Skip to main content

Advertisement

SpringerLink
Log in

The razor clam Sinonovacula constricta uses the strategy of conversion of toxic ammonia to glutamine in response to high environmental ammonia exposure

  • Original Article
  • Published:
Molecular Biology Reports Aims and scope Submit manuscript

Abstract

High ammonia can inhibit the survival and growth, and even cause mortality of razor clam (S. constricta). The accumulation of ammonia to lethal concentrations in some invertebrates may be partially prevented by converting some of the ammonia into glutamine (Gln). Glutamine dehydrogenase (GDH) and glutamine synthetase (GS) have been widely implicated a central role in response to ammonia stress. However, the molecular and physiological response of GDH and GS to ammonia alterations has not yet been determined in clams. To investigate the possible participatory role of GDH and GS genes in altered ammonia conditions, we have cloned their gene sequences and examined the mRNA expression and western blotting under ammonia exposure in S. constricta (ScGDH and ScGS), and detected the levels of GS and GDH, and the content of glutamate (Glu) and Gln. The full-length cDNA of ScGDH was 3924 bp, with a 1629 bp open reading frame (ORF) encoding a 542 amino-acid polypeptide. The complete cDNA sequence for ScGS had 2739 bp with an ORF of 1110 bp encoding 369 amino acids. To investigate ammonia detoxification strategies, the clams were exposed to ammonia for 96 h at four different concentrations (0, 100, 140, and 180 mg/L). Exposure to ammonia resulted in a significant increase of glutamate concentration and Gln in the haemocytes. GDH activity, GDH relative mRNA and protein expression, GS activity, GS relative mRNA and protein expression increased significantly and showed a pronounced time and dosage interaction in the liver. The results suggested that the protective strategies of Gln formation existed in S. constricta, which could convert ammonia to non- or less toxic nitrogenous compounds on the exposure of ammonia. Glutamate content in the haemocytes increased significantly, which is to ensure sufficient Glu to meet the needs for GS to catalyze the conversion of ammonia to Gln. We proposed that the induction of Glu synthesis-related genes and the subsequent formation of the active protein occurred in preparation for the increased capacity of the body to convert ammonia, into Gln. The results of this study suggested that GDH and GS play an important role in the synthesis of Gln, emphasizing, the protective strategies of Gln formation in S. constricta convert ammonia to nontoxic or less toxic nitrogenous compounds upon exposure to ammonia.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Wang XR, Xue QG, Mao XW, Dong YH, Li CH, Lin ZH (2018) Two I84 family protease inhibitors from Chinese razor clams, Sinonovacula constricta, expressed in response to environmental challenges. Fish Shellfish Immunol 75:149–157

    CAS  PubMed  Google Scholar 

  2. The ministry of agriculture and fishery of the People’s Republic of China (2018) Chinese fishery statistical yearbook 2018. China Agriculture Press, Beijing

    Google Scholar 

  3. Cheng CH, Ma HL, Su YL, Deng YQ, Feng J, Xie JW, Chen XL, Guo ZX (2019) Ammonia toxicity in the mud crab (Scylla paramamosain), the mechanistic insight from physiology to transcriptome analysiss. Ecotoxicol Environ Saf 179:9–16

    CAS  PubMed  Google Scholar 

  4. Xiao J, Li QY, Tu JP, Chen XL, Chen XH, Liu QY, Liu H, Zhou XY, Zhao YZ, Wang HL (2019) Stress response and tolerance mechanisms of ammonia exposure based on transcriptomics and metabolomics in Litopenaeus vannamei. Ecotoxicol Environ Saf 180:491–500

    CAS  PubMed  Google Scholar 

  5. Wright PA, Steele SL, Huitema A, Bernier NJ (2007) Induction of four glutamine synthetase genes in brain of rainbow trout in response to elevated environmental ammonia. J Exp Biol 210(16):2905–2911

    CAS  PubMed  Google Scholar 

  6. Rodela TM, Esbaugh AJ, Weihrauch D, Veauvy CM, McDonald MD, Gilmour KM, Walsh PJ (2012) Revisiting the effects of crowding and feeding in the gulf toadfish, Opsanus beta: the role of rhesus glycoproteins in nitrogen metabolism and excretion. J Exp Biol 215(2):301–313

    CAS  PubMed  Google Scholar 

  7. Si LJ, Pan LQ, Wang HD, Zhang X (2018) Identification of the role of Rh protein in ammonia excretion of swimming crab Portunus trituberculatus. J Exp Biol 221(20):1–12

    Google Scholar 

  8. Sinha AK, Diric M, Chan LP, Liew HJ, Kumar V, Blust R, Boeck GD (2012) Expression pattern of potential biomarker genes related to growth, ion regulation and stress in response to ammonia exposure, food deprivation and exercise in common carp (Cyprinus carpio). Aquat Toxicol 122–123(2):93–105

    PubMed  Google Scholar 

  9. Cheng CH, Yang FF, Liao SA, Miao YT, Ye CX, Wang AL (2015) Effect of acute ammonia exposure on expression of GH/IGF axis genes GHR1, GHR2 and IGF-1 in pufferfish (Takifugu obscurus). Fish Physiol Biochem 41(2):495–507

    CAS  PubMed  Google Scholar 

  10. Liang ZX, Liu R, Zhao DP, Wang LL, Sun MZ, Wang MQ, Song LS (2016) Ammonia exposure induces oxidative stress, endoplasmic reticulum stress and apoptosis in hepatopancreas of Pacific white shrimp (Litopenaeus vannamei). Fish Shellfish Immunol 54:523–528

    CAS  PubMed  Google Scholar 

  11. Zhang MZ, Li M, Wang RX, Qian YX (2018) Effects of acute ammonia toxicity on oxidative stress, immune response and apoptosis of juvenile yellow catfish Pelteobagrus fulvidraco and the mitigation of exogenous taurine. Fish Shellfish Immunol 79:313–320

    CAS  PubMed  Google Scholar 

  12. Li M, Gong S, Li Q, Yuan L, Meng F (2016) Wang R. Ammonia toxicity induces glutamine accumulation, oxidative stress and immunosuppression in juvenile yellow catfish Pelteobagrus fulvidraco. Comp Biochem Physiol C 183–184:1–6

    Google Scholar 

  13. Lu X, Luan S, Dai P, Meng XH, Cao BX, Luo K, Kong J (2018) iTRAQ-based comparative proteome analysis for molecular mechanism of defense against acute ammonia toxicity in Pacific white shrimp Litopenaeus vannamei. Fish Shellfish Immunol 74:52–61

    CAS  PubMed  Google Scholar 

  14. Schram E, Roques JAC, Abbink W, Spanings T, Vries PD, Bieman S, Vis HVD, Flik G (2010) The impact of elevated water ammonia concentration on physiology, growth and feed intake of African catfish (Clarias gariepinus). Aquaculture 306(1–4):108–115

    CAS  Google Scholar 

  15. Smutná M, Vorlová L, Svobodová Z (2002) Pathobiochemistry of ammonia in the internal environment of fish (Review). Acta Vet Brno 71(2):169–181

    Google Scholar 

  16. Tok CY, Chew SF, Ip YK (2011) Gene cloning and mRNA expression of glutamate dehydrogenase in the liver, brain, and intestine of the swamp eel, Monopterus albus (Zuiew), exposed to freshwater, terrestrial conditions, environmental ammonia, or salinity stress. Front Physiol 2(100):2

    Google Scholar 

  17. Braun MH, Perry SF (2010) Ammonia and urea excretion in the Pacific hagfish Eptatretus stoutii: evidence for the involvement of Rh and UT proteins. Comp Biochem Physiol A 157(4):405–415

    Google Scholar 

  18. Chew SF (2003) Urea synthesis in the African lungfish Protopterus dolloi – hepatic carbamoyl phosphate synthetase iii and glutamine synthetase are upregulated by 6 days of aerial exposure. J Exp Biol 206(20):3615–3624

    PubMed  Google Scholar 

  19. Ip YK, Chew SF, Wilson JM, Randall DJ (2004) Defences against ammonia toxicity in tropical air-breathing fishes exposed to high concentrations of environmental ammonia: a review. J Comp Physiol B 174(7):565–575

    CAS  PubMed  Google Scholar 

  20. Ip YK, Chew SF (2010) Ammonia production, excretion, toxicity, and defense in fish, a review. Front Physiol 1(134):1–20

    Google Scholar 

  21. Wang GH, Pan LQ, Ding YG (2014) Defensive strategies in response to environmental ammonia exposure of the sea cucumber Apostichopus japonicus: glutamine and urea formation. Aquaculture 432:278–285

    CAS  Google Scholar 

  22. Weihrauch D (2004) Ammonia excretion in aquatic and terrestrial crabs. J Exp Biol 207(26):4491–4504

    CAS  PubMed  Google Scholar 

  23. Weihrauch D, Wilkie MP, Walsh PJ (2009) Ammonia and urea transporters in gills of fish and aquatic crustaceans. J Exp Biol 212(17):1716–1730

    CAS  PubMed  Google Scholar 

  24. Liu SN, Pan LQ, Liu MQ, Yang LB (2014) Effects of ammonia exposure on nitrogen metabolism in gills and hemolymph of the swimming crab Portunus trituberculatus. Aquaculture 432:351–359

    CAS  Google Scholar 

  25. Qiu LG, Shi X, Yu SM, Han Q, Diao XP, Zhou HL (2018) Changes of ammonia-metabolizing enzyme activity and gene expression of two strains in shrimp Litopenaeus vannamei under ammonia stress. Front Physiol 9:211

    PubMed  PubMed Central  Google Scholar 

  26. Lim SL, Ip E, Jow A, Chew SF, Lim CB, Anderson PM, Ip YK (1999) The marble goby oxyeleotris marmoratus activates hepatic glutamine synthetase and detoxifies ammonia to glutamine during air exposure. J Exp Biol 202(3):237–245

    PubMed  Google Scholar 

  27. Banerjee B, Koner D, Bhuyan G, Saha N (2018) Differential expression of multiple glutamine synthetase genes in air-breathing Magur catfish, Clarias magur and their induction under hyper-ammonia stress. Gene 671:85–95

    CAS  PubMed  Google Scholar 

  28. Cooper AJL, Jeitner TM (2016) Central role of glutamate metabolism in the maintenance of nitrogen homeostasis in normal and hyperammonemic brain. Biomolecules 6(2):16

    PubMed Central  Google Scholar 

  29. Wang YR, Li E, Yu N, Wang XD, Cai CF, Tang BP, Chen LQ, Wormhoudt AV (2012) Characterization and expression of glutamate dehydrogenase in response to acute salinity stress in the Chinese mitten crab, Eriocheir sinensis. PLoS One 7(5):e37316

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Treberg JR, Banh S, Pandey U, Weihrauch D (2014) Intertissue differences for the role of glutamate dehydrogenase in metabolism. Neurochem Res 39(3):516–526

    CAS  PubMed  Google Scholar 

  31. Gaspar C, Silva-Marrero JI, Fàbregas A, Miñarrob M, Ticób JR, Baanante IV, Metón I (2018) Administration of chitosan-tripolyphosphate-DNA nanoparticles to knockdown glutamate dehydrogenase expression impairs transdeamination and gluconeogenesis in the liver. J Biotechnol S0168–1656(18):30629–30621

    Google Scholar 

  32. Wang P, Tan Z, Guan L, Tang S, Zhou C, Han X, Kang J, He Z (2015) Ammonia and amino acids modulates enzymes associated with ammonia assimilation pathway by ruminal microbiota in vitro. Livest Sci 178:130–139

    Google Scholar 

  33. Ding YT, Zang XN, Shi JW, Hou LL, He BX, Dong MM, Cong XM, Cao XX, Liu Z, Song XW, Huang XY, Zhang XC (2019) cDNA cloning of gs, gogat, and gdh from Haematococcus pluvialis and transcription and enzyme level analysis in different nitrogen concentration. J Appl Phycol 31(1):183–190

    CAS  Google Scholar 

  34. Hu R, Qu FF, Tang JZ, Zhao Q, Yan JP, Zhou ZG, Zhou Y, Liu Z (2017) Cloning, expression, and nutritional regulation of the glutamine synthetase gene in Ctenopharyngodon idellus. Comp Biochem Physiol B:S109649591730074X

  35. Pan LQ, Si LJ, Liu SN, Liu MQ, Wang GH (2018) Levels of metabolic enzymes and nitrogenous compounds in the swimming crab Portunus trituberculatus exposed to elevated ambient ammonia-N. J Ocean Univ China 17(4):957–966

    CAS  Google Scholar 

  36. Peng RB, Le KX, Wang PS, Wang Y, Han QX, Jiang XM (2017) Detoxification pathways in response to environmental ammonia exposure of the cuttlefish, Sepia pharaonis: glutamine and urea formation. J World Aquacult Soc 48(2):342–352

    CAS  Google Scholar 

  37. Ferretti JA, Calesso DF (2011) Toxicity of ammonia to surf clam (Spisula solidissima) larvae in saltwater and sediment elutriates. Mar Environ Res 71(3):189–194

    CAS  PubMed  Google Scholar 

  38. Wang XQ, Wang LL, Yao C, Qiu LM, Zhang H, Zhi Z (2012) Alternation of immune parameters and cellular energy allocation of Chlamys farreri under ammonia-N exposure and Vibrio anguillarum challenge. Fish Shellfish Immunol 32(5):741–749

    CAS  PubMed  Google Scholar 

  39. Montresor LC, Miranda-Filho KC, Paglia A, Luz DMR, Araújo JM, Silva MJDS, Gerhard L, Martinez CB, Vidigal THDA (2013) Short-term toxicity of ammonia, sodium hydroxide and a commercial biocide to golden mussel Limnoperna fortunei (Dunker, 1857). Ecotoxicol Environ Saf 92:150–154

    CAS  PubMed  Google Scholar 

  40. Zhao XL, Fu JP, Jiang LT, Zhang WW, Shao YN, Jin CH, Xiong JB, Li CH (2018) Transcriptome-based identification of the optimal reference genes as internal controls for quantitative RT-PCR in razor clam (Sinonovacula constricta). Genes Genomics 40(6):603–613

    CAS  PubMed  Google Scholar 

  41. Willett CS, Burton RS (2003) Characterization of the glutamate dehydrogenase gene and its regulation in a euryhaline copepod. Comp Biochem Physiol B 135(4):639–646

    PubMed  Google Scholar 

  42. Chakrapani V, Rasal KD, Mohapatra SD, Rasal AR, Jayasankar P, Barman HK (2017) Molecular characterization, computational analysis and transcript profiling of glutamate dehydrogenase (gdh) gene of Macrobrachium rosenbergii exposed to saline water. Gene Rep:S2452014417300377

  43. Liu HY, Sun WW, Tan BP, Chi SY, Dong XH, Yang QH (2012) Molecular cloning and expression of hepatopancreas glutamine synthetase in the Pacific white shrimp, Litopenaeus vannamei, induced by acute hypo-osmotic stress. Aquaculture 362–363(Complete):80–87

    Google Scholar 

  44. Lu ZJ, Qin ZD, Babu VS, Ye CK, Su GM, Li JB, Yang HY, Pan G, Lin L (2019) Expression and functional characterization of glutamine synthetase from giant freshwater prawn (Macrobrachium rosenbergii) under osmotic stress. Aquac Res 2635

  45. Bao YB, Li L, Ye MX, Dong YH, Jin WX, Lin ZH (2013) Expression of glutamine synthetase in Tegillarca granosa (Bivalvia, Arcidae) hemocytes stimulated by Vibrio parahaemolyticus and lipopolysaccharides. Genet Mol Res 12(2):1143–1154

    CAS  PubMed  Google Scholar 

  46. Geng ZX, Liu Q, Wang T, Ma S, Shan HW (2020) Changes in physiological parameters involved in glutamine and urea synthesis in Pacific white shrimp, Litopenaeus vannamei, fed Ampithoe sp. meal and exposed to ammonia stress. Aquac Res 51(7):1–10

    Google Scholar 

  47. Dong XX, Liu QG, Kan DQ, Zhao WH, Guo HS, Lv LL (2020) Effects of ammonia-N exposure on the growth, metabolizing enzymes, and metabolome of Macrobrachium rosenbergii. Ecotoxicol Environ Saf 189:110046

    CAS  PubMed  Google Scholar 

  48. Tang XB, Fu Y, Zhao YR, Pi J, Wang HQ (2020) Dietary α-Ketoglutarate supplementation alleviates harmful effects of high environmental ammonia on grass carp, Ctenopharyngodon idella. Aquac Res 51:1182–1189

    CAS  Google Scholar 

  49. Hong ML, Chen LQ, Sun XJ, Gu SZ, Zhang L, Chen Y (2007) Metabolic and immune responses in Chinese mitten-handed crab (Eriocheir sinensis) juveniles exposed to elevated ambient ammonia. Comp Biochem Physiol C 145(3):363–369

    Google Scholar 

  50. Peh WYX, Chew SF, Ching BY, Loong AM, Ip YK (2010) Roles of intestinal glutamate dehydrogenase and glutamine synthetase in environmental ammonia detoxification in the euryhaline four-eyed sleeper, Bostrychus sinensis. Aquat Toxicol 98(1):91–98

    CAS  PubMed  Google Scholar 

  51. Nissen JD, Lykke K, Bryk J, Stridh MH, Zaganas L, Skytt DM, Schousboe A, Bak LK, Enard W, Pääbo S, Waagepetersen HS (2017) Expression of the human isoform of glutamate dehydrogenase, hGDH2, augments TCA cycle capacity and oxidative metabolism of glutamate during glucose deprivation in astrocytes. Glia 65(3):474–488

    PubMed  Google Scholar 

  52. Zhang X, Pan LQ, Wei C, Tong RX, Li YF, Ding M, Wang HD (2020) Crustacean hyperglycemic hormone (CHH) regulates the ammonia excretion and metabolism in white shrimp, Litopenaeus vannamei under ammonia-N stress. Sci Total Environ 723:138128

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by National Key Research and Development Program of China (2018YFD0901405), Zhejiang Major Program of Science and Technology (2016C02055-9), Ningbo Major Project of Science and Technology (2019B10005), Modern Agro-industry Technology Research System (CARS-49) and Ningbo Top Discipline of Environmental Science and Engineering.

Author information

Authors and Affiliations

  1. Zhejiang Key Laboratory of Aquatic Germplasm Resource, College of Biological & Environmental Sciences, Zhejiang Wanli University, Ningbo, 315100, People’s Republic of China

    Huan Zhang, Gaigai Sun, Zhihua Lin, Hanhan Yao & Yinghui Dong

  2. Ninghai Marine Biological Seed Industry Research Institute, Zhejiang Wanli University, Ninghai, 315604, China

    Zhihua Lin

Authors
  1. Huan Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Gaigai Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Zhihua Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hanhan Yao
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yinghui Dong
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yinghui Dong.

Ethics declarations

Ethical approval

The S. constricta used in this work were collected from the genetic breeding research center of Zhejiang Wanli University, China. All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Zhejiang Wanli University, China.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhang, H., Sun, G., Lin, Z. et al. The razor clam Sinonovacula constricta uses the strategy of conversion of toxic ammonia to glutamine in response to high environmental ammonia exposure. Mol Biol Rep 47, 9579–9593 (2020). https://doi.org/10.1007/s11033-020-06018-w

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11033-020-06018-w

Keywords

Search

Navigation