Cell Stress and Chaperones

, Volume 24, Issue 3, pp 503–515 | Cite as

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

  • Qianqian Ge
  • Jian LiEmail author
  • Jiajia Wang
  • Zhengdao Li
  • Jitao Li
Original Paper


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.


Carbonic anhydrase Exopalaemon carinicauda pH stress Saline–alkaline stress Expression analysis RNA interference 



This project was financially supported by the National Key R & D Program of China (2018YFD0901302), the National Natural Science Foundation of China (No. 31702319), Central Public-interest Scientific Institution Basal Research Fund, CAFS (NO.2019ZD0603), China Agriculture Research System-48 (CARS-48), The Program of Shandong Leading Talent (LNJY2015002).

Authors’ contributions

Qianqian Ge and Jian Li conceived and designed the experiments; Qianqian Ge, Jiajia Wang and Zhengdao Li performed the experiments; Qianqian Ge and Jiajia Wang analyzed the data; Jitao Li contributed materials; Qianqian Ge drafted the manuscript; and Jian Li made a critical revision of the manuscript.

Compliance with ethical standards

Conflicts of interest

The authors declare that they have no conflict of interest.

Supplementary material

12192_2019_987_MOESM1_ESM.docx (7.2 mb)
ESM 1 (DOCX 7354 kb)


  1. Ali MY (2016) Characterization of the carbonic anhydrase gene family and other key osmoregulatory genes in Australian freshwater crayfish (genus Cherax). Doctoral dissertation, Queensland University of TechnologyGoogle Scholar
  2. Ali MY, Pavasovic A, Mather PB, Prentis PJ (2015a) Analysis, characterisation and expression of gill-expressed carbonic anhydrase genes in the freshwater crayfish Cherax quadricarinatus. Gene 564(2):176–187CrossRefGoogle Scholar
  3. Ali MY, Pavasovic A, Mather PB, Prentis PJ (2015b) Transcriptome analysis and characterisation of gill-expressed carbonic anhydrase and other key osmoregulatory genes in freshwater crayfish Cherax quadricarinatus. Data in brief 5:187–193CrossRefGoogle Scholar
  4. Ali MY, Pavasovic A, Mather PB, Prentis PJ (2015c) Expression patterns of two carbonic anhydrase genes, Na+/K+-ATPase and V-type H+-ATPase, in the freshwater crayfish, Cherax quadricarinatus, exposed to low pH and high pH. Aust J Zool 65(1):50–59CrossRefGoogle Scholar
  5. Cai YM, Chen T, Ren CH, Huang W, Jiang X, Gao Y, Huo D, Hu CQ (2017) Molecular characterization of pacific white shrimp (Litopenaeus vannamei) sodium bicarbonate cotransporter (NBC) and its role in response to pH stress. Fish Shellfish Immunol 64:226–233CrossRefGoogle Scholar
  6. Christianson DW, Fierke CA (1996) Carbonic anhydrase: evolution of the zinc binding site by nature and by design. Acc Chem Res 29(7):331–339CrossRefGoogle Scholar
  7. Esbaugh AJ, Tufts B (2006) The structure and function of carbonic anhydrase isozymes in the respiratory system of vertebrates. Respir Physiol Neurobiol 154:185–198CrossRefGoogle Scholar
  8. Frost SC, McKenna R (2013) Carbonic anhydrase: mechanism, regulation, links to disease, and industrial applications, vol 75. Springer Science & Business Media, DordrechtGoogle Scholar
  9. Ge Q, Liang J, Li J, Li J, Duan Y, Zhao F, Ren H (2015) Molecular cloning and expression analysis of Relish gene from the ridgetail white prawn Exopalaemon carinicauda. Fish Sci 81(4):699–711CrossRefGoogle Scholar
  10. Ge Q, Li J, Duan Y, Li J, Sun M, Zhao F (2016) Isolation of prawn (Exopalaemon carinicauda) lipopolysaccharide and beta-1, 3-glucan binding protein gene and its expression in responding to bacterial and viral infections. J Ocean Univ China 15(2):288–296CrossRefGoogle Scholar
  11. Gilmour KM (2012) New insights into the many functions of carbonic anhydrase in fish gills. Resp Physiol Neurobi 184(3):223–230CrossRefGoogle Scholar
  12. Gilmour KM, Perry SF (2009) Carbonic anhydrase and acid–base regulation in fish. J Exp Biol 212(11):1647–1661CrossRefGoogle Scholar
  13. Havird JC, Henry RP, Wilson AE (2013) Altered expression of Na+/K+-ATPase and other osmoregulatory genes in the gills of euryhaline animals in response to salinity transfer: a meta-analysis of 59 quantitative PCR studies over 10 years. Comp Biochem Physiol D 8(2):131–140Google Scholar
  14. Henry RP (1987) Membrane-associated carbonic anhydrase in gills of the blue crab, Callinectes sapidus. Am J Phys 252:R966–R971Google Scholar
  15. Henry RP, Garrelts EE, McCarty MM, Towle DW (2002) Differential induction of branchial carbonic anhydrase and Na+/K+ ATPase activity in the euryhaline crab, Carcinus maenas, in response to low salinity exposure. J Exp Zool A 292(7):595–603CrossRefGoogle Scholar
  16. Henry RP, Gehnrich S, Weihrauch D, Towle DW (2003) Salinity-mediated carbonic anhydrase induction in the gills of the euryhaline green crab, Carcinus maenas. Comp Biochem Physiol A 136:243–258CrossRefGoogle Scholar
  17. Henry RP, Lucu C, Onken HD, Weihrauch D (2012) Multiple functions of the crustacean gill: osmotic/ionic regulation, acid-base balance, ammonia excretion, and bioaccumulation of toxic metals. Front Physiol 3:431Google Scholar
  18. Liang JP (2013) Study on the technology of artificial breeding and expression of genes involving in reproducing of Exopalaemon carinicauda. Doctoral dissertation, Ocean university of China, Qingdao China (In Chinese)Google Scholar
  19. Lin T, Lai Q, Yao Z, Lu J, Zhou K, Wang H (2013) Combined effects of carbonate alkalinity and pH on survival, growth and haemocyte parameters of the Venus clam Cyclina sinensis. Fish Shellfish Immunol 35(2):525–531CrossRefGoogle Scholar
  20. Liu M, Liu S, Hu Y, Pan L (2015) Cloning and expression analysis of two carbonic anhydrase genes in white shrimp Litopenaeus vannamei, induced by pH and salinity stresses. Aquaculture 448:391–400CrossRefGoogle Scholar
  21. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(−delta delta C(T)) method. Methods 25:402–408CrossRefGoogle Scholar
  22. Martinez AS, Charmantier G, Compere P, Charmantier-Daures M (2005) Branchial chamber tissues in two caridean shrimps: the epibenthic Palaemon adspersus and the deep-sea hydrothermal Rimicaris exoculata. Tissue Cell 37:153–165CrossRefGoogle Scholar
  23. Muesch A, Hartmann E, Rohde K, Rubartelli A, Sitia R, Rapoport TA (1990) A novel pathway for secretory proteins? Trends Biochem Sci 15(3):86–88CrossRefGoogle Scholar
  24. Pan L, Hu D, Liu M, Hu Y, Liu S (2016) Molecular cloning and sequence analysis of two carbonic anhydrase in the swimming crab Portunus trituberculatus and its expression in response to salinity and pH stress. Gene 576(1):347–357CrossRefGoogle Scholar
  25. Perry SF, Furimsky M, Bayaa M, Georgalis T, Shahsavarani A, Nickerson JG, Moon TW (2003) Integrated responses of Na+/HCO3 cotransporters and V-type H+-ATPases in the fish gill and kidney during respiratory acidosis. Biochim Biophys Acta Bioenerg 1618(2):175–184CrossRefGoogle Scholar
  26. Pham D, Charmantier G, Boulo V, Wabete N, Ansquer D, Dauga C, Grousset E, Labreuche Y, Charmantier-Daures M (2016) Ontogeny of osmoregulation in the Pacific blue shrimp, Litopenaeus stylirostris (Decapoda, Penaeidae): deciphering the role of the Na+/K+-ATPase. Comp Biochem Physiol B 196:27–37CrossRefGoogle Scholar
  27. Pongsomboon S, Udomlertpreecha S, Amparyup P, Wuthisuthimethavee S, Tassanakajon A (2009) Gene expression and activity of carbonic anhydrase in salinity stressed Penaeus monodon. Comp Biochem Physiol A 152(2):225–233CrossRefGoogle Scholar
  28. Roy LA, Davis DA, Saoud IP, Henry RP (2007) Branchial carbonic anhydrase activity and ninhydrin positive substances in the Pacific white shrimp, Litopenaeus vannamei, acclimated to low and high salinities. Comp Biochem Physiol A 147(2):404–411CrossRefGoogle Scholar
  29. Santos LCF, Belli NM, Augusto A, Masui DC, Leone FA, McNamara JC et al (2007) Gill (Na+,K+)-ATPase in diadromous, freshwater palaemonid shrimps: species-specific kinetic characteristics and alpha-subunit expression. Comp Biochem Physiol A 148:178–188CrossRefGoogle Scholar
  30. Serrano L, Henry RP (2008) Differential expression and induction of two carbonic anhydrase isoforms in the gills of the euryhaline green crab, Carcinus maenas, in response to low salinity. Comp Biochem Physiol D 3(2):186–193Google Scholar
  31. Serrano L, Halanych KM, Henry RP (2007) Salinity-stimulated changes in expression and activity of two carbonic anhydrase isoforms in the blue crab Callinectes sapidus. J Exp Biol 210(13):2320–2332CrossRefGoogle Scholar
  32. Supuran CT, Scozzafava A, Casini A (2003) Carbonic anhydrase inhibitors. Med Res Rev 23(2):146–189CrossRefGoogle Scholar
  33. Syrjanen L, Tolvanen M, Hilvo M, Olatubosun A, Innocenti A, Scozzafava A, Leppiniemi J, Niederhauser B, Hytonen V, Gorr T, Parkkila S, Supuran C (2010) Characterization of the first beta-class carbonic anhydrase from an arthropod (Drosophila melanogaster) and phylogenetic analysis of beta-class carbonic anhydrases in invertebrates. BMC Biochem 11:28CrossRefGoogle Scholar
  34. Syrjänen L, Tolvanen M, Hilvo M, Olatubosun A, Innocenti A, Scozzafava A, Leppiniemi J, Niederhauser B, Hytönen VP, Gorr TA, Parkkila S (2010) Characterization of the first beta-class carbonic anhydrase from an arthropod (Drosophila melanogaster) and phylogenetic analysis of beta-class carbonic anhydrases in invertebrates. BMC Biochem 11(1):28CrossRefGoogle Scholar
  35. Vince JW, Reithmeier RA (2000) Identification of the carbonic anhydrase II binding site in the cl/HCO3 anion exchanger AE1. Biochemistry 39:5527–5533CrossRefGoogle Scholar
  36. Wang WN, Wang AL, Chen L, Liu Y, Sun RY (2002) Effects of pH on survival, phosphorus concentration, adenylate energy charge and Na+-K+ ATPase activities of Penaeus chinensis Osbeck juveniles. Aquat Toxicol 60(1):75–83CrossRefGoogle Scholar
  37. Wang X, Wang M, Jia Z, Qiu L, Wang L, Zhang A, Song L (2017) A carbonic anhydrase serves as an important acid-base regulator in pacific oyster Crassostrea gigas exposed to elevated CO2: implication for physiological responses of mollusk to ocean acidification. Mar Biotechnol 19(1):22–35CrossRefGoogle Scholar
  38. Xu WJ, Xie JJ, Hui S, Li CW (2010) Hematodinium infections in cultured ridgetail white prawns, Exopalaemon carinicauda, in eastern China. Aquaculture 300(1–4):25–31CrossRefGoogle Scholar
  39. Xu J, Li Q, Xu L, Wang S, Jiang Y, Zhao Z, Zhang Y, Li J, Sun X (2013a) Gene expression changes leading extreme alkaline tolerance in Amur ide (Leuciscus waleckii) inhabiting soda lake. BMC Genomics 14(1):682CrossRefGoogle Scholar
  40. Xu J, Ji P, Wang B, Zhao L, Wang J, Zhao Z, Li JT, Xu P, Sun X (2013b) Transcriptome sequencing and analysis of wild Amur Ide (Leuciscus waleckii) inhabiting an extreme alkaline-saline lake reveals insights into stress adaptation. PLoS One 8(4):e59703CrossRefGoogle Scholar
  41. Yao ZL, Wang H, Zhou K, Ying CQ, Lai QF (2010) Effects of water carbonate alkalinity and pH on survival rate of post-larval Litopenaeus vannamei. Chin J Ecol 5:019 (In Chinese) Google Scholar
  42. Yao ZL, Wang H, Chen L, Zhou K, Ying CQ, Lai QF (2012) Transcriptomic profiles of Japanese medaka (Oryzias latipes) in response to alkalinity stress. Genet Mol Res 11(3):2200–2246CrossRefGoogle Scholar
  43. Yao Z, Guo W, Lai Q, Shi J, Zhou K, Qi H, Lin T, Li Z, Wang H (2016) Gymnocypris przewalskii decreases cytosolic carbonic anhydrase expression to compensate for respiratory alkalosis and osmoregulation in the saline-alkaline Lake Qinghai. J Comp Physiol B 186(1):83–95CrossRefGoogle Scholar
  44. Zhou J, Wang WN, Wang AL, He WY, Zhou QT, Liu Y, Xu J (2009) Glutathione S-transferase in the white shrimp Litopenaeus vannamei: characterization and regulation under pH stress. Comp Biochem Physiol C 150(2):224–230Google Scholar

Copyright information

© Cell Stress Society International 2019

Authors and Affiliations

  • Qianqian Ge
    • 1
    • 2
  • Jian Li
    • 1
    • 2
    Email author
  • Jiajia Wang
    • 1
  • Zhengdao Li
    • 1
  • Jitao Li
    • 1
    • 2
  1. 1.Key Laboratory for Sustainable Utilization of Marine Fisheries Resources, Ministry of Agriculture and Rural Affairs, Yellow Sea Fisheries Research InstituteChinese Academy of Fishery SciencesQingdaoPeople’s Republic of China
  2. 2.Laboratory for Marine Fisheries Science and Food Production ProcessesQingdao National Laboratory for Marine Science and TechnologyQingdaoPeople’s Republic of China

Personalised recommendations