Skip to main content
SpringerLink
Log in

Freeze tolerance and the underlying metabolite responses in the Xizang plateau frog, Nanorana parkeri

  • Original Paper
  • Published:
Journal of Comparative Physiology B Aims and scope Submit manuscript

Abstract

The frog Nanorana parkeri (Dicroglossidae) is endemic to the Tibetan Plateau, and overwinters shallow pond within damp caves for up to 6 months. Herein, we investigate the freeze tolerance of this species and profile changes in liver and skeletal muscle metabolite levels using an untargeted LC–MS-based metabolomic approach to investigate molecular mechanisms that may contribute to freezing survival. We found that three of seven specimens of N. parkeri could survive after being frozen for 12 h at − 2.0 °C with 39.91% ± 5.4% (n = 7) of total body water converted to ice. Freezing exposure induced partial dehydration of the muscle, which contributed to decreasing the amount of freezable water within the muscle and could be protective for the myocytes themselves. A comparative metabolomic analysis showed that freezing elicited significant responses, and a total of 33 and 36 differentially expressed metabolites were identified in the liver and muscle, respectively. These metabolites mainly participate in alanine, aspartic acid and glutamic acid metabolism, arginine and proline metabolism, and D-glutamine and D-glutamate metabolism. After freezing exposure, the contents of ornithine, melezitose, and maltotriose rose significantly; these may act as cryoprotectants. Additionally, the content of 8-hydroxy-2-deoxyguanine, 7-Ketocholesterol and hypoxanthine showed a marked increase, suggesting that freezing induced oxidative stress in the frogs. In summary, N. parkeri can tolerate a brief and partial freezing of their body, which was accompanied by substantial changes in metabolomic profiles after freezing exposure.

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

  • An TZ, Iwakiri M, Edashige K, Sakurai T, Kasai M (2000) Factors affecting the survival of frozen–thawed mouse spermatozoa. Cryobiology 40:237–249

    CAS  PubMed  Google Scholar 

  • Anchordoguy T, Carpenter JF, Loomis SH, Crowe JH (1988) Mechanisms of interaction of amino acids with phospholipid bilayers during freezing. BBA-Biomembranes 946:299–306

    CAS  PubMed  Google Scholar 

  • Angelcheva L, Mishra Y, Antti H, Kjellsen TD, Funk C, Strimbeck RG, Schröder WP (2014) Metabolomic analysis of extreme freezing tolerance in Siberian spruce (Picea obovata). New Phytol 204:545–555

    CAS  PubMed  Google Scholar 

  • Bennett VA, Pruitt NL, Lee RE (1997) Seasonal changes in fatty acid composition associated with cold-hardening in third instar larvae of Eurosta solidaginis. J Comp Physiol B 167:249–255

    CAS  Google Scholar 

  • Cohen BD (1970) Guanidinosuccinic acid in uremia. Arch Intern Med 126:846–850

    CAS  Google Scholar 

  • Costanzo JP (2019) Overwintering adaptations and extreme freeze tolerance in a subarctic population of the wood frog, Rana sylvatica. J Comp Physiol B 189:1–15

    PubMed  Google Scholar 

  • Costanzo JP, do Amaral MCF, Rosendale AJ, Lee RE, (2013) Hibernation physiology, freezing adaptation and extreme freeze tolerance in a northern population of the wood frog. J Exp Biol 216:3461–3473

    CAS  PubMed  Google Scholar 

  • Costanzo JP, Lee RE (2008) Urea loading enhances freezing survival and postfreeze recovery in a terrestrially hibernating frog. J Exp Biol 211:2969–2975

    PubMed  Google Scholar 

  • Costanzo JP, Lee RE (2013) Avoidance and tolerance of freezing in ectothermic vertebrates. J Exp Biol 216:1961–1967

    PubMed  Google Scholar 

  • Costanzo JP, Baker PJ, Lee RE (2006) Physiological responses to freezing in hatchlings of freeze-tolerant and -intolerant turtles. J Comp Physiol B 176:697–707

    CAS  PubMed  Google Scholar 

  • Costanzo JP, Reynolds AM, do Amaral MCF, Rosendale AJ, Lee RE (2015) Cryoprotectants and extreme freeze tolerance in a subarctic population of the wood frog. PLoS ONE 10:e0117234

    PubMed  PubMed Central  Google Scholar 

  • Croes SA, Thomas RE (2000) Freeze tolerance and cryoprotectant synthesis of the Pacific tree frog Hyla regilla. Copeia 2000:863–868

    Google Scholar 

  • D’Alessandro A, Nemkov T, Bogren LK, Martin SL, Hansen KC (2016) Comfortably numb and back: plasma metabolomics reveals biochemical adaptations in the hibernating 13-lined ground squirrel. J Proteome Res 16:958–969

    PubMed  Google Scholar 

  • Dieni CA, Storey KB (2009) Creatine kinase regulation by reversible phosphorylation in frog muscle. Comp Biochem Physiol B 152:405–412

    PubMed  Google Scholar 

  • do Amaral MCF, Frisbie J, Goldstein DL, Krane CM (2018) The cryoprotectant system of Cope’s gray treefrog, Dryophytes chrysoscelis: responses to cold acclimation, freezing, and thawing. J Comp Physiol B 188:611–621

    PubMed  PubMed Central  Google Scholar 

  • Driedzic WR, Clow KA, Short CE, Ewart KV (2006) Glycerol production in rainbow smelt (Osmerus mordax) may be triggered by low temperature alone and is associated with the activation of glycerol-3-phosphate dehydrogenase and glycerol-3-phosphatase. J Exp Biol 209:1016–1023

    CAS  PubMed  Google Scholar 

  • Duman JG (2015) Animal ice-binding (antifreeze) proteins and glycolipids: an overview with emphasis on physiological function. J Exp Biol 218:1846–1855

    PubMed  Google Scholar 

  • Dunn WB, Ellis DI (2005) Metabolomics: current analytical platforms and methodologies. TrAC Trends Anal Chem 24:285–294

    CAS  Google Scholar 

  • Florant GL (1998) Lipid metabolism in hibernators: the importance of essential fatty acids. Am Zool 38:331–340

    CAS  Google Scholar 

  • Geiss L, do Amaral MCF, Frisbie J, Goldstein DL, Krane CM (2019) Postfreeze viability of erythrocytes from Dryophytes chrysoscelis. J Exp Zool A 331:308–313

    CAS  Google Scholar 

  • Hermes-Lima M, Storey KB (1993) Antioxidant defenses in the tolerance of freezing and anoxia by garter snakes. Am J Physiol Regul Integr Comp Physiol 265:R646–R652

    CAS  Google Scholar 

  • Izumi Y, Katagiri C, Sonoda S, Tsumuki H (2009) Seasonal changes of phospholipids in last instar larvae of rice stem borer Chilo suppressalis Walker (Lepidoptera: Pyralidae). Entomol Sci 12:376–381

    Google Scholar 

  • Joanisse DR, Storey KB (1996) Oxidative damage and antioxidants in Rana sylvatica, the freeze-tolerant wood frog. Am J Physiol Regul Integr Comp Physiol 271:R545–R553

    CAS  Google Scholar 

  • Koštál V, Berková P, Šimek P (2003) Remodelling of membrane phospholipids during transition to diapause and cold-acclimation in the larvae of Chymomyza costata (Drosophilidae). Comp Biochem Physiol B 135:407–419

    PubMed  Google Scholar 

  • Koštál V, Korbelová J, Rozsypal J, Zahradníčková H, Cimlová J, Tomčala A, Šimek P (2011) Long-term cold acclimation extends survival time at 0 °C and modifies the metabolomic profiles of the larvae of the fruit fly Drosophila melanogaster. PLoS ONE 6:e25025

    PubMed  PubMed Central  Google Scholar 

  • Lardon I, Eyckmans M, Vu TN, Laukens K, De Boeck G, Dommisse R (2013) 1H-NMR study of the metabolome of a moderately hypoxia-tolerant fish, the common carp (Cyprinus carpio). Metabolomics 9:1216–1227

    CAS  Google Scholar 

  • Larson DJ, Middle L, Vu H, Zhang W, Serianni AS, Duman J, Barnes BM (2014) Wood frog adaptations to overwintering in Alaska: new limits to freezing tolerance. J Exp Biol 217:2193–2200

    PubMed  Google Scholar 

  • Lee RE, Costanzo JP (1998) Biological ice nucleation and ice distribution in cold-hardy ectothermic animals. Annu Rev Physiol 60:55–72

    CAS  PubMed  Google Scholar 

  • Lewis JM, Ewart KV, Driedzic WR (2004) Freeze resistance in rainbow smelt (Osmerus mordax): seasonal pattern of glycerol and antifreeze protein levels and liver enzyme activity associated with glycerol production. Physiol Biochem Zool 77:415–422

    CAS  PubMed  Google Scholar 

  • Li Z, Agellon LB, Allen TM, Umeda M, Jewell L, Mason A, Vance DE (2006) The ratio of phosphatidylcholine to phosphatidylethanolamine influences membrane integrity and steatohepatitis. Cell Metab 3:321–331

    CAS  PubMed  Google Scholar 

  • Lu X, Ma X, Fan L, Hu Y, Lang Z, Li Z, Fang B, Guo W (2016) Reproductive ecology of a Tibetan frog Nanorana parkeri (Anura: Ranidae). J Nat Hist 50:2769–2782

    Google Scholar 

  • McCord JM (1985) Oxygen-derived free radicals in postischemic tissue injury. N Engl J Med 312:159–163

    CAS  PubMed  Google Scholar 

  • Michaud MR, Benoit JB, Lopez-Martinez G, Elnitsky MA, Lee RE, Denlinger DL (2008) Metabolomics reveals unique and shared metabolic changes in response to heat shock, freezing and desiccation in the Antarctic midge, Belgica antarctica. J Insect Physiol 54:645–655

    Google Scholar 

  • Nelson CJ, Otis JP, Martin SL, Carey HV (2009) Analysis of the hibernation cycle using LC-MS-based metabolomics in ground squirrel liver. Physiol Genomics 37:43–51

    CAS  PubMed  Google Scholar 

  • Nelson CJ, Otis JP, Carey HV (2010) Global analysis of circulating metabolites in hibernating ground squirrels. Comp Biochem Physiol D 5:265–273

    Google Scholar 

  • Niu Y, Cao W, Zhao Y, Zhai H, Zhao Y, Tang X, Chen Q (2018a) The levels of oxidative stress and antioxidant capacity in hibernating Nanorana parkeri. Comp Biochem Physiol A 219:19–27

    Google Scholar 

  • Niu Y, Wang J, Men S, Zhao Y, Lu S, Tang X, Chen Q (2018b) Urea and plasma ice-nucleating proteins promoted the modest freeze tolerance in Pleske’s high altitude frog Nanorana pleskei. J Comp Physiol B 188:599–610

    CAS  PubMed  Google Scholar 

  • Ooshiro Z, Hironaka Y, Hayashi S (1976) Preventive effect of sugars on denaturation of fish protein during frozen storage. Mem Fac Fish Kagoshima Univ 25:91–99

    CAS  Google Scholar 

  • Pruitt NL, Lu C (2008) Seasonal changes in phospholipid class and class-specific fatty acid composition associated with the onset of freeze tolerance in third-instar larvae of Eurosta solidaginis. Physiol Biochem Zool 81:226–234

    CAS  PubMed  Google Scholar 

  • Reynolds AM, Lee RE, Costanzo JP (2014) Membrane adaptation in phospholipids and cholesterol in the widely distributed, freeze-tolerant wood frog, Rana sylvatica. J Comp Physiol B 184:371–383

    CAS  PubMed  Google Scholar 

  • Rochfort S (2005) Metabolomics reviewed: a new “omics” platform technology for systems biology and implications for natural products research. J Nat Prod 68:1813–1820

    CAS  PubMed  Google Scholar 

  • Rubinsky B, Wong ST, Hong JS, Gilbert J, Roos M, Storey KB (1994) 1H magnetic resonance imaging of freezing and thawing in freeze-tolerant frogs. Am J Physiol Regul Integr Comp Physiol 266:R1771–R1777

    CAS  Google Scholar 

  • Schiller TM, Costanzo JP, Lee RE (2008) Urea production capacity in the wood frog (Rana sylvatica) varies with season and experimentally induced hyperuremia. J Exp Zool A 309:484–493

    Google Scholar 

  • Seet RC, Lee C-YJ, Lim EC, Tan JJ, Quek AM, Chong W-L, Looi W-F, Huang S-H, Wang H, Chan Y-H (2010) Oxidative damage in Parkinson disease: measurement using accurate biomarkers. Free Radical Bio Med 48:560–566

    CAS  Google Scholar 

  • Shi X, Mao Y, Knapton AD, Ding M, Rojanasakul Y, Gannett PM, Dalal N, Liu K (1994) Reaction of Cr (VI) with ascorbate and hydrogen peroxide generates hydroxyl radicals and causes DNA damage: role of a Cr (IV)-mediated Fenton-like reaction. Carcinogenesis 15:2475–2478

    CAS  PubMed  Google Scholar 

  • Storey KB (2015) Regulation of hypometabolism: insights into epigenetic controls. J Exp Biol 218:150–159

    PubMed  Google Scholar 

  • Storey KB, Storey JM (1984) Biochemical adaption for freezing tolerance in the wood frog, Rana sylvatica. J Comp Physiol B 155:29–36

    CAS  Google Scholar 

  • Storey KB, Storey JM (1986) Freeze tolerant frogs: cryoprotectants and tissue metabolism during freeze-thaw cycles. Can J Zool 64:49–56

    CAS  Google Scholar 

  • Storey KB, Storey JM (1988) Freeze tolerance in animals. Physiol Rev 68:27–84

    CAS  PubMed  Google Scholar 

  • Storey KB, Storey JM (2013) Molecular biology of freezing tolerance. Compr Physiol 3:1283–1308

    PubMed  Google Scholar 

  • Storey KB, Storey JM (2017) Molecular physiology of freeze tolerance in vertebrates. Physiol Rev 97:623–665

    CAS  PubMed  Google Scholar 

  • Storey KB, Baust JG, Storey JM (1981) Intermediary metabolism during low temperature acclimation in the overwintering gall fly larva, Eurosta solidaginis. J Comp Physiol B 144:183–190

    CAS  Google Scholar 

  • Sturm RM, Jones BR, Mulvana DE, Lowes S (2016) HRMS using a Q-Exactive series mass spectrometer for regulated quantitative bioanalysis: how, when, and why to implement. Bioanalysis 8:1709–1721

    CAS  PubMed  Google Scholar 

  • Sun Y-B, Xiong Z-J, Xiang X-Y, Liu S-P, Zhou W-W, Tu X-L, Zhong L, Wang L, Wu D-D, Zhang B-L (2015) Whole-genome sequence of the Tibetan frog Nanorana parkeri and the comparative evolution of tetrapod genomes. Proc Natl Acad Sci 112:E1257–E1262

    CAS  PubMed  Google Scholar 

  • Vesala L, Salminen TS, Koštál V, Zahradníčková H, Hoikkala A (2012) Myo-inositol as a main metabolite in overwintering flies: seasonal metabolomic profiles and cold stress tolerance in a northern drosophilid fly. J Exp Biol 215:2891–2897

    CAS  PubMed  Google Scholar 

  • Viant MR (2008) Recent developments in environmental metabolomics. Mol BioSyst 4:980–986

    CAS  PubMed  Google Scholar 

  • Voituron Y, Eugene M, Barré H (2003) Survival and metabolic responses to freezing by the water frog (Rana ridibunda). J Exp Zool A 299:118–126

    Google Scholar 

  • Voituron Y, Barré H, Ramløv H, Douady CJ (2009) Freeze tolerance evolution among anurans: frequency and timing of appearance. Cryobiology 58:241–247

    PubMed  Google Scholar 

  • Vr K, Berková P, Šimek P (2003) Remodelling of membrane phospholipids during transition to diapause and cold-acclimation in the larvae of Chymomyza costata (Drosophilidae). Comp Biochem Physiol B 135:407–419

    Google Scholar 

  • Wang G-D, Zhang B-L, Zhou W-W, Li Y-X, Jin J-Q, Shao Y, Yang H-c, Liu Y-H, Yan F, Chen H-M (2018) Selection and environmental adaptation along a path to speciation in the Tibetan frog Nanorana parkeri. Proc Natl Acad Sci 115:E5056–E5065

    CAS  PubMed  Google Scholar 

  • Williams E, Hazel J (1995) Restructuring of plasma membrane phospholipids in isolated hepatocytes of rainbow trout during brief in vitro cold exposure. J Comp Physiol B 164:600–608

    CAS  Google Scholar 

  • Willmore W, Storey KB (1997) Antioxidant systems and anoxia tolerance in a freshwater turtle Trachemys scripta elegans. Mol Cell Biochem 170:177–185

    CAS  PubMed  Google Scholar 

  • Xia J, Sinelnikov IV, Han B, Wishart DS (2015) MetaboAnalyst 3.0-making metabolomics more meaningful. Nucleic Acids Res 43:W251–W257

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

Funding for this research was provided by the National Natural Science Foundation of China (no. 31971416 and no. 32001110), the Open Project of State Key Laboratory of Plateau Ecology and Agriculture, Qinghai University (2020-KF-002) and the Project of Scientific Research Foundation of Dezhou University (2019xjrc315). We thank Biotree Biotechnology Co., Ltd. for assistance in the metabolomics analysis of samples.

Author information

Authors and Affiliations

  1. Department of Life Sciences, Dezhou University, Dezhou, China

    Yonggang Niu

  2. School of Life Sciences, Lanzhou University, Lanzhou, 730000, Gansu, China

    Yonggang Niu, Wangjie Cao, Jinzhou Wang, Jie He, Li Ding, Xiaolong Tang & Qiang Chen

  3. State Key Laboratory of Plateau Ecology and Agriculture, Qinghai University, Xining, China

    Yonggang Niu & Qiang Chen

  4. Department of Biology, Carleton University, Ottawa, ON, K1S 5B6, Canada

    Kenneth B. Storey

Authors
  1. Yonggang Niu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Wangjie Cao
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jinzhou Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jie He
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Kenneth B. Storey
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Li Ding
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Xiaolong Tang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Qiang Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Qiang Chen.

Additional information

Communicated by G. Heldmaier.

Publisher's Note

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

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 640 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Niu, Y., Cao, W., Wang, J. et al. Freeze tolerance and the underlying metabolite responses in the Xizang plateau frog, Nanorana parkeri. J Comp Physiol B 191, 173–184 (2021). https://doi.org/10.1007/s00360-020-01314-0

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00360-020-01314-0

Keywords

Search

Navigation