BACKGROUND: The ubiquitous occurrence of inducible Heat Shock Proteins (Hsps) up-regulation in response to cold-acclimation and/or to cold shock, including massive increase of Hsp70 mRNA levels, often led to hasty interpretations of its role in the repair of cold injury expressed as protein denaturation or misfolding. So far, direct functional analyses in Drosophila melanogaster and other insects brought either limited or no support for such interpretations. In this paper, we analyze the cold tolerance and the expression levels of 24 different mRNA transcripts of the Hsps complex and related genes in response to cold in two strains of D. melanogaster: the wild-type and the Hsp70- null mutant lacking all six copies of Hsp70 gene. PRINCIPAL FINDINGS: We found that larvae of both strains show similar patterns of Hsps complex gene expression in response to long-term cold-acclimation and during recovery from chronic cold exposures or acute cold shocks. No transcriptional compensation for missing Hsp70 gene was seen in Hsp70- strain. The cold-induced Hsps gene expression is most probably regulated by alternative splice variants C and D of the Heat Shock Factor. The cold tolerance in Hsp70- null mutants was clearly impaired only when the larvae were exposed to severe acute cold shock. No differences in mortality were found between two strains when the larvae were exposed to relatively mild doses of cold, either chronic exposures to 0°C or acute cold shocks at temperatures down to -4°C. CONCLUSIONS: The up-regulated expression of a complex of inducible Hsps genes, and Hsp70 mRNA in particular, is tightly associated with cold-acclimation and cold exposure in D. melanogaster. Genetic elimination of Hsp70 up-regulation response has no effect on survival of chronic exposures to 0°C or mild acute cold shocks, while it negatively affects survival after severe acute cold shocks at temperatures below -8°C.

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

Institute of Entomology Biology Centre of the Czech Academy of Sciences České Budějovice Czech Republic; Faculty of Science University of South Bohemia České Budějovice Czech Republic

Zobrazit více v PubMed

Lee RE Jr (2010) A primer on insect cold-tolerance In: Denlinger DL, Lee RE Jr, eds. Low Temperature Biology of Insects. Cambridge: Cambridge University Press, pp. 3–34.

Hochachka PW (1986) Defense strategies against hypoxia and hypothermia. Science 231: 234–241. PubMed

Rojas RR, Leopold RA (1996) Chilling injury in the housefly: evidence for the role of oxidative stress between pupariation and emergence. Cryobiol 33: 447–458.

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 PubMed

MacMillan HA, Sinclair BJ (2011) The role of the gut in insect chilling injury: cold-induced disruption of osmoregulation in the fall field cricket, PubMed DOI

MacMillan HA, Williams CM, Staples JF, Sinclair BJ (2012) Reestablishment of ion homeostasis during chill-coma recovery in the cricket PubMed DOI PMC

Privalov P (1990) Cold denaturation of proteins. Crit Rev Biochem Mol Biol 25: 281–305. PubMed

Tsai C-J, Maizel JV, Nussinov R (2002) The hydrophobic effect: A new insight from cold denaturation and two-state water structure. Crit Rev Biochem Mol Biol 37: 55–69. PubMed

Razvi A, Scholtz JM (2006) Lessons in stability from thermophilic proteins. Protein Sci 15: 1569–1578. PubMed PMC

Knight JD, Bale JS, Franks F, Mathias SF, Baust JG (1986) Insect cold hardiness—supercooling points and prefreeze mortality. CryoLett 7: 194–203.

Drobnis EZ, Crowe LM, Berger T, Anchordoguy TJ, Overstreet JW, Crowe JH (1993) Cold shock damage is due to lipid phase transition in cell membranes: a demonstration using sperm as a model. J Exp Zool 265: 432–437. PubMed

Muldrew K, Acker JP, Elliott JAV, McGann LE (2004) The water to ice transition: Implications for living cells In: Fuller B, Lane N, Benson EE, eds. Life in the Frozen State. Boca Raton: CRC Press, pp. 67–108.

Emerson KJ, Uyemura AM, McDaniel KL, Schmidt PS, Bradshaw WE, Holzapfel CM (2009) Environmental control of ovarian dormancy in natural populations of PubMed DOI

Koštál V, Korbelová J, Rozsypal J, Zahradníčková H, Cimlová J, Tomčala A et al. (2011) Long-term cold acclimation extends survival time at 0°C and modifies the metabolomic profiles of the larvae of the fruit fly PubMed DOI PMC

Colinet H, Hoffmann AA (2012) Comparing phenotypic effects and molecular correlates of developmental, gradual and rapid cold acclimation responses in

Lee RE Jr, Chen CP, Denlinger DL (1987) A rapid cold-hardening process in insects. Science 238: 1415–1417. PubMed

Lee RE Jr, Denlinger DL (2010) Rapid cold-hardening: Ecological significance and underpinning mechanisms In: Denlinger DL, Lee RE Jr, eds. Low Temperature Biology of Insects. Cambridge: Cambridge University Press, pp. 35–58.

Kelty JD, Lee RE Jr (2001) Rapid cold-hardening of PubMed

Teets NM, Denlinger DL (2013) Physiological mechanisms of seasonal and rapid cold-hardening in insects. Physiol Entomol 38, 105–116.

Denlinger DL (1991) Relationship between cold hardiness and diapause In: Lee RE Jr, Denlinger DL, eds. Insects at Low Temperature. New York and London: Chapman and Hall, pp. 174–198.

Koštál V (2006) Eco-physiological phases of insect diapause. J Insect Physiol 52: 113–127. PubMed

Ragland GJ, Denlinger DL, Hahn DA (2010) Mechanisms of suspended animation are revealed by transcript profiling of diapause in the flesh fly. Proc Natl Sci Acad USA 107: 14909–14914. 10.1073/pnas.1007075107 PubMed DOI PMC

Colinet H, Renault D, Charoy-Guevel B, Com E (2012) Metabolic and proteomic profiling of diapause in the aphid parasitoid PubMed DOI PMC

Hayward SAL (2014) Application of functional "Omics" in environmental stress physiology: Insights, limitations, and future challenges. Curr Opinion Insect Sci 4: 35–41. PubMed

Salt RW (1961) Principles of insect cold-hardiness. Annu Rev Entomol 6: 55–74.

Zachariassen KE (1985) Physiology of cold tolerance in insects. Physiol Rev 65: 799–832. PubMed

Sömme L (1982) Supercooling and winter survival in terrestrial arthropods. Comp Biochem Physiol A 73: 519–543.

Storey KB, Storey JM (1991) Biochemistry of cryoprotectants In: Lee R.E. Jr, Denlinger D.L., eds. Insects at Low Temperature. New York and London: Chapman and Hall, pp. 64–93.

Zachariassen KE, Kristiansen E (2000) Ice nucleation and antinucleation in nature. Cryobiol 41: 257–279. PubMed

Duman JG (2001) Antifreeze and ice nucleator proteins in terrestrial arthropods. Annu Rev Physiol 63: 327–357. PubMed

Koštál V (2010) Cell structural modifications in insects at low temperatures In: Denlinger DL, Lee RE Jr, eds. Low Temperature Biology of Insects. Cambridge: Cambridge University Press, pp. 116–140.

Yi SX, Moore CW, Lee RE Jr (2007) Rapid cold-hardening protects PubMed

Lalouette L, Williams CW, Hervant F, Sinclair B, Renault D (2011) Metabolic rate and oxidative stress in insects exposed to low temperature thermal fluctuations. Comp Biochem Physiol A 158: 229–234. 10.1016/j.cbpa.2010.11.007 PubMed DOI

Rinehart JP, Li A, Yocum GD, Robich RM, Hayward SAL, Denlinger DL (2007) Up-regulation of heat shock proteins is essential for cold survival during insect diapause. Proc Natl Acad Sci USA 104: 11130–11137. PubMed PMC

Koštál V, Tollarová-Borovanská M (2009) The 70 kDa heat shock protein assists during the reparation of chilling injury in the insect, PubMed DOI PMC

Hayward SAL, Murray PA, Gracey AY, Cossins AR (2007) Beyond the lipid hypothesis: Mechanisms underlying phenotypic plasticity in inducible cold tolerance In: Csermely P, Vigh L, eds. Molecular Aspects of the Stress Response: Chaperons, membranes and Networks. Austin: Landes Bioscience, pp. 132–142. PubMed

Ritossa F (1962) A new puffing pattern induced by temperature shock and DNP in

Lindquist S (1986) The heat shock response. Annu Rev Biochem 55: 1151–1191. PubMed

Lindquist S, Craig EA (1988) The heat shock proteins. Annu Rev Genet 22: 631–677. PubMed

Sorensen JG, Kristensen TN, Loeschke V (2003) The evolutionary and ecological role of heat shock proteins. Ecol Lett 6: 1025–1037.

Sorensen J, Nielsen MM, Kruhoffer M, Justensen J, Loeschke V (2005) Full genome expression analysis of the heat stress response in PubMed PMC

Czajka MC, Lee RE Jr (1990) A rapid cold-hardening response protecting against cold shock injury in PubMed

Koštál V., Šimek P., Zahradníčková H., Cimlová J., Štětina T. (2012). Conversion of the chill susceptible fruit fly larva ( PubMed DOI PMC

Hoffmann AA (2010) Physiological climatic limits in PubMed DOI

Kellermann V, Loeschke V, Hoffmann AA, Kristensen TG, Flojgaard C, David JR et al. (2012) Phylogenetic constraints in key functional traits behind species' climate niches: Patterns of desiccation and cold resistance across 95 PubMed DOI

Burton V, Mitchell HK, Young P, Petersen NS (1988) Heat shock protection against cold stress of PubMed PMC

Goto SG, Kimura MT (1998) Heat- and cold-shock responses and temperature adaptations in subtropical and temperate species of PubMed

Goto SG, Yoshida KM, Kimura MT (1998) Accumulation of PubMed

Sinclair BJ, Gibbs AG, Roberts SP (2007) Gene transcription during exposure to, and recovery from, cold and desiccation stress in PubMed

Colinet H, Lee SF, Hoffmann AA (2010) Temporal expression of heat shock genes during cold stress and recovery from chill coma in adult PubMed DOI

Colinet H, Lee SF, Hoffmann AA (2010) Knocking down expression of Hsp22 and Hsp23 by RNA interference affects recovery from chill coma in PubMed DOI

Nielsen MM, Overgaard J, Sorensen JG, Holmstrup M, Justensen J, Loeschke V (2005) Role of HSF activation for resistance to heat, cold and high-temperature knock-down. J Insect Physiol 51: 1320–1329. PubMed

Jedlicka P, Mortin MA, Wu C (1997) Multiple functions of PubMed PMC

Gong WJ, Golic KG (2004) Genomic deletions of the PubMed PMC

Lindsley D.L., Grell E.H. (1968). Genetic variations of

Feder ME, Krebs RA (1998) Natural and genetic engineering of the heat-shock protein Hsp70 in

Morgan TH (1910) Sex Limited Inheritance in PubMed

Gong WJ, Golic KG (2006) Loss of Hsp70 in PubMed PMC

Betterncourt BR, Hogan CC, Nimali M, Drohan BW (2008) Inducible and constitutive heat shock gene expression responds to modification of PubMed DOI PMC

Ponton F, Chapuis M-P, Pernice M, Sword G, Simpson SJ (2011) Evaluation of potential reference genes for reverse transcription-qPCR studies of physiological responses in PubMed DOI

Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29: e45 PubMed PMC

Yocum GD (2001) Differential expression of two HSP70 transcripts in response to cold shock, thermoperiod, and adult diapause in the Colorado potato beetle. J Insect Physiol 47: 1139–1145. PubMed

Hayward SAL, Pavlides SC, Tammariello SP, Rinehart JP, Denlinger DL (2005) Temporal expression patterns of diapause-associated genes in flesh fly pupae from the onset of diapause through post-diapause quiescence. J Insect Physiol 51: 631–640. PubMed

Yocum GD, Kemp WP, Bosch J, Knoblett JN (2006) Thermal history influences diapause development in the solitary bee PubMed

Rinehart JP, Hayward SAL, Elnitsky MA, Sandro LH, Lee RE Jr Denlinger DL (2006) Continuous up-regulation of heat shock proteins in larvae, but not adults, of a polar insect. Proc Natl Acad Sci U S A 103: 14223–14227. PubMed PMC

Parsell D A, Lindquist S (1993) The function of heat-shock proteins in stress tolerance: Degradation and reactivation of damaged proteins. Annu Rev Genet 27: 437–496. PubMed

Hartl FU (1996) Molecular chaperones in cellular protein folding. Nature 381: 571–580. PubMed

Palter KB, Watanabe M, Stinson L, Mahowald AP, Craig EA (1986) Expression and localization of PubMed PMC

Ish-Horowicz D, Pinchin SM, Gausz H, Gyurkovics H, Bencze G (1979) Deletion mapping of two PubMed

Vesala L, Salminen TS, Laiho A, Hoikkala A, Kankare M (2012) Cold tolerance and cold-induced modulation of gene expression in two PubMed DOI

Yocum GD, Joplin KH, Denlinger DL (1998) Upregulation of a 23 kDa small heat shock protein transcript during pupal diapause in the flesh fly, PubMed

Rinehart JP, Yocum GD, Denlinger DL (2000) Developmental upregulation of inducible hsp70 transcripts, but not the cognate form, during pupal diapause in the flesh fly, PubMed

Jaattela M, Wissing D, Kokholm K, Kallunki T, Egeblad M (1998) Hsp70 exerts its anti-apoptotic function downstream of caspase-3-like proteases. EMBO J 17: 6124–6134. PubMed PMC

Wallin RP, Lundquist A, More SH, von Bonin A, Kiessling R, Ljunggren HG (2002) Heat-shock proteins as activators of the innate immune system. Trends Immunol 23: 130–135. PubMed

Feder ME, Walser J-C (2005) The biological limitations of transcriptomics in elucidating stress and stress responses. J Evol Biol 18: 901–910. PubMed

Wu C (1995) Heat shock transcription factors: structure and regulation. Annu Rev Cell Dev Biol 11: 441–469. PubMed

Westwood JT, Clos J, Wu C (1991) Stress-induced oligomerization and chromosomal relocalization of heat-shock factor. Nature 353: 822–827. PubMed

Wisniewski J, Orosz A, Allada R, Wu C (1996) The C-terminal region of PubMed PMC

Pirkkala L, Nykanen P, Sistonen L (2001) Roles of the heat shock transcription factors in regulation of the heat shock response and beyond. FASEB J 15: 1118–1131. PubMed

Fujikake N, Nagai Y, Popiel HA, Kano H, Yamaguchi M, Toda T (2005) Alternative splicing regulates the transcriptional activity of PubMed

Krebs RA, Feder ME (1997) Tissue-specific variation in Hsp70 expression and thermal damage in PubMed

Krebs RA, Feder ME (1998) Hsp70 and larval thermotolerance in PubMed

Goto SG (2001) A novel gene that is up-regulated during recovery from cold shock in PubMed

Colinet H, Hoffmann AA (2011) Comparing phenotypic effects and molecular correlates of developmental, gradual and rapid cold acclimation responses in

Bing X, Zhang J, Sinclair BJ (2012) A comparison of PubMed DOI

Colinet H, Lee SF, Hoffmann AA (2010) Functional characterization of the PubMed DOI PMC

Udaka H, Percival-Smith A, Sinclair BJ (2013) Increased abundance of PubMed DOI

Agarwal S K, Guru SC, Heppner C, Erdos MR, Collins RM, Park SY et al. (1999) Menin interacts with the AP1 transcription factor JunD and represses JunD-activated transcription. Cell 96:143–152. PubMed

Papaconstantinou M, Wu Y, Pretorius HN, Singh N, Gianfelice G, Tanguay RM, Campos AR et al. (2005) Menin is a regulator of the stress response in PubMed PMC

Ermolenko DN, Makhatadze GI (2002) Bacterial cold-shock proteins. Cell Mol Life Sci 59: 1902–1913. PubMed PMC

Weber MHW, Fricke I, Doll N, Marahiel MA (2002) CSDBase: an interactive database for cold shock domain-containing proteins and the bacterial cold shock response. Nucleic Acids Res 30: 375–378. PubMed PMC

Coulson M, Robert S, Saint R (2005) PubMed PMC

Doong H, Vrailas A, Kohn EC (2002) What's in the BAG? A functional domain analysis of the BAG-family proteins. Cancer Lett 188: 25–32. PubMed

Colinet H, Hoffmann AA (2010) Gene and protein expression of PubMed DOI

Reference Gene Selection for Normalizing Gene Expression in Ips Sexdentatus (Coleoptera: Curculionidae: Scolytinae) Under Different Experimental Conditions

Recovery from supercooling, freezing, and cryopreservation stress in larvae of the drosophilid fly, Chymomyza costata