Change in overwintering mechanism of the Cucujid beetle, Cucujus clavipes

Overwintering larvae of the Cucujid beetle, Cucujus clavipes, were freeze tolerant, able to survive the freezing of their extracellular body fluids, during the winter of 1978–1979. These larvae had high levels of polyols (glycerol and sorbitol), thermal hysteresis proteins and haemolymph ice nucleators that prevented extensive supercooling (the supercooling points of the larvae were ? 10°C), thus preventing lethal intracellular ice formation. In contrast, C. clavipes larvae were freeze suspectible, died if frozen, during the winter of 1982–1983, but supercooled to ～ ? 30°C. The absence of the ice nucleators in the 1982–1983 larvae, obviously essential in the now freeze-susceptible insects, was the major detected difference in the larvae from the 2 years. However, experiments in which the larvae were artifically seeded at ? 10°C (the temperature at which the natural haemolymph ice nucleators produced spontaneous nucleation in the 1978–1979 freeze tolerant larvae) demonstrated that the absence of the ice nucleators was not the critical factor, or at least not the only critical factor, responsible for the loss of freeze tolerance in the 1982–1983 larvae. The lower lethal temperatures for the larvae were approximately the same during the 2 winters in spite of the change in overwintering strategy.     

Freeze tolerance in larvae of the winter-active Diamesa mendotae Muttkowski (Diptera: Chironomidae): a contrast to adult strategy for survival at low temperatures

The winter-active Diamesa mendotae Muttkowski (Diptera: Chironomidae) is freeze intolerant in the adult stage with a low mean supercooling point (SCP) of ~−20 °C. However, cold-hardiness strategies for immatures of this species are unknown. In this study, we measured SCP values for D. mendotae larvae, pupae and adults using surface-contact thermometry. In addition, the lower lethal temperature (LLT) was determined for the larval stage. The mean SCPs for larvae (−7.4 °C) and pupae (−9.1 °C) were relatively high compared to adults (−19.7 °C). Our results indicate that the larvae of D. mendotae are freeze tolerant with a LLT₉₉ (−25.4 °C), ~−10 °C lower than their minimum SCP (−15.6 °C). Freeze tolerance in these larvae may be a strategy to provide protection from short-term exposures to ice crystals or to permit diapause within frozen substrates. The change in cold-hardiness strategy from freeze tolerant to freeze intolerant between the larval and adult stages of this species is likely a result of the different habitats occupied by these two life stages.     

Complex bud architecture and cell‐specific chemical patterns enable supercooling of Picea abies bud primordia

Bud primordia of Picea abies, despite a frozen shoot, stay ice free down to ?50 °C by a mechanism termed supercooling whose biophysical and biochemical requirements are poorly understood. Bud architecture was assessed by 3D—reconstruction, supercooling and freezing patterns by infrared video thermography, freeze dehydration and extraorgan freezing by water potential measurements, and cell‐specific chemical patterns by Raman microscopy and mass spectrometry imaging. A bowl‐like ice barrier tissue insulates primordia from entrance by intrinsic ice. Water repellent and densely packed bud scales prevent extrinsic ice penetration. At ?18 °C, break‐down of supercooling was triggered by intrinsic ice nucleators whereas the ice barrier remained active. Temperature‐dependent freeze dehydration (?0.1 MPa K^?1) caused accumulation of extraorgan ice masses that by rupture of the shoot, pith tissue are accommodated in large voids. The barrier tissue has exceptionally pectin‐rich cell walls and intercellular spaces, and the cell lumina were lined or filled with proteins, especially near the primordium. Primordial cells close to the barrier accumulate di, tri and tetrasaccharides. Bud architecture efficiently prevents ice penetration, but ice nucleators become active inside the primordium below a temperature threshold. Biochemical patterns indicate a complex cellular interplay enabling supercooling and the necessity for cell‐specific biochemical analysis.     

Synchrotron X-Ray Visualisation of Ice Formation in Insects during Lethal and Non-Lethal Freezing

Although the biochemical correlates of freeze tolerance in insects are becoming well-known, the process of ice formation in vivo is subject to speculation. We used synchrotron x-rays to directly visualise real-time ice formation at 3.3 Hz in intact insects. We observed freezing in diapausing 3^rd instar larvae of Chymomyza amoena (Diptera: Drosophilidae), which survive freezing if it occurs above −14°C, and non-diapausing 3^rd instar larvae of C. amoena and Drosophila melanogaster (Diptera: Drosophilidae), neither of which survive freezing. Freezing was readily observed in all larvae, and on one occasion the gut was seen to freeze separately from the haemocoel. There were no apparent qualitative differences in ice formation between freeze tolerant and non-freeze tolerant larvae. The time to complete freezing was positively related to temperature of nucleation (supercooling point, SCP), and SCP declined with decreasing body size, although this relationship was less strong in diapausing C. amoena. Nucleation generally occurred at a contact point with the thermocouple or chamber wall in non-diapausing larvae, but at random in diapausing larvae, suggesting that the latter have some control over ice nucleation. There were no apparent differences between freeze tolerant and non-freeze tolerant larvae in tracheal displacement or distension of the body during freezing, although there was markedly more distension in D. melanogaster than in C. amoena regardless of diapause state. We conclude that although control of ice nucleation appears to be important in freeze tolerant individuals, the physical ice formation process itself does not differ among larvae that can and cannot survive freezing. This suggests that a focus on cellular and biochemical mechanisms is appropriate and may reveal the primary adaptations allowing freeze tolerance in insects.     

Intracellular freezing: effect of extracellular supercooling.

Human erythrocytes were frozen on the stage of a cryomicroscope at accurately controlled constant-cooling rates with varying degrees of extracellular supercooling. The formation of intracellular ice was detected by direct observation of the frozen cells through the microscope. A significant coupling effect was determined between the minimum cooling rate necessary to produce intracellular freezing and the extent of supercooling. Increased degrees of extracellular supercooling reduced the range of cooling rates for which water would freeze within the cell. Specific data points were obtained at ΔT_SC = 0, ?5, and ?12 °C for which the corresponding transition cooling rates were respectively ?845, ?800, and ?11 °C/min.An explanation for the occurrence of this phenomenon is presented based on the physiochemical processes that govern the freezing of a cell suspension.     

Cold adaptation of the terrestrial isopod,Porcellio scaber,to subnivean environments

The terrestrial isopod, Porcellio scaber, was susceptible to subzero temperature: both freezing and chilling were injurious. The level of cold hardiness against chilling and freezing showed different patterns in their seasonal variation. The lower lethal temperature causing 50% mortality, an indicator of the tolerance to chilling, ranged from-1.37°C in August to-4.58°C in December. The whole body supercooling point, the absolute limit of freeze avoidance, was kept at about-7°C throughout the year. The winter decrease in lower lethal temperature was concomitant with an accumulation of low molecular weight carbohydrates which are possible protective reagents against chilling injury, whereas the less seasonally variable supercooling point seemed to be associated with the year-round presence of gut content. Food derivatives may act as efficient ice nucleators. The different trend in seasonal changes between lower lethal temperature and supercooling point may be related to the microclimate of the hibernacula in subnivean environments, where the winter temperature became lower than the lower lethal temperature in the summer active phase, but remained higher than the summer supercooling point.Abbreviations LLT₅₀ lower lethal temperature inducing 50% mortality - SCP supercooling point - T _a ambient air temperature - T _s soil surface temperature     

Ice formation in the freeze-tolerant eutardigrades Adorybiotus coronifer and Amphibolus nebulosus studied by differential scanning calorimetry

Summary This paper describes ice formation in two freeze-tolerant eutardigrades Adorybiotus coronifer and Amphibolus nebulosus, both commonly found in Arctic areas, based on a study by differential scanning calorimetry, and results are compared with similar investigations on freeze-tolerant insects. Under Arctic conditions A. coronifer typically inhabits drought-resistant mosses and overwinters in a frozen or dry state. A. nebulosus inhabits moist mosses or algae and overwinters as frozen cysts or, in few cases, as eggs or adults. Both species show a moderate capacity for supercooling, with crystallization temperatures (T_c) of -6° to -7°C. No differences in T_c were observed between summer- and winter-acclimatized A. coronifer or between active and encysted A. nebulosus. Distinct differences in the heat stability of their ice-nucleating activity indicate that the compounds responsible for nucleation are chemically different in the two species. Ice formation progresses rapidly in both species, and crystallization of water probably ceases within 1 min of the nucleation. Ice constitutes 80–90% of total body water, even at temperatures just below T_c, in both species. Winter-acclimatized A. coronifer build up about 10% less ice than summer-acclimatized animals, and a similar reduction was induced by cold-acclimation of summer animals in the laboratory. No winter reduction of the melting point could be detected in A. coronifer, indicating little, if any, accumulation of low molecular weight cryoprotectives. We suggest that the reduction in ice content is functionally related to increased amounts of water kept unfrozen due to interactions with macromolecules.     

Freeze-tolerance adaptations,including haemolymph protein and lipoprotein nucleators,in the larvae of the cranefly Tipula trivittata

The terrestrial overwintering larvae of the cranefly Tipula trivittata were freeze tolerant (able to survive the freezing of their extracellular body fluids) throughout the winter and spring of 1982–1983 until they pupated in mid-May. The larvae were most cold tolerant (24 h lower lethal temperatures of ?25 to ?30°C) in late January and early February. Sorbitol, at a maximal concentration of ～0.4 M, was the only polyol determined to be present at high levels and sorbitol accounted for most of the seasonal fluctuation in osmotic concentration. Haemolymph inorganic ion (Na⁺, K⁺, Ca²⁺, Mg²⁺, Cl^?) concentrations did not vary seasonally.The supercooling points of the larvae remained constant at ?6 to ?7°C over the study period because of the presence of haemolymph ice nucleating factors. These ice nucleating factors consist not only of haemolymph proteins, as had been demonstrated previously in other insect species, but also lipoproteins.     

Field ecology of freezing: Linking microhabitat use with freezing tolerance in Litoria ewingii

A small number of vertebrate species, including some frogs, are freezing tolerant and survive ice forming in their bodies under ecologically relevant conditions. Habitat use information is critical for interpreting laboratory studies of freezing tolerance, but there is often little known about the winter habitat and behaviours of the species under study. This work describes microhabitats used by the freezing‐tolerant frog Litoria ewingii Duméril and Bibron 1841 and their temperature characteristics. In winter, L. ewingii used microhabitats with wood, located further away from water than in summer. Microhabitat temperature records showed that frog microhabitats regularly fell below the temperature at which frog body fluids freeze (?1°C), and cooled substantially more slowly than did the air temperature. Temperatures were highly variable between microhabitats, seasons and years, with a minimum of ?2.4°C and a maximum cooling rate of 0.77°C h^?1. Frozen frogs were observed to recover in the field, demonstrating freezing tolerance. Both the characteristics of microhabitats and their selection are important in ensuring freezing survival.     

The Siberian timberman Acanthocinus aedilis: a freeze-tolerant beetle with low supercooling points

Larvae of the Siberian timberman beetle Acanthocinus aedilis display a number of unique features, which may have important implications for the field of cold hardiness in general. Their supercooling points are scattered over a wide temperature range, and some individuals have supercooling points in the low range of other longhorn beetles. However, they differ from other longhorn beetles in being tolerant to freezing, and in the frozen state they tolerate cooling to below −37°C. In this respect they also differ from the European timberman beetles, which have moderate supercooling capacity and die if they freeze. The combination of freezing tolerance and low supercooling points is unusual and shows that freezing at a high subzero temperature is not an absolute requirement for freezing tolerance. Like other longhorn beetles, but in contrast to other freeze-tolerant insects, the larvae of the Siberian timberman have a low cuticular water permeability and can thus stay supercooled for long periods without a great water loss. This suggests that a major function of the extracellular ice nucleators of some freeze-tolerant insects may be to prevent intolerable water loss in insects with high cuticular water permeability, rather than to create a protective extracellular freezing as has generally been assumed. The freezing tolerance of the Siberian timberman larvae is likely to be an adaptation to the extreme winter cold of Siberia.     

Collapse temperature of solutions important for lyopreservation of living cells at ambient temperature

In this study, the collapse temperature was determined using the freeze‐drying microscopy (FDM) method for a variety of cell culture medium‐based solutions (with 0.05–0.8 M trehalose) that are important for long‐term stabilization of living cells in the dry state at ambient temperature (lyopreservation) by freeze‐drying. Being consistent with what has been reported in the literature, the collapse temperature of binary water‐trehalose solutions was found to be similar to the glass transition temperature (T′_g ～ ?30°C) of the maximally freeze‐concentrated trehalose solution (～80 wt% trehalose) during the freezing step of freeze‐drying, regardless of the initial concentration of trehalose. However, the effect of the initial trehalose concentration on the collapse temperature of the cell culture medium‐based trehalose solutions was identified to be much more significant, particularly when the trehalose concentration is less than 0.2 M (the collapse temperature can be as low as ?65°C). We also determined that cell density from 1 to 10 million cells/mL and ice seeding at high subzero temperatures (?4 and ?7°C) have negligible impact on the solution collapse temperature. However, ice seeding does significantly affect the ice crystal morphology formed during the freezing step and therefore the drying rate. Finally, bulking agents (mannitol) could significantly affect the collapse temperature only when trehalose concentration is low (<0.2 M). However, improving the collapse temperature by using a high concentration of trehalose might be preferred to the addition of bulking agents in the solutions for freeze‐drying of living cells. We further confirmed the applicability of the collapse temperature measured with small‐scale (2 µL) samples using the FDM system to freeze‐drying of large‐scale (1 mL) samples using scanning electron microscopy (SEM) data. Taken together, the results reported in this study should provide useful guidance to the development of optimal freeze‐drying protocols for lyopreservation of living cells at ambient temperature for easy maintenance and convenient wide distribution to end users, which is important to the eventual success of modern cell‐based medicine. Biotechnol. Bioeng. 2010;106: 247–259. © 2010 Wiley Periodicals, Inc.     

Supercooling,ice inoculation and freeze tolerance in the European common lizard,Lacerta vivipara

The European common lizard (Lacerta vivipara) is widely distributed throughout Eurasia and is one of the few Palaearctic reptiles occurring above the Arctic Circle. We investigated the cold-hardiness of L. vivipara from France which routinely encounter subzero temperatures within their shallow hibernation burrows. In the laboratory, cold-acclimated lizards exposed to subfreezing temperatures as low as -3.5°C could remain unfrozen (supercooled) for at least 3 weeks so long as their microenvironment was dry. In contrast, specimens cooled in contact with ambient ice crystals began to freeze within several hours. However, such susceptibility to inoculative freezing was not necessarily deleterious since L. vivipara readily tolerated the freezing of its tissues, with body surface temperatures as low as -3.0°C during trials lasting up to 3 days. Freezing survival was promoted by relatively low post-nucleation cooling rates (0.1°C·h^-1) and apparently was associated with an accumulation of the putative cryoprotectant, glucose. The cold-hardiness strategy of L. vivipara may depend on both supercooling and freeze tolerance capacities, since this combination would afford the greatest likelihood of surviving winter in its dynamic thermal and hydric microenvironment.Abbreviations bm body mass - SVL snout-vent length - T_b body surface temperature - T _c crystallization temperature     

Assessing the effects of low temperature on the establishment potential in Britain of the non-native biological control agent Eretmocerus eremicus

Abstract. Eretmocerus eremicus is a parasitoid wasp that is not native to Britain. It is a biological control agent of glasshouse whitefly and has recently been released under licence in Britain for the first time. This study assessed the effect of low temperature on the outdoor establishment potential of E. eremicus in Britain. The developmental threshold calculated by three linear methods was between 6.1° and 11.6 °C, with a degree‐day requirement per generation between 256.3 and 366.8° day^?1. The supercooling points of non‐acclimated and acclimated larvae were similar (approximately ?25 °C). Non‐acclimated and acclimated larvae were subject to considerable pre‐freeze mortality, with lethal temperature (LTemp₅₀₎ values of ?16.3 and ?21.3 °C, respectively. Lethal time experiments indicated a similar lack of cold tolerance with 50% mortality of both non‐acclimated and acclimated larvae after 7 days at ?5 °C, 10 days at 0 °C and 13 days at 5 °C. Field trials showed that neither non‐acclimated nor acclimated larvae survived longer than 1 month when exposed to naturally fluctuating winter temperatures. These results suggest that releasing E. eremicus into British greenhouses would pose minimal risk because typical British winter temperatures would be an effective barrier against establishment in the wild.     

Mechanisms underlying insect freeze tolerance

Freeze tolerance – the ability to survive internal ice formation – has evolved repeatedly in insects, facilitating survival in environments with low temperatures and/or high risk of freezing. Surviving internal ice formation poses several challenges because freezing can cause cellular dehydration and mechanical damage, and restricts the opportunity to metabolise and respond to environmental challenges. While freeze‐tolerant insects accumulate many potentially protective molecules, there is no apparent ‘magic bullet’ – a molecule or class of molecules that appears to be necessary or sufficient to support this cold‐tolerance strategy. In addition, the mechanisms underlying freeze tolerance have been minimally explored. Herein, we frame freeze tolerance as the ability to survive a process: freeze‐tolerant insects must withstand the challenges associated with cooling (low temperatures), freezing (internal ice formation), and thawing. To do so, we hypothesise that freeze‐tolerant insects control the quality and quantity of ice, prevent or repair damage to cells and macromolecules, manage biochemical processes while frozen/thawing, and restore physiological processes post‐thaw. Many of the molecules that can facilitate freeze tolerance are also accumulated by other cold‐ and desiccation‐tolerant insects. We suggest that, when freezing offered a physiological advantage, freeze tolerance evolved in insects that were already adapted to low temperatures or desiccation, or in insects that could withstand small amounts of internal ice formation. Although freeze tolerance is a complex cold‐tolerance strategy that has evolved multiple times, we suggest that a process‐focused approach (in combination with appropriate techniques and model organisms) will facilitate hypothesis‐driven research to understand better how insects survive internal ice formation.     

Insect antifreezes and ice-nucleating agents

Cold-tolerant, freeze-susceptible insects (those which die if frozen) survive subzero temperatures by proliferating antifreeze solutes which lower the freezing and supercooling points of their body fluids. These antifreezes are of two basic types. Lowmolecular-weight polyhydroxy alcohols and sugars depress the freezing point of water on a colligative basis, although at higher concentrations these solutes may deviate from linearity. Recent studies have shown that these solutes lower the supercooling point of aqueous solutions approximately two times more than they depress the freezing point. Consequently, if a freeze-susceptible insect accumulates sufficient glycerol to lower the freezing point by 5 °C, then the glycerol should depress the insect's supercooling point by 10 °C.Some cold-tolerant, freeze-susceptible insects produce proteins which produce a thermal hysteresis (a difference between the freezing and melting point) of several degrees in the body fluids. These thermal hysteresis proteins (THPs) are similar to the antifreeze proteins and glycoproteins of polar marine teleost fishes. The THPs lower the freezing, and presumably the supercooling, point by a noncolligative mechanism. Consequently, the insect can build up these antifreezes, and thereby gain protection from freezing, without the disruptive increases in osmotic pressure which accompany the accumulation of polyols or sugars. Therefore the THPs can be more easily accumulated and maintained during warm periods in anticipation of subzero temperatures. It is not surprising then that photoperiod, as well as temperature, is a critical environmental cue in the control of THP levels in insects.Some species of freeze-tolerant insects also produce THPs. This appears somewhat odd, since most freeze-tolerant insects produce ice nucleators which function to inhibit supercooling and it is therefore not clear why such an insect would produce antifreeze proteins. It is possible that the THPs have an alternate function in these species. However, it also appears that the THPs function as antifreezes during those periods of the year when these insects are not freeze tolerant (i.e., early autumn and spring) but when subzero temperatures could occur. In addition, at least one freeze-tolerant insect which produces THPs, Dendroides canadensis, typically loses freeze tolerance during midwinter thaws and then regains tolerance. The THPs could be important during those periods when Dendroides loses freeze tolerance by making the insect less susceptible to sudden temperature decreases.Comparatively little is known of the biochemistry of insect THPs. However, comparisons of those few insect THPs which have been purified with the THPs of fishes show some interesting differences. The insect THPs lack the large alanine component commonly found in the fish THPs. In addition, the insect THPs generally contain greater percentages of hydrophilic amino acids than do those of the fish. Perhaps the most interesting insect THPs are those from Tenebrio molitor which have an extremely large cysteine component (28% in one THP). Studies on the primary and higher-order structure of the insect THPs need to be carried out so that more critical comparisons with the fish THPs can be made. This may provide important insights into the mechanisms of freezing point and supercooling point depression exhibited by these molecules. In addition, comparative studies of the freezing and supercooling point depressing activities of the various THPs, in relation to their structures, should prove most interesting.It has become increasingly apparent over the last few years that most freeze-tolerant insects, unlike freeze-susceptible species, inhibit supercooling by accumulating ice-nucleating agents in their hemolymph. These nucleators function to ensure that ice formation occurs in the extracellular fluid at fairly high temperatures, thereby minimizing the possibility of formation of lethal intracellular ice. Little is known of the nature of the insect ice-nucleating agents. Those few which have been studied are heat sensitive and nondialyzable and are inactivated by proteolytic enzymes, thus indicating that they are proteinaceous. Studies on the structure-function relationships of these unique molecules should be done.     

Intracellular ice formation in insects: Unresolved after 50 years?

Many insects survive internal ice formation. The general model of freeze tolerance is of extracellular ice formation (EIF) whereby ice formation in the haemocoel leads to osmotic dehydration of the cells, whose contents remain unfrozen. However, survivable intracellular ice formation (IIF) has been reported in fat body and certain other cells of some insects. Although the cellular location of ice has been determined only in vitro, several lines of evidence suggest that IIF occurs in vivo. Both cell-to-cell propagation of intracellular ice and inoculation from the haemocoel may be important, although the route of ice into the cell is unclear. It is unclear why some cells survive IIF and others do not, but it is suggested that the shape, size, and low water content of fat body cells may predispose them towards surviving ice formation. We speculate that IIF may reduce water loss in some freeze tolerant species, but there are too few data to build a strong conceptual model of the advantages of IIF. We suggest that new developments in microscopy and other forms of imaging may allow investigation of the cellular location of ice in freeze tolerant insects in vivo.     

Modelling exposure to selected temperature during pregnancy: the limitations of squamate viviparity in a cool-climate environment

Behavioural thermoregulation is important for the success of cool‐climate lizards, and a basis of the cold‐climate hypothesis for the evolution of viviparity in squamate reptiles. The temperature (T_sel) selected by pregnant females in a thermal gradient is considered to be optimal for embryonic development; however, exposure to T_sel throughout pregnancy has been difficult to estimate in small‐bodied lizards as temperature‐sensitive telemetry is impractical. In addition, the value of maternal thermophily during pregnancy is controversial: some studies have shown elevated T_sel, whereas others have found lowered T_sel or no change during pregnancy. We estimated indirectly the overall exposure to T_sel during the 4–5 months of pregnancy of the cool‐climate, sub‐alpine species Oligosoma maccanni (McCann's skink, 3–6 g) from southern New Zealand. The thermal environment available to skinks was modelled using temperature loggers inside validated copper models in basking and retreat sites. Pregnant skinks were able to achieve mean T_sel (28.9 °C) in the field very infrequently (4–15% of each month during the final 4 months of pregnancy). In field thermoregulatory studies, pregnant females did not bask more frequently and did not show altered field body temperature compared with non‐pregnant adults, suggesting that all skinks (whether pregnant or not) thermoregulate maximally whenever conditions allow. Further research on cool‐climate lizards should address the significance for offspring phenotypes of low and variable exposure to T_sel during pregnancy, as well as the significance of temperatures for embryos in maternal bodies (viviparity) versus nest sites (oviparity) arising from differences in maternal body size. © 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 96 , 541–552.     

Host‐mediated shift in the cold tolerance of an invasive insect

While many insects cannot survive the formation of ice within their bodies, a few species can. On the evolutionary continuum from freeze‐intolerant (i.e., freeze‐avoidant) to freeze‐tolerant insects, intermediates likely exist that can withstand some ice formation, but not enough to be considered fully freeze tolerant. Theory suggests that freeze tolerance should be favored over freeze avoidance among individuals that have low relative fitness before exposure to cold. For phytophagous insects, numerous studies have shown that host (or nutrition) can affect fitness and cold‐tolerance strategy, respectively, but no research has investigated whether changes in fitness caused by different hosts of polyphagous species could lead to systematic changes in cold‐tolerance strategy. We tested this relationship with the invasive, polyphagous moth, Epiphyas postvittana (Walker). Host affected components of fitness, such as larval survivorship rates, pupal mass, and immature developmental times. Host species also caused a dramatic change in survival of late‐instar larvae after the onset of freezing—from less than 8% to nearly 80%. The degree of survival after the onset of freezing was inversely correlated with components of fitness in the absence of cold exposure. Our research is the first empirical evidence of an evolutionary mechanism that may drive changes in cold‐tolerance strategies. Additionally, characterizing the effects of host plants on insect cold tolerance will enhance forecasts of invasive species dynamics, especially under climate change.     

Overwintering adaptations of the stag beetle,Ceruchus piceus: removal of ice nucleators in the winter to promote supercooling

Summary Overwintering larvae and adults of the stag beetle,Ceruchus piceus, are freeze sensitive (i.e. cannot survive internal freezing). The most commonly described cold adaptation of freeze susceptible insects involves the production of antifreezes to promote supercooling, butCeruchus piceus larvae produced only low levels of antifreezes in the winter. However, by removing ice nucleators from the gut and hemolymph in the winter the larvae were able to depress their supercooling points from approximately –7°C in the summer to near –25°C in mid-winter. The ice nucleators present in the non-winter hemolymph were identified as lipoproteins. One of these lipoproteins with ice nucleator activity was purified using flotation ultracentrifugation and anion exchange (DEAE-Sephadex) chromatography.Removal of ice nucleators to promote supercooling in winter may be energetically preferable to costly production and maintenance of high, of-ten molar, concentrations of antifreeze. Obviously the ice nucleator must normally perform a function which the insect can spare over the winter. Hemolymph lipoproteins, which generally function in lipid transport, may fit this criterion during the winter period of reduced metabolic activity.Abbreviations LP I very low density lipoprotein - LP II low density lipoprotein - PAGE polyacrylamide gel electrophoresis - SCP supercooling point     

Adaptations to cold in Canadian arctic insects

The survival of insects that inhabit Canadian arctic regions depends on a number of factors which have important ecological, behavioral, physiological, and biochemical components. The ability to withstand low winter temperatures is one of the most conspicuous adaptations of northern insects and the one most closely studied in the laboratory. Most species studied so far conform to one or other of the two major overwintering strategies, namely, frost susceptibility, the ability to avoid freezing by supercooling to a considerable degree, or frost tolerance, the survival of actual ice formation within the body. The Arctic beetle, Pytho americanus Kirby, is frost tolerant in both larval and adult stages, a situation which would be congruous with its northern distribution and allow it to spread its life cycle over a number of growing seasons. The main biochemical correlates during the cold-hardening process in this species are increasing glycerol and decreasing glycogen concentrations. In addition to its normally assumed roles in cryoprotection there is evidence to suggest that glycerol may further serve to minimize dehydration in the overwintering insect by increasing the level of bound water. P. americanus larvae and adults have narrow supercooling ranges and maintain their supercooling points in the region of ?4 to ?8 °C. It is hypothesized that these elevated supercooling points are a result of the presence in the hemolymph of nucleating agents which ensure ice formation at high subzero temperatures.Low temperature tolerance strategies of some other arctic and alpine species have been examined and compared with those of relatives from more southerly latitudes. P. americanus has been collected in the Canadian Rockies at elevations of over 6000′, and its frost-tolerant attributes are identical to those of the population collected in the Arctic. A closely related species, P. deplanatus, from the Rockies, however, although it too exhibits frost tolerance in the larval stage, differs markedly from P. americanus in its ability to depress its supercooling range to ?54 °C. It appears that P. deplanatus does not have the ability to synthesize ice-nucleating agents and, therefore, can overwinter in a supercooled condition. Two congeneric species of willow leaf gall sawflies (Pontania spp.), one from Tuktoyaktuk, N.W.T., and the other from southern Vancouver Island have also been compared and contrasted. Pontania sp. on Salix glauca (Tuk., ca. 70 °N) is frost tolerant in its larval stage, has relatively high supercooling points (ca. ?9.0 °C), but does not accumulate glycerol. Pontania sp. from Salix lasiandra (Victoria, ca. 48 °N) has almost identical overwintering properties, indicating the close phylogenetic affinities of cold tolerance in this genus rather than independent adaptation to widely different climatic conditions. Some of the lowest supercooling points ever recorded are from willow stem gall forming insects. Rhabdophaga sp. (Cecidomyiidae) forms potato galls on the stems of Salix lanata in the Inuvik area, N.W.T. After low temperature acclimation, supercooling points down to ?66 °C have been recorded from individual larvae. This is a record, and it indicates that we may be dealing with a system in which most water is in a metabolically bound state. Glycerol levels reach 20% of the fresh body weight during this period. Diastrophus kincaidii Cynipidae) forms stem galls on Thimble Berry (Rubus parviflorus) on southern Vancouver Island. Both of the forementioned species overwinter as larvae in their galls and are, therefore, exposed to ambient air temperatures. A more benign winter climate on Vancouver Island is reflected in the fact that D. kincaidii has supercooling points only in the ?30 to ?33 °C range at the peak of low temperature acclimation, and glycerol levels just below 4% of fresh body weight. Both species are frost susceptible and depend on their supercooling abilities to survive low winter temperatures.