SummaryInsects are highly successful animals inhabiting marine, freshwater and terrestrial habitats from the equator to the poles. As a group, insects have limited ability to regulate their body temperature and have thus required a range of strategies to support life in thermally stressful environments, including behavioural avoidance through migration and seasonal changes in cold tolerance. With respect to overwintering strategies, insects have traditionally been divided into two main groups: freeze tolerant and freeze avoiding, although this simple classification is underpinned by a complex of interacting processes, i.e. synthesis of ice nucleating agents, cryoprotectants, antifreeze proteins and changes in membrane lipid composition. Also, in temperate and colder climates, the overwintering ability of many species is closely linked to the diapause state, which often increases cold tolerance ahead of temperature-induced seasonal acclimatisation. Importantly, even though most species can invoke one or both of these responses, the majority of insects die from the effects of cold rather than freezing. Most studies on the effects of a changing climate on insects have focused on processes that occur predominantly in summer (development, reproduction) and on changes in distributions rather than winter survival per se. For species that routinely experience cold stress, a general hypothesis would be that predicted temperature increases of 1°C to 5°C over the next 50-100 years would increase winter survival in some climatic zones. However, this is unlikely to be a universal effect. Negative impacts may occur if climate warming leads to a reduction or loss of winter snow cover in polar and sub-polar areas, resulting in exposure to more severe air temperatures, increasing frequency of freeze-thaw cycles and risks of ice encasement. Likewise, whilst the dominant diapause-inducing cue (photoperiod) will be unaffected by global climate change, higher temperatures may modify normal rates of development, leading to a decoupling of synchrony between diapause-sensitive life-cycle stages and critical photoperiods for diapause induction. In terms of climate warming and potential heat stress, the most recent predictions of summer temperatures in Europe of 40°C or higher in 50-75 years, are close to the current upper lethal limit of some insects. Long-term data sets on insect distributions and the timing of annual migrations provide strong evidence for 'positive' responses to higher winter temperatures over timescales of the past 20-50 years in North America, Europe and Asia.

Diapause, the dormancy common to overwintering insects, evokes a unique pattern of gene expression. In the flesh fly, most, but not all, of the fly's heat shock proteins (Hsps) are up-regulated. The diapause up-regulated Hsps include two members of the Hsp70 family, one member of the Hsp60 family (TCP-1), at least four members of the small Hsp family, and a small Hsp pseudogene. Expression of an Hsp70 cognate, Hsc70, is uninfluenced by diapause, and Hsp90 is actually down-regulated during diapause, thus diapause differs from common stress responses that elicit synchronous up-regulation of all Hsps. Up-regulation of the Hsps begins at the onset of diapause, persists throughout the overwintering period, and ceases within hours after the fly receives the signal to reinitiate development. The up-regulation of Hsps appears to be common to diapause in species representing diverse insect orders including Diptera, Lepidoptera, Coleoptera, and Hymenoptera as well as in diapauses that occur in different developmental stages (embryo, larva, pupa, adult). Suppressing expression of Hsp23 and Hsp70 in flies by using RNAi did not alter the decision to enter diapause or the duration of diapause, but it had a profound effect on the pupa's ability to survive low temperatures. We thus propose that up-regulation of Hsps during diapause is a major factor contributing to cold-hardiness of overwintering insects.cold tolerance ͉ overwintering ͉ stress proteins ͉ RNAi

Antarctica's terrestrial environment is a challenge to which very few animals have adapted. The largest, free-living animal to inhabit the continent year-round is a flightless midge, Belgica antarctica. Larval midges survive the lengthy austral winter encased in ice, and when the ice melts in summer, the larvae complete their 2-yr life cycle, and the wingless adults form mating aggregations while subjected to surprisingly high substrate temperatures. Here we report a dichotomy in survival strategies exploited by this insect at different stages of its life cycle. Larvae constitutively up-regulate their heat shock proteins (small hsp, hsp70, and hsp90) and maintain a high inherent tolerance to temperature stress. High or low temperature exposure does not further upregulate these genes nor does it further enhance thermotolerance. Such ''preemptive'' synthesis of hsps is sufficient to prevent irreversible protein aggregation in response to a variety of common environmental stresses. Conversely, adults exhibit no constitutive up-regulation of their hsps and have a lower intrinsic tolerance to high temperatures, but their hsps can be thermally activated, resulting in enhanced thermotolerance. Thus, the midge larvae, but not the adults, have adopted the unusual strategy of expressing hsps continuously, possibly to facilitate proper protein folding in a cold habitat that is more thermally stable than that of the adults but a habitat subjected frequently to freeze-thaw episodes and bouts of pH, anoxic, and osmotic stress.Antarctica ͉ cold tolerance ͉ protein aggregation ͉ temperature stress ͉ thermotolerance

Rapid cold-hardening (RCH) is well known to increase the tolerance of chilling or cold shock in a diverse array of invertebrate systems at both organismal and cellular levels. Here, we report a novel role for RCH by showing that RCH also increases freezing tolerance in an Antarctic midge, Belgica antarctica (Diptera, Chironomidae). The RCH response of B. antarctica was investigated under two distinct physiological states: summer acclimatized and cold acclimated. Summer-acclimatized larvae were less cold tolerant, as indicated by low survival following exposure to -10°C for 24·h; by contrast, nearly all coldacclimated larvae survived -10°C, and a significant number could survive -15°C. Cold-acclimated larvae had higher supercooling points than summer larvae. To evaluate the RCH response in summer-acclimatized midges, larvae and adults, maintained at 4°C, were transferred to -5°C for 1·h prior to exposures to -10, -15 or -20°C. RCH significantly increased survival of summer-acclimatized larvae frozen at -10°C for 1·h compared with larvae receiving no cold-hardening treatment, but adults, which live for only a week or so in the austral summer, lacked the capacity for RCH. In coldacclimated larvae, RCH significantly increased freeze tolerance to both -15 and -20°C. Similarly, RCH significantly increased cellular survival of fat body, Malpighian tubules and gut tissue from cold-acclimated larvae frozen at -20°C for 24·h. These results indicate that RCH not only protects against non-freezing injury but also increases freeze tolerance.

Protection of poikilothermic animals from seasonal cold is widely regarded as being causally linked to changes in the unsaturation of membrane phospholipids, yet in animals this proposition remains formally untested. We have now achieved this by the genetic manipulation of lipid biosynthesis of Caenorhabditis elegans independent of temperature. Worms transferred from 25°C to 10°C develop over several days a much-increased tolerance of lethal cold (0°C) and also an increased phospholipid unsaturation, as in higher animal models. Of the three C. elegans ⌬9-desaturases, transcript levels of fat-7 only were up-regulated by cold transfer. RNAi suppression of fat-7 caused the induction of fat-5 desaturase, so to control desaturase expression we combined RNAi of fat-7 with a fat-5 knockout. These fat-5/fat-7 manipulated worms displayed the expected negative linear relationship between lipid saturation and cold tolerance at 0°C, an outcome confirmed by dietary rescue. However, this change in lipid saturation explains just 16% of the observed difference between cold tolerance of animals held at 25°C and 10°C. Thus, although the manipulated lipid saturation affects the tolerable thermal window, and altered ⌬9-desaturase expression accounts for cold-induced lipid adjustments, the effect is relatively small and none of the lipid manipulations were sufficient to convert worms between fully cold-sensitive and fully cold-tolerant states. Critically, transfer of 10°C-acclimated worms back to 25°C led to them restoring the usual cold-sensitive phenotype within 24 h despite retaining a lipid profile characteristic of 10°C worms. Other nonlipid mechanisms of acquired cold protection clearly dominate inducible cold tolerance.⌬9-acyl desaturase ͉ lipid composition ͉ RNAi

SUMMARYDuring winter, larvae of the Antarctic midge, Belgica antarctica (Diptera, Chironomidae), must endure 7-8 months of continuous subzero temperatures, encasement in a matrix of soil and ice, and severely desiccating conditions. This environment, along with the fact that larvae possess a high rate of water loss and are extremely tolerant of desiccation, may promote the use of cryoprotective dehydration as a strategy for winter survival. This study investigates the capacity of larvae to resist inoculative freezing and undergo cryoprotective dehydration at subzero temperatures. Slow cooling to -3°C in an environment at equilibrium with the vapor pressure of ice reduced larval water content by ~40% and depressed the body fluid melting point more than threefold to -2.6°C. This melting point depression was the result of the concentration of existing solutes (i.e. loss of body water) and the de novo synthesis of osmolytes. By day 14 of the subzero exposure, larval survival was still >95%, suggesting larvae have the capacity to undergo cryoprotective dehydration. However, under natural conditions the use of cryoprotective dehydration may be constrained by inoculative freezing as result of the insectʼs intimate contact with environmental ice. During slow cooling within a substrate of frozen soil, the ability of larvae to resist inoculative freezing and undergo cryoprotective dehydration was dependent upon the moisture content of the soil. As detected by a reduction of larval water content, the percentage of larvae that resisted inoculative freezing increased with decreasing soil moisture. These results suggest that larvae of the Antarctic midge have the capacity to resist inoculative freezing at relatively low soil moisture contents and likely undergo cryoprotective dehydration when exposed to subzero temperatures during the polar winter.

delayed adult eclosion noted in samples desiccated for more than 3·days at <5% relative humidity/25°C. In diapausing pupae, hsp23 and hsp70 transcripts are already highly expressed and are not further upregulated by desiccation stress. Both of the constitutive Hsps investigated, Hsp90 and Hsc70, were unresponsive to desiccation in both nondiapausing and diapausing pupae. However, both Hsp90 and Hsc70 were upregulated upon rehydration in nondiapausing and diapausing pupae. These results indicate distinct roles for the different Hsps during desiccation stress and rehydration/stress recovery. The response to desiccation recovery (rehydration) is similar to the Hsp response to cold recovery identified in S. crassipalpis: Hsp90 and Hsc70 are upregulated in both cases.

No abstract