Background: Many insect species have evolved the ability to survive extracellular freezing. The search for the underlying principles of their natural freeze tolerance remains hampered by our poor understanding of the mechanistic nature of freezing damage itself. Objectives: Here, in search of potential primary cellular targets of freezing damage, we compared mitochondrial responses (changes in morphology and physical integrity, respiratory chain protein functionality, and mitochondrial inner membrane (IMM) permeability) in freeze-sensitive vs. freeze-tolerant phenotypes of the larvae of the drosophilid fly, Chymomyza costata. Methods: Larvae were exposed to freezing stress at -30°C for 1 h, which is invariably lethal for the freeze-sensitive phenotype but readily survived by the freeze-tolerant phenotype. Immediately after melting, the metabolic activity of muscle cells was assessed by the Alamar Blue assay, the morphology of muscle mitochondria was examined by transmission electron microscopy, and the functionality of the oxidative phosphorylation system was measured by Oxygraph-2K microrespirometry. Results: The muscle mitochondria of freeze-tolerant phenotype larvae remained morphologically and functionally intact after freezing stress. In contrast, most mitochondria of the freeze-sensitive phenotype were swollen, their matrix was diluted and enlarged in volume, and the structure of the IMM cristae was lost. Despite this morphological damage, the electron transfer chain proteins remained partially functional in lethally frozen larvae, still exhibiting strong responses to specific respiratory substrates and transferring electrons to oxygen. However, the coupling of electron transfer to ATP synthesis was severely impaired. Based on these results, we formulated a hypothesis linking the observed mitochondrial swelling to a sudden loss of barrier function of the IMM.

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

Zobrazit více v PubMed

Abate M., Festa A., Falco M., Lombardi A., Luce A., Grimaldi A., et al. (2020). Mitochondria as playmakers of apoptosis, autophagy and senescence. Semin. Cell Dev. Biol. 98, 139–153. 10.1016/j.semcdb.2019.05.022 PubMed DOI

Abdelwahid E., Yokokura T., Krieser R. J., Balasundaram S., Fowle W. H., White K. (2007). Mitochondrial disruption in Drosophila apoptosis. Dev. Cell 12, 793–806. 10.1016/j.devcel.2007.04.004 PubMed DOI

Asahina E. (1970). Frost resistance in insects. Adv. Insect Physiol. 6, 1–49. 10.1016/S0065-2806(08)60109-5 DOI

Ballantyne J. S., Storey K. B. (1985). Characterization of mitochondria isolated from the freezing-tolerant larvae of the goldenrod gall fly (Eurosta solidaginis): substrate preferences, salt effects, and pH effects on warm-and cold-acclimated animals. Can. J. Zool. 63, 373–379. 10.1139/z85-057 DOI

Bayley J. S., Klepke M. J., Pedersen T. H., Overgaard J. (2019). Cold acclimation modulates voltage gated Ca2+ channel currents and fiber excitability in skeletal muscles of Locusta migratoria . J. Insect Physiol. 114, 116–124. 10.1016/j.jinsphys.2019.03.003 PubMed DOI

Bayley J. S., Sørensen J. G., Moos M., Koštál V., Overgaard J. (2020). Cold acclimation increases depolarization resistance and tolerance in muscle fibers from a chill-susceptible insect, Locusta migratoria . Am. J. Physiol.-Regul., Integr. Comp. Physiol. 319, R439–R447. 10.1152/ajpregu.00068.2020 PubMed DOI

Bayley J. S., Winther C. B., Andersen M. K., Grønkjær C., Nielsen O. B., Pedersen T. H., et al. (2018). Cold exposure causes cell death by depolarization-mediated Ca2+ overload in a chill-susceptible insect. Proc. Natl. Acad. Sci. U. S. A. 115, E9737–E9744. 10.1073/pnas.1813532115 PubMed DOI PMC

Bernardi P., Gerle C., Halestrap A. P., Jonas E. A., Karch J., Mnatsakanyan N., et al. (2023). Identity, structure, and function of the mitochondrial permeability transition pore: controversies, consensus, recent advances, and future directions. Cell Death Differ. 30, 1869–1885. 10.1038/s41418-023-01187-0 PubMed DOI PMC

Bernardi P., von Stockum S. (2012). The permeability transition pore as a Ca2+ release channel: new answers to an old question. Cell Calcium 52, 22–27. 10.1016/j.ceca.2012.03.004 PubMed DOI PMC

Braut-Boucher F., Aubery M. (2017). “Fluorescent molecular probes,” in Encyclopedia of specroscopy and spectrometry. Editors Lindon J. C., Tranter G. E., Koppenaal D. W. (Elsevier; ), 661–669. ISBN: 978-0-12-803224-4.

Camus M. F., Wolff J. N., Sgrò C. M., Dowling D. K. (2017). Experimental support that natural selection has shaped the latitudinal distribution of mitochondrial haplotypes in Australian Drosophila melanogaster . Mol. Biol. Evol. 34, 2600–2612. 10.1093/molbev/msx184 PubMed DOI

Chapman D. (1975). Phase transitions and fluidity characteristics of lipids and cell membranes. Q. Rev. Biophys. 8, 185–235. 10.1017/S0033583500001797 PubMed DOI

Chinopoulos C. (2018). Mitochondrial permeability transition pore: back to the drawing board. Neurochem. Int. 117, 49–54. 10.1016/j.neuint.2017.06.010 PubMed DOI

Chowański S., Lubawy J., Paluch-Lubawa E., Spochacz M., Rosiński G., Słocińska M. (2017). The physiological role of fat body and muscle tissues in response to cold stress in the tropical cockroach Gromphadorhina coquereliana . PLoS One 12, e0173100. 10.1371/journal.pone.0173100 PubMed DOI PMC

Chung D. J., Schulte P. M. (2020). Mitochondria and the thermal limits of ectotherms. J. Exp. Biol. 223, jeb227801. 10.1242/jeb.227801 PubMed DOI PMC

Colinet H., Renault D., Roussel D. (2017). Cold acclimation allows Drosophila flies to maintain mitochondrial functioning under cold stress. Insect biochem. Mol. Biol. 80, 52–60. 10.1016/j.ibmb.2016.11.007 PubMed DOI

Collins S. D., Allenspach A. L., Lee R. E., Jr (1997). Ultrastructural effects of lethal freezing on brain, muscle and Malpighian tubules from freeze-tolerant larvae of the gall fly, Eurosta solidaginis . J. Insect Physiol. 43, 39–45. 10.1016/s0022-1910(96)00073-x PubMed DOI

Des Marteaux L. E., Štětina T., Koštál V. (2018). Insect fat body cell morphology and response to cold stress is modulated by acclimation. J. Exp. Biol. 221, jeb189647. 10.1242/jeb.189647 PubMed DOI

Gasanoff E. S., Yaguzhinsky L. S., Garab G. (2021). Cardiolipin, non-bilayer structures and mitochondrial bioenergetics: relevance to cardiovascular disease. Cells 10, 1721. 10.3390/cells10071721 PubMed DOI PMC

Ghadially F. N. (1988). “Mitochondria,” in Ultrastructural pathology of the cell and matrix (Elsevier; ), 191–328. ISBN: 9781483192086.

Giorgio V., Guo L., Bassot C., Petronilli V., Bernardi P. (2018). Calcium and regulation of the mitochondrial permeability transition. Cell Calcium 70, 56–63. 10.1016/j.ceca.2017.05.004 PubMed DOI

Grgac R., Rozsypal J., Des Marteaux L., Štětina T., Koštál V. (2022). Stabilization of insect cell membranes and soluble enzymes by accumulated cryoprotectants during freezing stress. Proc. Natl. Acad. Sci. U. S. A. 119, e2211744119. 10.1073/pnas.2211744119 PubMed DOI PMC

Halestrap A. P. (2009). What is the mitochondrial permeability transition pore? J. Mol. Cell. Cardiol. 46, 821–831. 10.1016/j.yjmcc.2009.02.021 PubMed DOI

Haworth R. A., Hunter D. R. (1979). The Ca2+-induced membrane transition in mitochondria: II. Nature of the Ca2+ trigger site. Arch. Biochem. Biophys. 195, 460–467. 10.1016/0003-9861(79)90372-2 PubMed DOI

Hazel J. R. (1995). Thermal adaptation in biological membranes: is homeoviscous adaptation the explanation? Annu. Rev. Physiol. 57, 19–42. 10.1146/annurev.ph.57.030195.000315 PubMed DOI

Heinrich E. C., Gray E. M., Ossher A., Meigher S., Grun F., Bradley T. J. (2017). Aerobic function in mitochondria persists beyond death by heat stress in insects. J. Therm. Biol. 69, 267–274. 10.1016/j.jtherbio.2017.08.009 PubMed DOI

Hothorn T., Bretz F., Westfall P. (2008). Simultaneous inference in general parametric models. Biom. J. 50, 346–363. 10.1002/bimj.200810425 PubMed DOI

Hunter D. R., Haworth R. A. (1979). The Ca2+-induced membrane transition in mitochondria: I. The protective mechanisms. Arch. Biochem. Biophys. 195, 453–459. 10.1016/0003-9861(79)90371-0 PubMed DOI

Javadov S., Chapa-Dubocq X., Makarov V. (2018). Different approaches to modeling analysis of mitochondrial swelling. Mitochondrion 38, 58–70. 10.1016/j.mito.2017.08.004 PubMed DOI PMC

Joanisse D. R., Storey K. B. (1994). Mitochondrial enzymes during overwintering in two species of cold-hardy gall insects. Insect biochem. Mol. Biol. 24, 145–150. 10.1016/0965-1748(94)90080-9 DOI

Jørgensen L. B., Hansen A. M., Willot Q., Overgaard J. (2023). Balanced mitochondrial function at low temperature is linked to cold adaptation in Drosophila species. J. Exp. Biol. 226, jeb245439. 10.1242/jeb.245439 PubMed DOI

Kaasik A., Safiulina D., Zharkovsky A., Veksler V. (2007). Regulation of mitochondrial matrix volume. Am. J. Physiol. Cell Physiol. 292, C157–C163. 10.1152/ajpcell.00272.2006 PubMed DOI

Kadamur G., Ross E. M. (2013). Mammalian phospholipase C. Annu. Rev. Physiol. 75, 127–154. 10.1146/annurev-physiol-030212-183750 PubMed DOI

Klein P., Müller‐Rischart A. K., Motori E., Schönbauer C., Schnorrer F., Winklhofer K. F., et al. (2014). Ret rescues mitochondrial morphology and muscle degeneration of Drosophila Pink1 mutants. EMBO J. 33, 341–355. 10.1002/embj.201284290 PubMed DOI PMC

Koštál V. (2006). Eco-physiological phases of insect diapause. J. Insect Physiol. 52, 113–127. 10.1016/j.jinsphys.2005.09.008 PubMed DOI

Koštál V. (2010). “Cell structural modifications in insects at low temperature,” in Low temperature biology of insects. Editors Denlinger D. L., Lee R. (Cambridge: Cambridge University Press; ), 116–140. ISBN: 978-0-521-88635-2.

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. 10.1016/S1096-4959(03)00117-9 PubMed DOI

Koštál V., Noguchi H., Shimada K., Hayakawa Y. (1998). Developmental changes in dopamine levels in larvae of the fly Chymomyza costata: comparison between wild-type and mutant-nondiapause strains. J. Insect Physiol. 44, 605–614. 10.1016/S0022-1910(98)00043-2 PubMed DOI

Koštál V., Vambera J., Bastl J. (2004). On the nature of pre-freeze mortality in insects: water balance, ion homeostasis and energy charge in the adults of Pyrrhocoris apterus . J. Exp. Biol. 207, 1509–1521. 10.1242/jeb.00923 PubMed DOI

Krans J. L., Parfitt K. D., Gawera K. D., Rivlin P. K., Hoy R. R. (2010). The resting membrane potential of Drosophila melanogaster larval muscle depends strongly on external calcium concentration. J. Insect Physiol. 56, 304–313. 10.1016/j.jinsphys.2009.11.002 PubMed DOI

Kučera L., Moos M., Štětina T., Korbelová J., Vodrážka P., Des Marteaux L., et al. (2022). A mixture of innate cryoprotectants is key for freeze tolerance and cryopreservation of a drosophilid fly larva. J. Exp. Biol. 225, jeb243934. 10.1242/jeb.243934 PubMed DOI

Kukal O., Duman J. G., Serianni A. S. (1989). Cold-induced mitochondrial degradation and cryoprotectant synthesis in freeze-tolerant arctic caterpillars. J. Comp. Physiol. B 158, 661–671. 10.1007/BF00693004 PubMed DOI

Kuznetsov A. V., Veksler V., Gellerich F. N., Saks V., Margreiter R., Kunz W. S. (2008). Analysis of mitochondrial function in situ in permeabilized muscle fibers, tissues and cells. Nat. Protoc. 3, 965–976. 10.1038/nprot.2008.61 PubMed DOI

Lebenzon J. E., Denezis P. W., Mohammad L., Mathers K. E., Turnbull K. F., Staples J. F., et al. (2022). Reversible mitophagy drives metabolic suppression in diapausing beetles. Proc. Natl. Acad. Sci. U. S. A. 119, e2201089119. 10.1073/pnas.2201089119 PubMed DOI PMC

Lebenzon J. E., Overgaard J., Jørgensen L. B. (2023). Chilled, starved or frozen: insect mitochondrial adaptations to overcome the cold. Curr. Opin. Insect Sci. 58, 101076. 10.1016/j.cois.2023.101076 PubMed DOI

Lee R. E., Jr. (2010). “A primer on insect cold-tolerance,” in Low temperature biology of insects. Editors Denlinger D. L., Lee R. E. J. (Cambridge: Cambridge University Press; ), 3–34. ISBN: 978-0-521-88635-2.

Levin D., Danks H., Barber S. (2003). Variations in mitochondrial DNA and gene transcription in freezing‐tolerant larvae of Eurosta solidaginis (Diptera: tephritidae) and Gynaephora groenlandica (Lepidoptera: lymantriidae). Insect Mol. Biol. 12, 281–289. 10.1046/j.1365-2583.2003.00413.x PubMed DOI

Lubawy J., Chowański S., Adamski Z., Słocińska M. (2022). Mitochondria as a target and central hub of energy division during cold stress in insects. Front. Zool. 19, 1. 10.1186/s12983-021-00448-3 PubMed DOI PMC

Lubawy J., Chowański S., Colinet H., Słocińska M. (2023). Mitochondrial metabolism and oxidative stress in the tropical cockroach under fluctuating thermal regimes. J. Exp. Biol. 226, jeb246287. 10.1242/jeb.246287 PubMed DOI

MacMillan H. A. (2019). Dissecting cause from consequence: a systematic approach to thermal limits. J. Exp. Biol. 222, jeb191593. 10.1242/jeb.191593 PubMed DOI

MacMillan H. A., Baatrup E., Overgaard J. (2015). Concurrent effects of cold and hyperkalaemia cause insect chilling injury. Proc. R. Soc. B 282, 20151483. 10.1098/rspb.2015.1483 PubMed DOI PMC

Mazur P. (1984). Freezing of living cells: mechanisms and implications. Am. J. Physiol. 247, C125–C142. 10.1152/ajpcell.1984.247.3.C125 PubMed DOI

McDonald A. E., Pichaud N., Darveau C. A. (2018). “Alternative” fuels contributing to mitochondrial electron transport: importance of non-classical pathways in the diversity of animal metabolism. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 224, 185–194. 10.1016/j.cbpb.2017.11.006 PubMed DOI

McMullen D. C., Storey K. B. (2008). Mitochondria of cold hardy insects: responses to cold and hypoxia assessed at enzymatic, mRNA and DNA levels. Insect biochem. Mol. Biol. 38, 367–373. 10.1016/j.ibmb.2007.12.003 PubMed DOI

Menail H. A., Cormier S. B., Ben Youssef M., Jørgensen L. B., Vickruck J. L., Morin P., Jr, et al. (2022). Flexible thermal sensitivity of mitochondrial oxygen consumption and substrate oxidation in flying insect species. Front. Physiol. 13, 897174. 10.3389/fphys.2022.897174 PubMed DOI PMC

Menze M. A., Hutchinson K., Laborde S. M., Hand S. C. (2005). Mitochondrial permeability transition in the crustacean Artemia franciscana: absence of a calcium-regulated pore in the face of profound calcium storage. Am. J. Physiol. –Regul. Integr. Comp. Physiol. 289, R68–R76. 10.1152/ajpregu.00844.2004 PubMed DOI

Modesti L., Danese A., Angela Maria Vitto V., Ramaccini D., Aguiari G., Gafà R., et al. (2021). Mitochondrial Ca2+ signaling in health, disease and therapy. Cells 10, 1317. 10.3390/cells10061317 PubMed DOI PMC

Muldrew K., Acker J. P., Elliott J. A., McGann L. E. (2004). “The water to ice transition: implications for living cells,” in Life in the frozen state. Editors Fuller B. J., Lane N., Benson E. E. (Boca Raton, FL: CRC Press; ), 67–108. ISBN: 0-415-24700-4.

Newmeyer D. D., Ferguson-Miller S. (2003). Mitochondria: releasing power for life and unleashing the machineries of death. Cell 112, 481–490. 10.1016/S0092-8674(03)00116-8 PubMed DOI

Nunnari J., Suomalainen A. (2012). Mitochondria: in sickness and in health. Cell 148, 1145–1159. 10.1016/j.cell.2012.02.035 PubMed DOI PMC

Overgaard J., MacMillan H. A. (2017). The integrative physiology of insect chill tolerance. Annu. Rev. Physiol. 79, 187–208. 10.1146/annurev-physiol-022516-034142 PubMed DOI

Pearce R. S. (2001). Plant freezing and damage. Ann. Bot. 87, 417–424. 10.1006/anbo.2000.1352 DOI

Pichaud N., Messmer M., Correa C. C., Ballard J. W. O. (2013). Diet influences the intake target and mitochondrial functions of Drosophila melanogaster males. Mitochondrion 13, 817–822. 10.1016/j.mito.2013.05.008 PubMed DOI

Putney J. W., Tomita T. (2012). Phospholipase C signaling and calcium influx. Adv. Biol. Regul. 52, 152–164. 10.1016/j.advenzreg.2011.09.005 PubMed DOI PMC

Quinn P. (1985). A lipid-phase separation model of low-temperature damage to biological membranes. Cryobiol 22, 128–146. 10.1016/0011-2240(85)90167-1 PubMed DOI

Raison J., Lyons J., Mehlhorn R., Keith A. (1971). Temperature-induced phase changes in mitochondrial membranes detected by spin labeling. J. Biol. Chem. 246, 4036–4040. 10.1016/S0021-9258(18)62136-2 PubMed DOI

Rampersad S. N. (2012). Multiple applications of Alamar Blue as an indicator of metabolic function and cellular health in cell viability bioassays. Sensors 12, 12347–12360. 10.3390/s120912347 PubMed DOI PMC

R Core Team (2023). R: a language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing. Available at: https://www.R-project.org/.

Riihimaa A. J., Kimura M. T. (1988). A mutant strain of Chymomyza costata (Diptera: drosophilidae) insensitive to diapause‐inducing action of photoperiod. Physiol. Entomol. 13, 441–445. 10.1111/j.1365-3032.1988.tb01128.x DOI

Rozsypal J., Moos M., Šimek P., Koštál V. (2018). Thermal analysis of ice and glass transitions in insects that do and do not survive freezing. J. Exp. Biol. 221, 170464. 10.1242/jeb.170464 PubMed DOI

Schenkel L. C., Bakovic M. (2014). Formation and regulation of mitochondrial membranes. Int. J. Cell Biol. 2014, 709828. 10.1155/2014/709828 PubMed DOI PMC

Schlame M., Blais S., Edelman-Novemsky I., Xu Y., Montecillo F., Phoon C. K., et al. (2012). Comparison of cardiolipins from Drosophila strains with mutations in putative remodeling enzymes. Chem. Phys. Lipids 165, 512–519. 10.1016/j.chemphyslip.2012.03.001 PubMed DOI

Simard C. J., Pelletier G., Boudreau L. H., Hebert-Chatelain E., Pichaud N. (2018). Measurement of mitochondrial oxygen consumption in permeabilized fibers of Drosophila using minimal amounts of tissue. JoVE J. Vis. Expts., e57376. 10.3791/57376 PubMed DOI PMC

Sinclair B. J. (1999). Insect cold tolerance: how many kinds of frozen? Eur. J. Entomol. 96, 157–164.

Sinclair B. J., Renault D. (2010). Intracellular ice formation in insects: unresolved after 50 years? Comp. Biochem. Physiol. A 155, 14–18. 10.1016/j.cbpa.2009.10.026 PubMed DOI

Smith P. E., Krohn R. I., Hermanson G., Mallia A., Gartner F., Provenzano M., et al. (1985). Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 76–85. 10.1016/0003-2697(85)90442-7 PubMed DOI

Sokolova I. (2018). Mitochondrial adaptations to variable environments and their role in animals’ stress tolerance. Integr. Comp. Biol. 58, 519–531. 10.1093/icb/icy017 PubMed DOI

Štětina T., Des Marteaux L., Koštál V. (2020). Insect mitochondria as targets of freezing-induced injury. Proc. R. Soc. B 287, 20201273. 10.1098/rspb.2020.1273 PubMed DOI PMC

Storey K. B., Storey J. M. (1988). Freeze tolerance in animals. Physiol. Rev. 68, 27–84. 10.1152/physrev.1988.68.1.27 PubMed DOI

Storey K. B., Storey J. M. (2013). Molecular biology of freezing tolerance. Compr. Physiol. 3, 1283–1308. 10.1002/cphy.c130007 PubMed DOI

Storey K. B., Storey J. M. (2017). Molecular physiology of freeze tolerance in vertebrates. Physiol. Rev. 97, 623–665. 10.1152/physrev.00016.2016 PubMed DOI

Teets N. M., Marshall K. E., Reynolds J. A. (2023). Molecular mechanisms of winter survival. Annu. Rev. Entomol. 68, 319–339. 10.1146/annurev-ento-120120-095233 PubMed DOI

Toxopeus J., Des Marteaux L. E., Sinclair B. J. (2019). How crickets become freeze tolerant: the transcriptomic underpinnings of acclimation in Gryllus veletis . Comp. Biochem. Physiol. D. Genom. Proteom. 29, 55–66. 10.1016/j.cbd.2018.10.007 PubMed DOI

Toxopeus J., Sinclair B. J. (2018). Mechanisms underlying insect freeze tolerance. Biol. Rev. 93, 1891–1914. 10.1111/brv.12425 PubMed DOI

van Venetië R., Verkleij A. J. (1982). Possible role of non-bilayer lipids in the structure of mitochondria. A freeze-fracture electron microscopy study. Biochim. Biophys. Acta –Biomembr. 692, 397–405. 10.1016/0005-2736(82)90390-X PubMed DOI

Verkleij A. J. (1984). Lipidic intramembranous particles. Biochim. Biophys. Acta – Rev. Biomembr. 779, 43–63. 10.1016/0304-4157(84)90003-0 PubMed DOI

Von Stockum S., Basso E., Petronilli V., Sabatelli P., Forte M. A., Bernardi P. (2011). Properties of Ca2+ transport in mitochondria of Drosophila melanogaster . J. Biol. Chem. 286, 41163–41170. 10.1074/jbc.M111.268375 PubMed DOI PMC

Wiplfler B., Schneeberg K., Löffler A., Hünefeld F., Meier R., Beutel R. G. (2013). The skeletomuscular system of the larva of Drosophila melanogaster (Drosophilidae, Diptera) – a contribution to the morphology of a model organism. Arthr. Struct. Devel. 42, 47–68. 10.1016/j.asd.2012.09.005 PubMed DOI