Thermal-hysteresis-factors in overwintering insects

The haemolymph of 9 species of insects found overwintering under the bark of dead trees in northern Indiana contained factors which produced a thermal hysteresis (a difference between the freezing and melting points) of several degrees. These thermal-hysteresis-factors were common in overwintering beetles, but rare in non-Coleoptera, and are similar to the macromolecular antifreezes of polar marine teleost fishes. The factors were found in both freeze-susceptible and freeze-tolerant species, and their function in freeze-susceptible insects appears to be to depress the supercooling points and therefore the lower lethal temperatures of the insects. However, the function of the factors in freeze-tolerant species is not clear. Possible functions are discussed.     

The inhibition of ice nucleators by insect antifreeze proteins is enhanced by glycerol and citrate

Antifreeze proteins depress the freezing point of water while not affecting the melting point, producing a characteristic difference in freezing and melting points termed thermal hysteresis. Larvae of the beetle Dendroides canadensis accumulate potent antifreeze proteins (DAFPs) in their hemolymph and gut, but to achieve high levels of thermal hysteresis requires enhancers, such as glycerol. DAFPs have previously been shown to inhibit the activity of bacterial and hemolymph protein ice nucleators, however, the effect was not large and therefore the effectiveness of the DAFPs in promoting supercooling of the larvae in winter was doubtful. However, this study demonstrates that DAFPs, in combination with the thermal hysteresis enhancers glycerol (1 M) or citrate (0.5 M), eliminated the activity of hemolymph protein ice nucleators and Pseudomonas syringae ice-nucleating active bacteria, and lowered the supercooling points (nucleation temperatures) of aqueous solutions containing these ice nucleators to those of water or buffer alone. This shows that the DAFPs, along with glycerol, play a critical role in promoting hemolymph supercooling in overwintering D. canadensis. Also, DAFPs in combination with enhancers may be useful in applications which require inhibition of ice nucleators.     

Winter survival of an adult bark beetle Ips acuminatus Gyll

Freezing-susceptible adult Ips acuminatus hibernate underneath bark of Scots pine. The beetles lower their supercooling points from ?20 to ?34°C due to accumulation of low molecular weight antifreezes. The capability of specimens to supercool to about ?20°C in the absence of cryoprotective solutes during winter, seemed to be at least partially attributable to the presence of a thermal hysteresis factor at 3–4°C.Using a GC-MS-COM technique, a unique combination of accumulated solutes present only in specimens demonstrating supercooling points below ?20°C was identified as ethylene glycol, mannitol, sorbitol and dulcitol. Not previously found in nature, ethylene glycol was the major solute (90%) synthesized at sub-zero temperatures. Exposure to ?10°C was an effective cue to accumulation of ethylene glycol and nearly 5 times as effective in promoting sorbitol synthesis than was ?5°C. When low molecular weight substances were lost at high temperatures, they were not re-synthesized in beetles re-exposed to sub-zero temperature. The supercooling point was closely related to both the concentration of ethylene glycol and to the haemolymph melting point. Attempts to correlate changes in sorbitol concentrations to changes in supercooling points were not conclusive.Proliferation of thermal hysteresis was observed in the beginning of November. A melting-hysteresis freezing point differential of about 3.6°C was demonstrated in the haemolymph of beetles during December. No thermal hysteresis was demonstrated in the haemolymph of positive phototactic beetles or in the outdoor beetles in May. The combination of high temperature and long photoperiod appeared to be a more effective cue to the final loss of thermal hysteresis than was high temperature alone.     

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.     

Effects of diet and water supply on energy intake and water loss in a mygalomorph spider in a fluctuating environment of the central Andes

The metabolic and water evaporation strategies in spiders may be part of a set of physiological adaptations to tolerate low or unpredictable food availability, buffering spiders against environmental fluctuations such as those of the high mountains of the central Andes.The aim of this study is to analyze experimentally the variations in metabolic rate and the rate of evaporative water with food and/or water restriction in a high mountain mygalomorph spider population (Paraphysa sp.).We found that the low metabolism of this spider was not affected by water restriction, but its metabolism was depressed after 3 weeks of food deprivation. The spider did not show seasonal metabolic changes but it presented seasonal changes in the rate of evaporative water loss at high temperatures.Females with egg sacs reduced their metabolic rate and evaporative water at high temperatures.These findings constitute a set of possible adaptations to a highly fluctuating Mediterranean environment, which is completely covered with snow for many months and then progresses rapidly to a very dry climate with high temperatures.     

Foraging at the thermal limit: burrowing spiders (Seothyra,Eresidae) in the Namib desert dunes

Summary In the Namib Desert dunes, the web of Seothyra sp. (Eresidae) comprises sticky silk lining the edges of a horizontal mat on the sand surface. The spider sits in a silk-lined burrow attached to the mat. Arthropods become entangled in the sticky silk of the mat and are attacked and pulled into the burrow by the spider. We investigated the influence of sand surface temperature on the activity of spiders during the summer. We determined the range of thermal conditions encountered by spiders, their temperature tolerance and the influence of temperature on foraging activity and prey handling behavior. The environmental temperatures available to Seothyra vary from 17–33° C at the coolest time of day to 33–73° C at the hottest. When prevented from retreating into burrows, spiders showed signs of thermal stress at about 49° C, whereas unrestrained spiders continued to forage at web temperatures above 65° C by moving between the hot surface mat and the cooler burrow. Spiders responded quicker to prey stimuli during the hot hours of the day and completed prey capture sequences in significantly less time at surface temperatures above 49° C than below. Furthermore, captured arthropods succumbed more quickly at high surface temperatures. Our study supports the hypothesis that web design and thermoregulatory behaviors enable Seothyra to hunt under extreme thermal conditions.     

Cold hardiness in the scorpion, Paruroctonus aquilonalis.

Lower lethal temperatures represented by supercooling points were measured for the scorpion Paruroctonus aquilonalis (Stahnke) following exposure to laboratory regimes of photoperiod and temperature, as well as to ambient conditions. Analyses for the presence of glycerol and sorbitol in hemolymph, and of body water content were also performed.Laboratory results indicated that neither decreasing photoperiod nor decreasing temperature influenced supercooling point in a meaningful way. However, supercooling points did decrease significantly during the fall and winter among scorpions maintained outdoors, indicating development of cold hardiness. Neither glycerol nor sorbitol were detected, nor were the slight seasonal decreases found in water content considered sufficient to influence supercooling point.Ability to avoid winter freezing in P. aquilonalis appears primarily conferred by substantial seasonal depression of the supercooling point and is temporally associated with cessation of feeding in the fall. Supercooling responses were compared with those of another scorpion, Diplocentrus spitzeri, and differences tentatively were explained in terms of feeding activity.     

Temporal activity of spiders and earwigs during winter in apple trees under a Mediterranean climate

It is well known that spiders are present in high numbers in orchards and may contribute to biocontrol. Some recent studies in central Europe further showed that some spiders are active year-round and consume pests even in winter. Using cardboard traps laid every two weeks, we carried out a survey to determine which spider and earwig species are active from September to May in an experimental, pesticide-free, apple orchard under a Mediterranean climate. We observed that spider activity was never completely absent. The structure of the spider communities showed a marked seasonality in three periods (so-called ‘autumn’, ‘winter’ and ‘spring’). Only two spider genera, Philodromus and Trachelas, were highly active in winter (percentage of catches during this season above 40%) and six others (Lathys, Clubiona, Gnaphosa, Theridion, Phrurolithus) had moderate activity (between 20 and 40%). The two earwig species had different patterns of winter activity with Forficula auricularia almost absent whereas F. pubescens was moderately active on trees. Spider community abundance, diversity and evenness significantly decreased between autumn and winter and remained low in the following spring probably because the attractiveness of the traps is much lower at this time of year due to mild temperatures and the presence of leaves on the trees. Winter-active spiders could contribute to pest biocontrol during the cold season and we advocate that the use of broad-spectrum pesticides at the end of winter, as classically applied in orchards, may be counter-productive for pest control.     

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.     

Comparative analysis of overwintering physiology in nine species of semi‐aquatic bugs (Heteroptera: Gerromorpha)

In semi‐aquatic bugs (Heteroptera: Gerromorpha), the strategies of overwintering in a cryothermic state (i.e. at body temperatures below the equilibrium freezing point) remain largely unexplored. The present study provides an analysis of the ecophysiological aspects of overwintering in nine gerromorphan species. All nine species avoid ice formation by means of a more or less extensive supercooling of their body fluids. There is a tight correlation between the supercooling point (SCP) and the lower lethal temperature. Different species use different physiological adjustments to increase the likelihood of survival in a supercooled state. These include stabilization of the supercooled state by active antifreeze factors that cause thermal hysteresis between equilibrium melting and freezing points, the accumulation of low‐molecular weight sugars and polyols with putative cryoprotective functions, or by having a relatively high body fluid osmolality, combined with a low level of hydration. The majority of species under study overwinter only as adults, whereas Velia caprai Tamanini can overwinter either as an adult or in the egg stage. The supercooling capacity of V. caprai adults is insufficient to prevent the risk of lethal freezing. The adults therefore survive only opportunistically in suitable microhabitats, and/or during mild winters. The survival of V. caprai in winter is assured by extensive supercooling and having overwintering eggs that are highly cold tolerant.     

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.     

The thermal biology of Geolycosa godeffroyi and other burrow inhabiting Lycosidae (Araneae) in Australia

Summary Using both field and laboratory data a detailed examination of behavioural thermoregulation is presented for Geolycosa godeffroyi (Koch), a burrow inhabiting Australian wolf spider; comparative data are given for Schizocosa leuckartii (Thorell), Venatrix fuscus (Hogg), Lycosa speciosa Koch, and two undescribed species of Lycosa. Egg sac and spider body temperatures were recorded either by implanted thermocouples or biotelemetry. The burrows permitted the spiders access to a wide range of temperature throughout the day but the spiders maintained temperatures above the upper limit of this range except in the middle of the day in summer. In winter the burrows protected the spiders from extreme cold. G. godeffroyi thermoregulated throughout the year and could achieve temperatures of 38°C when the global radiation exceeded about 260 mW cm^-2 d^-1. The activity regimes deduced from temperature recording were in accord with those derived using field actographs and photographic recording. The spiders thermoregulated in a manner similar to that used by behaviourally thermoregulating lizards and did so whenever conditions were suitable. They maintained temperatures between 32 and 36°C but the preferred temperature was reduced by lack of food or water, in egg sacs or females carrying egg sacs and in mature males to about 30°C. The data do not support the concept of a refractory zone but suggest two or more set points required for specific physiological or ecological purposes.     

Cold tolerance of alpine,Arctic, and Antarctic Collembola and mites

Because of their dominant role in the fauna of alpine, Arctic and Antarctic locations Collembola and mites are of particular interest regarding adaptations to low temperatures. No freezing-tolerant species have been found in these groups of terrestrial arthropods, and it appears that all species depend entirely on supercooling to survive the lower temperatures of their habitats. While summer animals have high supercooling points, an increase in supercooling ability occurs during autumn and early winter, and can be explained as a two-step process. Initially gut content has to be eliminated to avoid heterogeneous nucleation at high subzero temperatures due to foreign nucleating agents. Second, supercooling is further enhanced through accumulation of glycerol or other lowmolecular cryoprotective substances. Further studies are needed on the ability of such animals to avoid inoculative freezing in their microhabitats.     

Interspecific predation as a mortality factor among overwintering spiders

Summary Results from field experiments indicate that predation occurs among spruce-living spiders during winter in SW Sweden. Field observations of natural activity showed that Philodromus spp and Pityohyphantes phrygianus together make up 80% of the spiders active on spruce in winter. They are therefore potential predators on other overwintering spiders. Laboratory experiments were performed at +4° C to assess the importance of such predation between spiders. Small spiders (length <2.5 mm) had a mean winter mortality of 58% when kept together with large spiders (2.5 mm) which had a mean mortality of 3% only. Among the small spiders the Erigninae spp seemed to be more vulnerable to predation than other taxonomic groups. Predation also occurred when large P. phrygianus were kept together, but such predation caused mortality of less importance to the spider populations than the mortality among small spiders. Differences in spider density and food availability did not change this pattern. Considerable weight increase occurred in subadult P. phrygianus when fed during winter. This suggests that winter foraging specimens increase their fitness. Interspecific predation among spiders is suggested to be an important mortality factor in natural populations at high spider densities in November and December, when the ambient temperature often is above 0° C and when the density of large spiders is not yet substantially reduced by bird predation.     

Physiological responses to supercooling and hypoxia in the hatchling painted turtle, Chrysemys picta

We investigated physiological responses to supercooling in hatchling painted turtles (Chrysemys picta) which remain in their natal nests over winter and therefore may become exposed to subzero temperatures. These turtles are freeze tolerant but also must rely on supercooling to survive exposure to the lower temperatures occurring in nests during winter. We compared whole-body concentrations of lactate, glucose, glycerol, and ATP in turtles chilled at 0 degrees C, -4 degrees C, or -6 degrees C for 5 days, or at 6 degrees C for 19 days. In a companion experiment, we measured metabolite concentrations in turtles exposed to a hypoxic environment for 1 day, 4 days, or 8 days. Supercooling and hypoxia exposure were both associated with an increase in concentrations of lactate and glucose and a decrease in glycerol concentrations (albeit no change in the ATP pool), suggesting that supercooling induces functional hypoxia. We conclude that hypoxia tolerance may be an important pre-adaptation for surviving exposure to subzero temperatures in hatchling C. picta.     

Effect of thermal acclimation on preferred temperatures in two mygalomorph spiders inhabiting contrasting habitats

Variations in the preferred temperatures during the rest periods of Grammostola rosea Walckenaer and Paraphysa parvula Pocock, two mygalomorph spiders occupying different habitats in central Chile, are analyzed. The former inhabits arid and semi‐arid lowland near plant communities, composed of shrubs (evergreens with small leathery leaves) and small trees; the latter is found in the central mountains of the Chilean Andes, above 2000 m.a.s.l. The preferred temperatures of these spiders at different times of day and exposure to cold (15 °C) and warm (25 °C) acclimation temperatures are compared. Body mass does not affect the preferred temperature of the larger spider G. rosea, although P. parvula, a spider with half of the body mass of G. rosea, shows a decrease in preferred temperature with body mass. This can be explained by a higher plasticity and thermal sensitivity of the smaller species as result of increased surface : volume ratio. The preferred temperature increases with the hour of the day under both acclimation conditions in P. parvula and in cold‐acclimated G. rosea, which is likely associated with crepuscular and nocturnal behaviour in both species. Grammostola rosea shows temperature preferences lower than those of P. parvula under both acclimation conditions. The increase of the acclimation temperature from 15 to 25 °C results in an increment of 2–3 °C in the preferred temperature of P. parvula but only 0.2 °C in that of G. rosea. Two contrasting lifestyle strategies are found: a small mygalomorph spider with phenotypic plasticity and adaptation to the fluctuating environment of high altitude, and a large mygalomorph spider with higher thermal inertia adapted to the more stable environment of lowlands.     

Physiology of cold hardiness in cocoons of five earthworm taxa (Lumbricidae: Oligochaeta)

Earthworm cocoons are mostly found in the uppermost soil layers and are therefore often exposed to low temperatures during winter. In the present study, cocoons of five taxa of earthworms were investigated for their tolerance to freezing, melting points of cocoon fluids and dehydration of cocoons when exposed to a frozen environment. Embryos of the taxa investigated were freeze intolerant. The melting points of fully hydrated cocoon fluids were high (above –0.3°C) and thermal hysteresis factors were absent. Exposure to a frozen environment caused the cocoons to dehydrate drastically and dehydrated cocoons showed significantly lower super-cooling points than fully hydrated cocoons, reducing the risk of freezing for dehydrated cocoons. It is proposed therefore that the cold-hardiness strategy of the earthworm cocoons is based on dehydration upon exposure to subzero temperatures in the frozen environment. Cocoons of three surface-dwelling taxa, Dendrobaena octaedra, Dendrodrilus rubidus tenuis and Dendrodrilus rubidus norvegicus had lower supercooling points and survived frost exposure better than cocoons of two deeper-dwelling taxa, Aporrectodea caliginosa and Allolobophora chlorotica. One of the investigated taxa, D. r. norvegicus, was collected from a cold alpine habitat. However, it was not more cold hardy than the closely related D. r. tenuis collected from a lowland temperate habitat. D. octaedra was the most cold hardy taxon, its cocoons being able to withstand –8°C for 3 months and –13.5°C for 2 weeks in frozen soil.Abbreviations dw dry weight - fw fresh weight - SCP supercooling point     

Factors affecting low temperature survival ofCinara pilicornis eggs on Sitka spruce

OviparousC. pilicornis deposited eggs in October and early November, the dry weight, but not the fresh weight, decreasing during winter. Diapause terminated in early March, eggs hatched in early May. Mean egg supercooling points varied during the winter, the variation being related to the temperature in the period preceding sample collection. Both glycerol and mannitol were isolated from the eggs. Mannitol levels decreased to zero by mid-winter, levels increasing after diapause termination. Glycerol levels increased up to diapause termination and thereafter decreased, none being present just prior to egg hatch. Neither glycerol nor mannitol were related to supercoolingability, the variation being related to low temperatures which possibly induced changes in the levels of an unknown factor.     

Experimental studies on the cold tolerance of Alaskozetes antarcticus

The cold tolerance mechanism of the Antarctic terrestrial mite Alaskozetes antarcticus (Michael) was investigated in cultured animals. Freezing is fatal in this species and winter survival occurs by means of supercooling, which is enhanced by the presence of glycerol in the body. There is an inverse, linear relationship between the concentration of glycerol and the supercooling point, which may be as low as ?30°C. Feeding detracts from supercooling ability by providing ice nucleators in the gut which initiate freezing at relatively high sub-zero temperatures. Experiments on the effects of various environmental factors showed that low temperature acclimation gave rise to increased glycerol concentrations and suppressed feeding, while desiccation also stimulated glycerol production. Photoperiod had no effect on cold tolerance in this species. The juvenile instars of A. antarcticus were found to possess a greater degree of low temperature tolerance than adults.     

Divergent mechanisms of frost-hardiness in two populations of the gall fly, Eurosta solidaginsis

Two populations of the gall fly Eurosta solidaginsis utilize different strategies to endure seasonal exposure to temperatures below freezing. Both populations are freezing tolerant. In north temperate populations, supercooling points rise from ?10.2°C to ?6.2°C following exposures to temperatures below freezing. This level is maintained throughout winter and ensures frequent and prolonged periods of tissue freezing. South temperate populations depress the supercooling point to ?14.2°C during autumn and early winter, and this depression precludes extracellular ice formation during periods of supra-optimal temperature fluctuations. During mid-winter, supercooling points rise to the same level as in northern groups.Both populations accumulate three principal cryoprotective agents following first frost exposures (glycerol, sorbitol and trehalose). Cryoprotectants levels do not peak in northern populations until 4–6 weeks after first frost. In southern populations the accumulation profile is characterized by a high initial rate of synthesis, a protective overshoot and pronounced seasonal fluctuations. The relative survival advantages of each strategy are discussed.