (p < 0.05) among different saline–alkaline treatments at the same time point

Fig. 6

Effect of prawns receiving EcCAc and EcCAg dsRNA on the expression levels of osmoregulatory genes. Prawns injected with GFP dsRNA was set as control group. Significant difference relative to GFP control was indicated with asterisk symbol (p < 0.05) at the same time point