Insect cold tolerance depends on their ability to withstand or repair perturbations in cellular homeostasis caused by low temperature stress. Decreased oxygen availability (hypoxia) can interact with low temperature tolerance, often improving insect survival. One mechanism proposed for such responses is that whole-animal cold tolerance is set by a transition to anaerobic metabolism. Here, we provide a test of this hypothesis in an insect model system (Thaumatotibia leucotreta) by experimental manipulation of oxygen availability while measuring metabolic rate, critical thermal minimum (CTmin), supercooling point and changes in 43 metabolites in moth larvae at three key timepoints (before, during and after chill coma). Furthermore, we determined the critical oxygen partial pressure below which metabolic rate was suppressed (c. 4.5 kPa). Results showed that altering oxygen availability did not affect (non-lethal) CTmin nor (lethal) supercooling point. Metabolomic profiling revealed the upregulation of anaerobic metabolites and alterations in concentrations of citric acid cycle intermediates during and after chill coma exposure. Hypoxia exacerbated the anaerobic metabolite responses induced by low temperatures. These results suggest that cold tolerance of T. leucotreta larvae is not set by oxygen limitation, and that anaerobic metabolism in these larvae may contribute to their ability to survive in necrotic fruit.

Department of Conservation Ecology and Entomology Centre for Invasion Biology Stellenbosch University Private Bag X1 Matieland 7602 South Africa

Institute of Entomology Biology Centre of the Czech Academy of Sciences České Budějovice Czech Republic

Section for Genetics Ecology and Evolution Department of Bioscience Aarhus University Ny Munkegade 116 DK 8000 Aarhus C Denmark

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Chown S. L. & Nicolson S. W. Insect Physiological Ecology: mechanisms and patterns (Oxford University Press, 2004).

Harrison J. F., Woods H. A. & Roberts S. P. Ecological and Environmental Physiology of Insects (Oxford University Press, 2012).

Hoback W. W. & Stanley D. W. Insects in hypoxia. J. Insect Physiol. 47, 533–542 (2001). PubMed

Dillon M. E. & Frazier M. R. PubMed

Holter P. & Spangenberg A. Oxygen uptake in coprophilous beetles (

Callier V., Hand S. C., Campbell J. B., Biddulph T. & Harrison J. F. Developmental changes in hypoxic exposure and responses to anoxia in PubMed

Terblanche J. S. In The Insects - Structure and Function (eds Simpson S. J. & Douglas A. E.) 588–618 (2012).

Hazell S. P. & Bale J. S. Low temperature thresholds: Are chill coma and CTmin synonymous? J. Insect Physiol. 57, 1085–1089 (2011). PubMed

MacMillan H. A. & Sinclair B. J. Mechanisms underlying insect chill-coma. J. Insect Physiol. 57, 12–20 (2011). PubMed

Goller B. Y. F. & Esch H. Comparative study of chill-coma temperatures and muscle potentials in insect flight muscles. J. Exp. Biol. 150, 221–231 (1990).

Pörtner H. O. Climate change and temperature-dependent biogeography: oxygen limitation of thermal tolerance in animals. Naturwissenschaften 88, 137–146 (2001). PubMed

Klok C. J., Sinclair B. J. & Chown S. L. Upper thermal tolerance and oxygen limitation in terrestrial arthropods. J. Exp. Biol. 207, 2361–2370 (2004). PubMed

Stevens M. M., Jackson S., Bester S. A., Terblanche J. S. & Chown S. L. Oxygen limitation and thermal tolerance in two terrestrial arthropod species. J. Exp. Biol. 213, 2209–2218 (2010). PubMed

Verberk W. C. E. P., Sommer U., Davidson R. L. & Viant M. R. Anaerobic metabolism at thermal extremes: a metabolomic test of the oxygen limitation hypothesis in an aquatic insect. Integr. Comp. Biol. 53, 609–619 (2013). PubMed PMC

Verberk W. C. E. P. PubMed PMC

Boardman L. & Terblanche J. S. Oxygen safety margins set thermal limits in an insect model system. J. Exp. Biol. 218, 1677–1685 (2015). PubMed

Hetz S. K. & Bradley T. J. Insects breathe discontinuously to avoid oxygen toxicity. Nature 433, 516–519 (2005). PubMed

Terblanche J. S., Clusella-Trullas S. & Chown S. L. Phenotypic plasticity of gas exchange pattern and water loss in PubMed

Williams C. M., Pelini S. L., Hellmann J. J. & Sinclair B. J. Intra-individual variation allows an explicit test of the hygric hypothesis for discontinuous gas exchange in insects. Biol. Lett. 6, 274–277 (2010). PubMed PMC

Huang S.-P., Talal S., Ayali A. & Gefen E. The effect of discontinuous gas exchange on respiratory water loss in grasshoppers (Orthoptera: Acrididae) varies across an aridity gradient. J. Exp. Biol. 218, 2510–2517 (2015). PubMed

Lee R. E., Chen C. P. & Denlinger D. L. A rapid cold-hardening process in insects. Science (80-) 238, 1415–1417 (1987). PubMed

Coulson S. J. & Bale J. S. Anoxia induces rapid cold hardening in the housefly

Yocum G. D. & Denlinger D. L. Anoxia blocks thermotolerance and the induction of rapid cold hardening in the flesh fly, Sarcophaga crassipalpis. Physiol. Biochem. Zool. 19, 152– 158 (1994).

Nilson T. L., Sinclair B. J. & Roberts S. P. The effects of carbon dioxide anesthesia and anoxia on rapid cold-hardening and chill coma recovery in PubMed PMC

Storey K. B. & Storey J. M. In Advances in Low Temperature Biology (ed Steponkus P. L.) 101–140 (JAI Press, 1992).

Boardman L., Sorensen J. G., Johnson S. A. & Terblanche J. S. Interactions between controlled atmospheres and low temperature tolerance: a review of biochemical mechanisms. Front. Physiol. 2, 92 (2011). PubMed PMC

Boardman L., Sørensen J. G. & Terblanche J. S. Physiological and molecular mechanisms associated with cross tolerance between hypoxia and low temperature in PubMed

Michaud M. R. & Denlinger D. L. Shifts in the carbohydrate, polyol, and amino acid pools during rapid cold-hardening and diapause-associated cold-hardening in flesh flies ( PubMed

Overgaard J. PubMed

Colinet H., Larvor V., Laparie M. & Renault D. Exploring the plastic response to cold acclimation through metabolomics. Funct. Ecol. 26, 711–722 (2012).

Williams C. M. PubMed PMC

MacMillan H. A., Williams C. M., Staples J. F. & Sinclair B. J. Reestablishment of ion homeostasis during chill-coma recovery in the cricket PubMed PMC

Robert Michaud M. PubMed

Lighton J. R. B. Hot hypoxic flies: Whole-organism interactions between hypoxic and thermal stressors in

Verberk W. C. E. P. & Bilton D. T. Can oxygen set thermal limits in an insect and drive gigantism? Plos One 6, e22610 (2011). PubMed PMC

Boardman L., Grout T. G. & Terblanche J. S. False codling moth

Lighton J. R. B. & Turner R. J. Thermolimit respirometry: an objective assessment of critical thermal maxima in two sympatric desert harvester ants, PubMed

Basson C. H. & Terblanche J. S. Metabolic responses of PubMed

Boardman L., Sørensen J. G. & Terblanche J. S. Physiological responses to fluctuating thermal and hydration regimes in the chill susceptible insect, Thaumatotibia leucotreta. J. Insect Physiol. 59, 781–794 (2013). PubMed

Lighton J. R. B. & Lovegrove B. G. A temperature-induced switch from diffusive to convective ventilation in the honeybee. J. Exp. Biol. 154, 509–516 (1990).

Koštál V. PubMed PMC

Hallman G. J. & Denlinger D. L. Temperature sensitivity in insects and application in integrated pest management. (Westview Press, Inc., 1998).

Fields P. G. & White N. D. G. Alternatives to methyl bromide treatmerts for stored-product and quarantine insect. Annu. Rev. Entomol. 47, 331–359 (2002). PubMed

Ben-Yehoshua S., Robertson R. N. & Biale J. B. Respiration & internal atmosphere of avocado fruit. Plant Physiol. 38, 194–201 (1962). PubMed PMC

Grové T., De Beer M. S. & Joubert P. H. Developing a systems approach for PubMed

Makarieva A. M., Gorshkov V. G., Li B.-L. & Chown S. L. Size- and temperature-independence of minimum life-supporting metabolic rates. Funct. Ecol. 20, 83–96 (2006).

Hochachka P. W. In Plant Life Under Oxygen Deprivation (eds Jackson M. B., Davies D. D. & Lambers H.) 121–128 (SPB Academic Publishing, 1991).

Yeager D. P. & Ultsch G. R. Physiological regulation and conformation: a BASIC program for the determination of critical points. Physiol. Zool. 62, 888–907 (1989).

Pörtner H. O. & Grieshaber, M K. In The vertebrate gas transport cascade: adaptations to environment and mode of life (ed. Bicudo J. E. P. W.) 330–357 (CRC Press, Boca Raton, Florida, 1993).

Greenlee K. J. & Harrison J. F. Respiratory changes throughout ontogeny in the tobacco hornworm caterpillar, Manduca sexta. J. Exp. Biol. 208, 1385–1392 (2005). PubMed

Zhou S., Criddle R. S. & Mitcham E. J. Metabolic response of Platynota stultana pupae during and after extended exposure to elevated CO PubMed

Herreid C. F. Hypoxia in invertebrates. Comp. Biochem. Physiol. Part A Physiol. 67, 311–320 (1980).

Clusella-Trullas S. & Chown S. L. Investigating onychophoran gas exchange and water balance as a means to inform current controversies in arthropod physiology. J. Exp. Biol. 211, 3139–3146 (2008). PubMed

Marshall D. J., Bode M. & White C. R. Estimating physiological tolerances - a comparison of traditional approaches to nonlinear regression techniques. J. Exp. Biol. 216, 2176–2182 (2013). PubMed

Van Voorhies W. A. Metabolic function in PubMed PMC

Lease H. M., Klok C. J., Kaiser A. & Harrison J. F. Body size is not critical for critical PO2 in scarabaeid and tenebrionid beetles. J. Exp. Biol. 215, 2524–2533 (2012). PubMed

Harrison J. PubMed

Terblanche J. S., Marais E., Hetz S. K. & Chown S. L. Control of discontinuous gas exchange in PubMed

Groenewald B., Chown S. L. & Terblanche J. S. A hierarchy of factors influence discontinuous gas exchange in the grasshopper PubMed

Lee R. E., Costanzo J. P. & Mugnano J. A. Regulation of supercooling and ice nucleation in insects. Eur. J. Entomol. 93, 405–418 (1996).

MacMillan H. A. & Sinclair B. J. The role of the gut in insect chilling injury: cold-induced disruption of osmoregulation in the fall field cricket, Gryllus pennsylvanicus. J. Exp. Biol. 214, 726–734 (2011). PubMed

Malmendal A. PubMed

Wade A. M. & Tucker H. N. Antioxidant characteristics of L-histidine. J. Nutr. Biochem. 9, 308–315 (1998).

Lemire J. PubMed

Görlach A. In Hypoxia and cancer (ed. Melillo M.) 65–90 (Springer Science + Business Media B.V., 2014).

Overgaard J., Sørensen J. G., Petersen S. O., Loeschcke V. & Holmstrup M. Changes in membrane lipid composition following rapid cold hardening in PubMed

Goto S. G., Udaka H., Ueda C. & Katagiri C. Fatty acids of membrane phospholipids in PubMed

Pujol-Lereis L. M., Fagali N. S., Rabossi A., Catalá Á. & Quesada-Allué L. A. Chill-coma recovery time, age and sex determine lipid profiles in PubMed

Slotsbo S.

Vereshtchagin S. M., Sytinsky I. A. & Tyshchenko V. P. The effect of γ-aminobutyric acid and β-alanine on bioelectrical activity of nerve ganglia of the pine moth caterpillar (

Joanisse D. R. & Storey K. B. Oxidative stress and antioxidants in overwintering larvae of cold-hardy goldenrod gall insects. J. Exp. Biol. 199, 1483–1491 (1996). PubMed

Carpenter J., Bloem S. & Hofmeyr H. In Area-Wide Control of Insect Pests (eds Vreysen M., Robinson A. & Hendrichs J.) 351–359 (Springer Netherlands, 2007).

Sinclair B. J., Jaco Klok C., Scott M. B., Terblanche J. S. & Chown S. L. Diurnal variation in supercooling points of three species of Collembola from Cape Hallett, Antarctica. J. Insect Physiol. 49, 1049–1061 (2003). PubMed

Lighton J. R. B. & Schilman P. E. Oxygen reperfusion damage in an insect. Plos One 2, e1267 (2007). PubMed PMC

Benjamini Y. & Hochberg Y. Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing. J. R. Stat. Soc. Ser. B 57, 289–300 (1995).

Xia J., Mandal R., Sinelnikov I. V., Broadhurst D. & Wishart D. S. MetaboAnalyst 2.0-a comprehensive server for metabolomic data analysis. Nucleic Acids Res. 40, W127–W133 (2012). PubMed PMC

Xia J., Sinelnikov I. V., Han B. & Wishart D. S. MetaboAnalyst 3.0—making metabolomics more meaningful. Nucleic Acids Res. 43, W251–W257 (2015). PubMed PMC

Colinet H., Larvor V., Bical R. & Renault D. Dietary sugars affect cold tolerance of