Abstract
Several earthworm species are known to be able to withstand freezing. At the biochemical level, this ability is based on cryoprotectant accumulation as well as several other mechanisms. In this study, we used 1H NMR to investigate metabolomic changes in two freeze-tolerant earthworm taxa, Dendrobaena octaedra and one of the genetic lineages of Eisenia sp. aff. nordenskioldi f. pallida. A total of 45 metabolites were quantified. High concentrations of glucose were present in frozen tissues of both taxa. No other putative cryoprotectants were found. We detected high levels of glycolysis end products and succinate in frozen animals, indicating the activation of glycolysis. Concentrations of many other substances also significantly increased. On the whole, metabolic change in response to freezing was much more pronounced in the specimens of Eisenia sp. aff. nordenskioldi f. pallida, including signs of nucleotide degradation.
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Data Availability
Raw NMR spectra, the descriptions of specimens and samples, metabolite concentrations and the preliminary metabolomic analysis are available at our Animal Metabolite Databaserepository, Experiment ID 269 (https://amdb.online/amdb/experiments/269/). All other data are available from the corresponding author upon request.
References
Bayley M, Overgaard J, Høj AS et al (2010) Metabolic changes during estivation in the common earthworm Aporrectodea caliginosa. Physiol Biochem Zool 83:541–550. https://doi.org/10.1086/651459
Berman DI, Leirikh AN (1985) The ability of earthworm Eisenia nordenskioldi (Eisen) (Lumbricidae, Oligochaeta) to resist minus temperatures. Dokl Akad Nauk SSSR 285:1258–1261 ([In Russian])
Berman DI, Meshcheryakova EN (2013) Ranges and cold hardiness of two earthworm subspecies (Eisenia nordenskioldi, Lumbricidae, Oligochaeta). Biol Bull 40:719–727. https://doi.org/10.1134/S1062359013090021
Berman DI, Meshcheryakova EN, Alfimov AV, Leirikh AN (2001) Distribution of the earthworm Dendrobaena octaedra (Lumbricidae, Oligochaeta) in the Northern Holarctic is restricted by its insufficient freeze tolerance. Doklady Biological Sci 377:145–148
Berman DI, Mescheryakova EN, Leirikh AN, Kurenshchikov DK (2010) Geographic range and cold hardiness of the earthworm Drawida ghilarovi (Oligochaeta, Moniligastridae). Biol Bull 37:895–904. https://doi.org/10.1134/S1062359010090049
Berman DI, Bulakhova NA, Meshcheryakova EN (2016) Cold hardiness and range of the earthworm Eisenia sibirica (Oligochaeta, Lumbricidae). Contemp Probl Ecol 9:45–52. https://doi.org/10.1134/S1995425516010030
Berman DI, Bulakhova NA, Meshcheryakova EN, Shekhovtsov SV (2019) Cold resistance and the distribution of genetic lineages of the earthworm Eisenia nordenskioldi (Oligochaeta, Lumbricidae). Biol Bull 46:430–437. https://doi.org/10.1134/S1062359019050042
Brown JN, Samuelsson L, Bernardi G et al (2014) Aqueous and lipid nuclear magnetic resonance metabolomic profiles of the earthworm Aporrectodea caliginosa show potential as an indicator species for environmental metabolomics. Environ Toxicol Chem 33:2313–2322. https://doi.org/10.1002/etc.2680
Bundgaard A, James AM, Gruszczyk AV et al (2019) Metabolic adaptations during extreme anoxia in the turtle heart and their implications for ischemia-reperfusion injury. Sci Rep 9:1–10. https://doi.org/10.1038/s41598-019-39836-5
Bundy JG, Ramløv H, Holmstrup M (2003) Multivariate metabolic profiling using 1H nuclear magnetic resonance spectroscopy of freeze-tolerant and freeze-intolerant earthworms exposed to frost. CryoLetters 24:347–358
Calderon S, Holmstrup M, Westh P, Overgaard J (2009) Dual roles of glucose in the freeze-tolerant earthworm Dendrobaena octaedra: cryoprotection and fuel for metabolism. J Exp Biol 212:859–866. https://doi.org/10.1242/jeb.026864
Chinopoulos C (2019) Succinate in ischemia: where does it come from? Int J Biochem Cell Biol 115:105580. https://doi.org/10.1016/j.biocel.2019.105580
Chouchani ET, Pell VR, Gaude E et al (2014) Ischaemic accumulation of succinate controls reperfusion injury through mitochondrial ROS. Nature 515:431–435. https://doi.org/10.1038/nature13909
Davies PL (2014) Ice-binding proteins: a remarkable diversity of structures for stopping and starting ice growth. Trends Biochem Sci 39:548–555. https://doi.org/10.1016/j.tibs.2014.09.005
Duman JG (2015) Animal ice-binding (antifreeze) proteins and glycolipids: an overview with emphasis on physiological function. J Exp Biol 218:1846–1855. https://doi.org/10.1242/jeb.116905
Fernández R, Almodóvar A, Novo M et al (2012) Adding complexity to the complex: new insights into the phylogeny, diversification and origin of parthenogenesis in the Aporrectodea caliginosa species complex (Oligochaeta, Lumbricidae). Mol Phylogenet Evol 64:368–379. https://doi.org/10.1016/j.ympev.2012.04.011
Hawes TC, Hines A, Viant MR et al (2008) Metabolomic fingerprint of cryo-stress in a freeze tolerant insect. CryoLetters 29:505–515
Hochachka PW, Somero GN (2002) Biochemical adaptation: mechanism and process in physiological evolution. Oxford University Press, Oxford
Holmstrup M (2003) Overwintering adaptations in earthworms. Pedobiologia 47:504–510. https://doi.org/10.1078/0031-4056-00220
Holmstrup M, Overgaard J (2007) Freeze tolerance in Aporrectodea caliginosa and other earthworms from Finland. Cryobiology 55:80–86. https://doi.org/10.1016/j.cryobiol.2007.06.001
Holmstrup M, Costanzo JP, Lee RE Jr (1999) Cryoprotective and osmotic responses to cold acclimation and freezing in freeze-tolerant and freeze-intolerant earthworms. J Comp Physiol B 169:207–214. https://doi.org/10.1007/s003600050213
Holmstrup M, Overgaard J, Bindesbøl A-M et al (2007) Adaptations to overwintering in the earthworm Dendrobaena octaedra: genetic differences in glucose mobilisation and freeze tolerance. Soil Biol Biochem 39:2640–2650
Holmstrup M, Slotsbo S, Henriksen PG, Bayley M (2016) Earthworms accumulate alanine in response to drought. Comp Biochem Physiol Part A Mol Integr Physiol 199:8–13. https://doi.org/10.1016/j.cbpa.2016.04.015
Jones OAH, Dias DA (2014) Environmental Metabolomics of Soil Organisms. In: eMagRes. John Wiley & Sons, Ltd, Chichester, UK, pp 1–12
Larson DJ, Middle L, Vu H et al (2014) Wood frog adaptations to overwintering in Alaska: new limits to freezing tolerance. J Exp Biol 72:1420–1425. https://doi.org/10.1242/jeb.101931
Lundheim R (2002) Physiological and ecological significance of biological ice nucleators. Philos Trans R Soc London Ser B Biol Sci 357:937–943. https://doi.org/10.1098/rstb.2002.1082
Marshall CB, Fletcher GL, Davies PL (2004) Hyperactive antifreeze protein in a fish. Nature 429:153–153. https://doi.org/10.1038/429153a
Meshcheryakova EN, Berman DI (2014) Cold hardiness and geographic distribution of earthworms (Oligochaeta, Lumbricidae, Moniligastridae). Entomol Rev 94:486–497. https://doi.org/10.1134/S0013873814040046
Nagana Gowda GA, Djukovic D, Bettcher LF et al (2018) NMR-guided mass spectrometry for absolute quantitation of human blood metabolites. Anal Chem 90:2001–2009. https://doi.org/10.1021/acs.analchem.7b04089
Overgaard J, Slotsbo S, Holmstrup M, Bayley M (2007) Determining factors for cryoprotectant accumulation in the freeze-tolerant earthworm, Dendrobaena octaedra. J Exp Zool Part A Ecol Genet Physiol 307A:578–589. https://doi.org/10.1002/jez.412
Overgaard J, Tollarova M, Hedlund K et al (2009) Seasonal changes in lipid composition and glycogen storage associated with freeze-tolerance of the earthworm, Dendrobaena octaedra. J Comp Physiol B 179:569–577. https://doi.org/10.1007/s00360-009-0341-9
Pérez-Losada M, Ricoy M, Marshall JC, Domínguez J (2009) Phylogenetic assessment of the earthworm Aporrectodea caliginosa species complex (Oligochaeta: Lumbricidae) based on mitochondrial and nuclear DNA sequences. Mol Phylogenet Evol 52:293–302. https://doi.org/10.1016/j.ympev.2009.04.003
Rasmussen L, Holmstrup M (2002) Geographic variation of freeze-tolerance in the earthworm Dendrobaena octaedra. J Comp Physiol B Biochem 172:691–698. https://doi.org/10.1007/s00360-002-0298-4
Rogatzki MJ, Ferguson BS, Goodwin ML, Gladden LB (2015) Lactate is always the end product of glycolysis. Front Neurosci 9:22. https://doi.org/10.3389/fnins.2015.00022
Severin S, Solovyeva G (1989) Praktikum po biohimii. MSU, Moscow ([In Russian])
Shekhovtsov SV, Berman DI, Bazarova NE et al (2016) Cryptic genetic lineages in Eisenia nordenskioldi pallida (Oligochaeta, Lumbricidae). Eur J Soil Biol 75:151–156. https://doi.org/10.1016/j.ejsobi.2016.06.004
Shekhovtsov SV, Berman DI, Golovanova EV, Peltek SE (2017) Genetic diversity of the earthworm Eisenia nordenskioldi (Lumbricidae, Annelida). Vavilovskii Zhurnal Genet Selektsii 21:588–595. https://doi.org/10.18699/VJ17.24-o. ([In Russian])
Shekhovtsov SV, Berman DI, Bulakhova NA et al (2018) Phylogeography of Eisenia nordenskioldi nordenskioldi (Lumbricidae, Oligochaeta) from the north of Asia. Polar Biol 41:237–247. https://doi.org/10.1007/s00300-017-2184-2
Shekhovtsov SV, Golovanova EV, Ershov NI et al (2020a) Phylogeny of the Eisenia nordenskioldi complex based on mitochondrial genomes. Eur J Soil Biol. https://doi.org/10.1016/j.ejsobi.2019.103137
Shekhovtsov SV, Bulakhova NA, Tsentalovich YP et al (2020b) Metabolic response of the Siberian wood frog Rana amurensis to extreme hypoxia. Sci Rep 10:14604. https://doi.org/10.1038/s41598-020-71616-4
Shekhovtsov SV, Bulakhova NA, Tsentalovich YP et al (2021a) Biochemical response to freezing in the Siberian salamander Salamandrella keyserlingii. Biology 10:1172. https://doi.org/10.3390/biology10111172
Shekhovtsov SV, Ermolov SA, Poluboyarova TV et al (2021b) Morphological differences between genetic lineages of the peregrine earthworm : Aporrectodea caliginosa (Savigny, 1826). Acta Zool Acad Sci Hungaricae 67:235–246. https://doi.org/10.17109/AZH.67.3.235.2021
Shekhovtsov SV, Shipova AA, Bulakhova NA, Berman DI (2022a) Differentiation within the Drawida ghilarovi complex (Moniligastridae: Annelida) revealed by multigene transcriptomic dataset analysis. Eur J Soil Biol 111:103411. https://doi.org/10.1016/j.ejsobi.2022.103411
Shekhovtsov SV, Bulakhova NA, Tsentalovich YP et al (2022b) Metabolomic analysis reveals that the moor frog Rana arvalis uses both glucose and glycerol as cryoprotectants. Animals 12:1286. https://doi.org/10.3390/ani12101286
Slotsbo S, Hansen LM, Jordaens K et al (2012) Cold tolerance and freeze-induced glucose accumulation in three terrestrial slugs. Comp Biochem Physiol Part A Mol Integr Physiol 161:443–449. https://doi.org/10.1016/j.cbpa.2012.01.002
Storey KB, Storey JM (1996) Natural freezing survival in animals. Annu Rev Ecol Syst 27:365–386. https://doi.org/10.1146/annurev.ecolsys.27.1.365
Storey KB, Storey JM (2017) Molecular physiology of freeze tolerance in vertebrates. Physiol Rev 97:623–665. https://doi.org/10.1152/physrev.00016.2016
Tárnoky K, Nagy S (1963) Spectrophotometric determination of glycogen with o-toluidine. Clin Chim Acta 8:627–628. https://doi.org/10.1016/0009-8981(63)90116-5
Toxopeus J, Sinclair BJ (2018) Mechanisms underlying insect freeze tolerance. Biol Rev 93:1891–1914. https://doi.org/10.1111/brv.12425
Voituron Y, Barré H, Ramløv H, Douady CJ (2009) Freeze tolerance evolution among anurans: frequency and timing of appearance. Cryobiology 58:241–247. https://doi.org/10.1016/j.cryobiol.2009.01.001
Vsevolodova-Perel TS (1997) The earthworms of the fauna of Russia. Nauka, Moscow
Wishart DS, Feunang YD, Marcu A et al (2018) HMDB 4.0: the human metabolome database for 2018. Nucleic Acids Res 46:D608–D617. https://doi.org/10.1093/nar/gkx1089
Zelentsova EA, Yanshole LV, Tsentalovich YP et al (2022) The application of quantitative metabolomics for the taxonomic differentiation of birds. Biology 11:1089. https://doi.org/10.3390/biology11071089
Zhang YF, Ganin GN, Atopkin DM, Wu DH (2020) Earthworm Drawida (Moniligastridae) molecular phylogeny and diversity in Far East Russia and Northeast China. Eur Zool J 87:180–191. https://doi.org/10.1080/24750263.2020.1741705
Funding
This study was supported by the State Budget Projects no. 1021060307698-5 (Faunogenesis and adaptive strategies of poikilothermic animals in extreme environments of the North) and no. FWNR-2022-0022.
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DIB and SVS jointly designed the study. ENM, NAB, and DIB collected the animals and performed freezing experiments. YPT and EAZ performed NMR analyses. KIS determined glycogen content. SVS, YPT, EAZ, and DIB analyzed the data. SVS wrote the manuscript with input from all authors. All authors read and approved the final manuscript.
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Shekhovtsov, S.V., Zelentsova, E.A., Bulakhova, N.A. et al. Biochemical response of two earthworm taxa exposed to freezing. J Comp Physiol B 193, 391–400 (2023). https://doi.org/10.1007/s00360-023-01500-w
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DOI: https://doi.org/10.1007/s00360-023-01500-w