Metabolic and molecular stress responses of gilthead seam bream Sparus aurata during exposure to low ambient temperature: an analysis of mechanisms underlying the winter syndrome

The winter syndrome in the gilthead sea bream Sparus aurata indicates that the species is exposed to critically low temperatures in Mediterranean aquaculture in winter. The present study of metabolic patterns and molecular stress responses during cold exposure was carried out to investigate this “disease”, in light of the recent concept of oxygen and capacity limited thermal tolerance. The metabolic profile of fuel oxidation was examined by determining the activities of the enzymes hexokinase (HK), aldolase (Ald), pyruvate kinase (PK), l-lactate dehydrogenase (l-LDH), citrate synthase (CS), malate dehydrogenase (MDH) and 3-hydroxyacyl CoA dehydrogenase (HOAD) in heart, red and white muscle after exposure to temperatures of 10, 14 and 18°C. Especially, the increase in LDH activity combined with the accumulation of l-lactate in tissues indicates that temperatures below 14°C are critical for Sparus aurata and stimulate the anaerobic component of metabolism. Increase in the activity of HOAD suggests that oxidation of free fatty acids might contribute to ATP turnover at low temperatures. The expression of Hsp70 and Hsp90 in all tissues examined revealed a cellular stress response during cooling below 18°C. In the light of winter temperatures in S. aurata cultures around 10°C, our data suggest that the fish are exposed to stressful conditions at the low end of their thermal tolerance window. These conditions likely impair the aerobic capacity of the fish, compromise the rates of growth and reproduction and may contribute to elicit pathological conditions.     

Accumulation of lactate by supercooled hatchlings of the painted turtle (Chrysemys picta): implications for overwinter survival

Hatchlings of the North American painted turtle (Chrysemys picta) typically spend their first winter of life inside the shallow, subterranean nest where they completed embryogenesis the preceding summer. Neonates at northern localities consequently may be exposed during winter to subzero temperatures and frozen soil. Hatchlings apparently survive exposure to such conditions by supercooling, but the physiological consequences of this adaptive strategy have not been examined. We measured lactate in hatchling painted turtles after exposure to each of three temperatures (0 °C, −4 °C, and −8 °C) for three time periods (5 days, 15 days, and 25 days) to determine the extent to which overwintering hatchlings might rely on anaerobic metabolism to regenerate ATP. Whole-body lactate increased with increasing duration of exposure and decreasing temperature, and the highest levels were associated with the group that experienced the highest mortality. These results indicate that animals may develop a considerable lactic acidosis during a winter in which temperatures fall below 0 °C for weeks or months and that accumulation of lactate may contribute to mortality of overwintering animals. Accepted: 20 October 1999     

Cold tolerance of hatched and unhatched second stage juveniles of the potato cyst-nematode Globodera rostochiensis

Perry R. N. and Wharton D. A. 1985. Cold tolerance of hatched and unhatched second stage juveniles of the potato cyst-nematode, Globodera rostochiensis. International Journal for Parasitology15: 441–445. Hatched second-stage juveniles of Globodera rostochiensis can survive sub-zero temperatures by supercooling when not in contact with water. When frozen in water free juveniles cannot survive ice seeding across the cuticle and concomitant freezing of their body contents. Unhatched juveniles can survive in water, probably because the egg-shell protects the juvenile from ice seeding across from the medium; in this state juveniles survive by supercooling.     

Chilling slows anaerobic metabolism to improve anoxia tolerance of insects

Background

Insects are renowned for their ability to survive anoxia. Anoxia tolerance may be enhanced during chilling through metabolic suppression.

Aims

Here, the metabolomic response of insects to anoxia, both with and without chilling, for different durations (12–36 h) was examined to assess the potential cross-tolerance mechanisms.

Results

Chilling during anoxia (cold anoxia) significantly improved survival relative to anoxia at warmer temperatures. Reduced intermediate metabolites and increased lactic acid, indicating a switch to anaerobic metabolism, were characteristic of larvae in anoxia.

Conclusions

Anoxia tolerance was correlated survival improvements after cold anoxia were correlated with a reduction in anaerobic metabolism.

     

Overwintering Strategy and Mechanisms of Cold Tolerance in the Codling Moth (Cydia pomonella)

Background

The codling moth (Cydia pomonella) is a major insect pest of apples worldwide. Fully grown last instar larvae overwinter in diapause state. Their overwintering strategies and physiological principles of cold tolerance have been insufficiently studied. No elaborate analysis of overwintering physiology is available for European populations.

Principal Findings

We observed that codling moth larvae of a Central European population prefer to overwinter in the microhabitat of litter layer near the base of trees. Reliance on extensive supercooling, or freeze-avoidance, appears as their major strategy for survival of the winter cold. The supercooling point decreases from approximately −15.3°C during summer to −26.3°C during winter. Seasonal extension of supercooling capacity is assisted by partial dehydration, increasing osmolality of body fluids, and the accumulation of a complex mixture of winter specific metabolites. Glycogen and glutamine reserves are depleted, while fructose, alanine and some other sugars, polyols and free amino acids are accumulated during winter. The concentrations of trehalose and proline remain high and relatively constant throughout the season, and may contribute to the stabilization of proteins and membranes at subzero temperatures. In addition to supercooling, overwintering larvae acquire considerable capacity to survive at subzero temperatures, down to −15°C, even in partially frozen state.

Conclusion

Our detailed laboratory analysis of cold tolerance, and whole-winter survival assays in semi-natural conditions, suggest that the average winter cold does not represent a major threat for codling moth populations. More than 83% of larvae survived over winter in the field and pupated in spring irrespective of the overwintering microhabitat (cold-exposed tree trunk or temperature-buffered litter layer).     

Effects of ice-seeding temperature and intracellular trehalose contents on survival of frozen Saccharomyces cerevisiae cells

Freezing tolerance is an important characteristic for baker’s yeast, Saccharomyces cerevisiae, as it is used to make frozen dough. The ability of yeast cells to survive freezing is thought to depend on various factors. The purpose of this work was to study the viability of yeast cells during the freezing process. We examined factors potentially affecting their survival, including the growth phase, ice-seeding temperature, intracellular trehalose content, freezing period, and duration of supercooling. The results showed that the ice-seeding temperature significantly affected cell viability. In the stationary phase, trehalose accumulation did not affect the viability of yeast cells after brief freezing, although it did significantly affect the viability after prolonged freezing. In the log phase, the ice-seeding temperature was more important for cell survival than the presence of trehalose during prolonged freezing. The importance of increasing the extracellular ice-seeding temperature was verified by comparing frozen yeast survival rates in a freezing test with ice-seeding temperatures of −5 °C and −15 °C. We also found that the cell survival rates began to increase at 3 h of supercooling. The yeast cells may adapt to subzero temperatures and/or acquire tolerance to freezing stress during the supercooling.     

Does cold tolerance plasticity correlate with the thermal environment and metabolic profiles of a parasitoid wasp?

Tolerance of ectotherm species to cold stress is highly plastic according to thermal conditions experienced prior to cold stress. In this study, we investigated how cold tolerance varies with developmental temperature (at 17, 25 and 30 °C) and whether developmental temperature induces different metabolic profiles. Experiments were conducted on the two populations of the parasitoid wasp, Venturia canescens, undergoing contrasting thermal regimes in their respective preferential habitat (thermally variable vs. buffered). We predicted the following: i) development at low temperatures improves the cold tolerance of parasitoid wasps, ii) the shape of the cold tolerance reaction norm differs between the two populations, and iii) these phenotypic variations are correlated with their metabolic profiles. Our results showed that habitat origin and developmental acclimation interact to determine cold tolerance and metabolic profiles of the parasitoid wasps. Cold tolerance was promoted when developmental temperatures declined and population originating from variable habitat presented a higher cold tolerance. Cold tolerance increases through the accumulation of metabolites with an assumed cryoprotective function and the depression of metabolites involved in energy metabolism. Our data provide an original example of how intraspecific cold acclimation variations correlate with metabolic response to developmental temperature.     

Natural freeze-tolerance in hatchling painted turtles?

Hatchlings of the North American painted turtle (Family Emydidae: Chrysemys picta) typically spend their first winter of life inside a shallow, subterranean hibernaculum (the natal nest) where life-threatening conditions of ice and cold commonly occur. Although a popular opinion holds that neonates exploit a tolerance for freezing to survive the rigors of winter, hatchlings are more likely to withstand exposure to ice and cold by avoiding freezing altogether-and to do so without the benefit of an antifreeze. In the interval between hatching by turtles in late summer and the onset of wintery weather in November or December, the integument of the animals becomes highly resistant to the penetration of ice into body compartments from surrounding soil, and the turtles also purge their bodies of catalysts for the formation of ice. These two adjustments, taken together, enable the animals to supercool to temperatures below those that they routinely experience in nature. However, cardiac function in hatchlings is diminished at subzero temperatures, thereby compromising the delivery of oxygen to peripheral tissues and eliciting an increase in reliance by those tissues on anaerobic metabolism for the provision of ATP. The resulting increase in production of lactic acid may disrupt acid/base balance and lead to death even in animals that remain unfrozen. Although an ability to undergo supercooling may be key to survival by overwintering turtles in northerly populations, a similar capacity to resist inoculation and undergo supercooling characterizes animals from a population near the southern limit of distribution, where winters are relatively benign. Thus, the suite of characters enabling hatchlings to withstand exposure to ice and cold may have been acquired prior to the northward dispersal of the species at the end of the Pleistocene, and the characters may not have originated as adaptations specifically to the challenges of winter.     

Pyruvate Formate-Lyase Is Essential for Fumarate-Independent Anaerobic Glycerol Utilization in the Enterococcus faecalis Strain W11

Although anaerobic glycerol metabolism in Enterococcus faecalis requires exogenous fumarate for NADH oxidation, E. faecalis strain W11 can metabolize glycerol in the absence of oxygen without exogenous fumarate. In this study, metabolic end product analyses and reporter assays probing the expression of enzymes involved in pyruvate metabolism were performed to investigate this fumarate-independent anaerobic metabolism of glycerol in W11. Under aerobic conditions, the metabolic end products of W11 cultured with glycerol were similar to those of W11 cultured with glucose. However, when W11 was cultured anaerobically, most of the glucose was converted to l-lactate, but glycerol was converted to ethanol and formate. During anaerobic culture with glycerol, the expression of the l-lactate dehydrogenase and pyruvate dehydrogenase E1αβ genes in W11 was downregulated, whereas the expression of the pyruvate formate-lyase (Pfl) and aldehyde/alcohol dehydrogenase genes was upregulated. These changes in the expression levels caused the change in the composition of end products. A pflB gene disruptant (Δpfl mutant) of W11 could barely utilize glycerol under anaerobic conditions, but the growth of the Δpfl mutant cultured with either glucose or dihydroxyacetone (DHA) under anaerobic conditions was the same as that of W11. Glucose metabolism and DHA generates one NADH molecule per pyruvate molecule, whereas glycerol metabolism in the dehydrogenation pathway generates two NADH molecules per pyruvate molecule. These findings demonstrate that NADH generated from anaerobic glycerol metabolism in the absence of fumarate is oxidized through the Pfl-ethanol fermentation pathway. Thus, Pfl is essential to avoid the accumulation of excess NADH during fumarate-independent anaerobic glycerol metabolism.     

Seasonal changes of free amino acids and thermal hysteresis in overwintering heteropteran insect, Pyrrhocoris apterus

Overwintering adults of Pyrrhocoris apterus do not tolerate freezing of their body fluids and rely on a supercooling strategy and seasonal accumulation of polyols to survive at subzero body temperatures. We sampled the adults monthly in the field during the cold season 2008-2009 and found active thermal hysteresis factors (THFs) in hemolymph of winter-sampled adults. The hysteresis between the equilibrium melting and freezing points ranged from 0.18°C to 0.30°C. No signs of THFs activity were found in the autumn- and spring-sampled insects. The total free amino acid pool almost doubled during winter time. The sum concentrations of 27 free amino acids ranged between 35 and 40mM in whole body water and 40-45mM in hemolymph during December-February. Two amino acids, Pro and α-Ala most significantly contributed to the seasonal increase, while Gln showed the most dramatic seasonal decrease. Moderate levels of amino acid accumulation in overwintering P. apterus suggest that they are by-products of protein degradation and pentose pathway activity during the state of metabolic suppression imposed by diapause and low body temperature. Potential colligative effects of accumulated amino acids, extending the supercooling capacity of overwintering P. apterus, are negligible. Non-colligative effects require further study.     

Formation and Fate of Fermentation Products in Hot Spring Cyanobacterial Mats

The fate of representative fermentation products (acetate, propionate, butyrate, lactate, and ethanol) in hot spring cyanobacterial mats was investigated. The major fate during incubations in the light was photoassimilation by filamentous bacteria resembling Chloroflexus aurantiacus. Some metabolism of all compounds occurred under dark aerobic conditions. Under dark anaerobic conditions, only lactate was oxidized extensively to carbon dioxide. Extended preincubation under dark anaerobic conditions did not enhance anaerobic catabolism of acetate, propionate, or ethanol. Acetogenesis of butyrate was suggested by the hydrogen sensitivity of butyrate conversion to acetate and by the enrichment of butyrate-degrading acetogenic bacteria. Accumulation of fermentation products which were not catabolized under dark anaerobic conditions revealed their importance. Acetate and propionate were the major fermentation products which accumulated in samples collected at temperatures ranging from 50 to 70°C. Other organic acids and alcohols accumulated to a much lesser extent. Fermentation occurred mainly in the top 4 mm of the mat. Exposure to light decreased the accumulation of acetate and presumably of other fermentation products. The importance of interspecies hydrogen transfer was investigated by comparing fermentation product accumulation at a 65°C site, with naturally high hydrogen levels, and a 55°C site, where active methanogenesis prevented significant hydrogen accumulation. There was a greater relative accumulation of reduced products, notably ethanol, in the 65°C mat.     

Effects of temperature acclimation on cardiorespiratory performance of the Antarctic notothenioid Trematomus bernacchii

Notothenioid fishes of the Southern Ocean have evolved under cold and stable temperatures for millions of years. Due to rising temperatures in the Southern Ocean, investigating thermal limits and the capacities for inducing a temperature acclimation response in notothenioids has become of increasing interest. Here, we investigated effects of temperature acclimation on cardiorespiratory responses and cardiac and skeletal muscle energy metabolism in a benthic Antarctic notothenioid, Trematomus bernacchii. We acclimated specimens to ?1, 2 and 4.5 °C for 14 days and quantified heart rates and ventilation rates during an acute increase in temperature. Ventilation rates showed an effect of acclimation both at initial steady-state acclimation conditions and during an acute temperature increase, suggesting a partial thermal compensatory response. However, acclimation did not affect heart rates at steady-state acclimation conditions and the temperatures at which onset of cardiac arrhythmia occurred, suggesting lack of inducible thermal tolerance in cardiac performance. Citrate synthase (CS), lactate dehydrogenase (LDH) and 3-hydroxyacyl dehydrogenase activities in skeletal muscle tissues suggested acclimation-induced shifts in metabolic fuel preferences, and a marked increase in LDH activity with acclimation to 4.5 °C showed an increase in anaerobic metabolism. In heart tissue, CS and LDH activities decreased with acclimation to 4.5 °C, suggesting reduced cardiac ATP production. Overall, the data suggest a partial acclimatory response to temperature by T. bernacchii and support the hypothesis that reduced cardiac acclimatory capacity may play a role in limiting the thermal plasticity of T. bernacchii.     

Resistance to desiccation in aquatic invasive snails and implications for their overland dispersal

At least 30 species of nonindigenous freshwater snails have invaded North America. The risk of these snails invading new lakes depends upon their ability to survive overland transport. We first reviewed published laboratory experiments using freshwater snails, which show numerous species are able to tolerate days of air exposure. We then tested tolerance to drying of three species of invasive aquatic snails that are widespread in Wisconsin: Bithynia tentaculata, Cipangopaludina chinensis, and Viviparus georgianus. In a series of seven experiments, we simulated boater transport by placing snails individually in mesh bags, hung outdoors, and confined in a screen tent. The screen roof allowed exposure to both sun and rain, and an on-site weather station recorded temperature, precipitation, and humidity. All three species exhibited high survivorship, with some individuals alive at the end of most experiments: 42 days for B. tentaculata and V. georgianus and 63 days for C. chinensis. Viable young were released by C. chinensis after 54 days of exposure. Overall, our results indicate that these invasive snails should readily survive long periods of transport overland, indicating a need for continued vigilance of recreational boaters entering lakes.     

Strategies for exploration of freeze responsive gene expression: advances in vertebrate freeze tolerance

Winter survival for many cold-blooded species involves freeze tolerance, the capacity to endure the freezing of a high percentage of total body water as extracellular ice. The wood frog (Rana sylvatica) is the primary model animal used for studies of vertebrate freeze tolerance and current studies in my lab are focused on the freeze-induced changes in gene expression that support freezing survival. Using cDNA library screening, we have documented the freeze-induced up-regulation of a number of genes in wood frogs including both identifiable genes (fibrinogen, ATP/ADP translocase, and mitochondrial inorganic phosphate carrier) and novel proteins (FR10, FR47, and Li16). All three novel proteins share in common the presence of hydrophobic regions that may indicate that they have an association with membranes, but apart from that each shows unique tissue distribution patterns, stimulation by different signal transduction pathways and responses to two of the component stresses of freezing, anoxia, and dehydration. The new application of cDNA array screening technology is opening up a whole new world of possibilities in the search for molecular mechanisms that underlie freezing survival. Array screening of hearts from control versus frozen frogs hints at the up-regulation of adenosine receptor signaling for the possible mediation of metabolic rate suppression, hypoxia inducible factor mediated adjustments of anaerobic metabolism, natriuretic peptide regulation of fluid dynamics, enhanced glucose transporter capacity for cryoprotectant accumulation, defenses against the accumulation of advanced glycation end products, and improved antioxidant defenses as novel parts of natural freeze tolerance that remain to be explored.     

Marenzelleria cf. wireni (Polychaeta: Spionidae) from the Tay estuary. Metabolic response to severe hypoxia and hydrogen sulphide

The appearance of the spionid polychaete Marenzelleria spp. in the Tay estuary (Scotland) was first reported in 1984. Since estuaries are known as environments where abiotic conditions fluctuate widely, a laboratory study was conducted aimed to elucidate how this introduced species deals with low oxygen concentrations and exposure to hydrogen sulphide. Experimental evidence reveals that during severe hypoxia Marenzelleria cf. wireni switched to anaerobic pathways known from other marine invertebrates. Under the influence of sulphide (1 mmol l^-1) accumulation of anaerobic end products is more pronounced. It is assumed that in the presence of sulphide, due to its inhibition of aerobic respiration, the worms have to switch to anaerobic energy production directly after the onset of hypoxia. Seasonal differences in the metabolic reaction were found. It was shown that in specimens that were filled with gametes (winter), accumulation of end products of anaerobic metabolism was more pronounced than in immature worms (summer). The amount of succinate, an indicator for anaerobic energy production for instance, was nearly twice as high in the winter specimens. Since the amount of thiosulphate (a typical sulphide detoxification product) in the tissues is relatively low, it is suggested that Marenzelleria cf. wireni also accumulates other detoxification products. Sulphite, however, also known as a detoxification product, was found only in traces. Field measurements provide evidence that the potential of Marenzelleria cf. wireni to survive low oxygen and high sulphide concentrations is clearly higher than that normally needed in the Tay estuary.     

Individual variation in metabolism and thermal tolerance in juvenile qingbo (Spinibarbus sinensis)

Studies of individual variation in the physiological performance of animals and their relationship with metabolism may provide insight into how selection influences diversity in phenotypic traits. Thus, the aims of the present study were to investigate variation in thermal tolerance and its relationship with individual metabolism in juvenile qingbo (Spinibarbus sinensis). To fulfill our goal, we first measured the resting metabolic rate (RMR), maximum metabolic rate (MMR), metabolic scope (MS, MMR–RMR) and excess post-exercise oxygen consumption (EPOC) of 40 fish at 25 °C. We then measured the critical thermal minimum (CT_min), lethal thermal minimum (LT_min), critical thermal maximum (CT_max), and lethal thermal maximum (LT_max) of 20 fish. Both MMR and MS were positively correlated with the metabolic recovery rate (MRR) (p = 0.001), indicating that high aerobic metabolic performance individuals possessed an advantage for the recovery of anaerobic metabolism. However, the negative correlation between EPOC and MRR (p = 0.017) indicated a slow recovery of the metabolism of high anaerobic metabolic capacity individuals. The RMR was positively correlated with CT_min and LT_min, whereas all of the metabolic rate parameters (RMR, MMR, and MS) were negatively correlated with CT_max and LT_max (p < 0.05), indicating that high aerobic metabolic performance individuals have a weakened thermal tolerance. These results suggested that there is a trade-off between aerobic metabolic performance and thermal tolerance.     

The response of two submerged macrophytes and periphyton to elevated temperatures in the presence and absence of snails: a microcosm approach

Global warming may affect snail–periphyton–macrophyte relationships in lakes with implications also for water clarity. We conducted a 40-day aquaria experiment to elucidate the response of submerged macrophytes and periphyton on real and artificial plants to elevated temperatures (3°C) under eutrophic conditions, with and without snails present. With snails, the biomass and length of Vallisneria spinulosa leaves increased more at the high temperature, and at both temperatures growth was higher than in absence of snails. The biomass of periphyton on V. spinulosa as well as on artificial plants was higher at the highest temperature in the absence but not in the presence of snails. The biomass of Potamogeton crispus (in a decaying state) declined in all treatments and was not affected by temperature or snails. While total snail biomass did not differ between temperatures, lower abundance of adults (size >1 cm) was observed at the high temperatures. We conclude that the effect of elevated temperature on the snail–periphyton–macrophyte relationship in summer differs among macrophyte species in active growth or senescent species in subtropical lakes and that snails, when abundant, improve the chances of maintaining actively growing macrophytes under eutrophic conditions, and more so in a warmer future with potentially denser growth of periphyton.     

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.     

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.     

Low-temperature resistance in Polylepis tarapacana, a tree growing at the highest altitudes in the world

The Polylepis tarapacana forests found in Bolivia are unique with respect to their altitudinal distribution (4200–5200 m). Given the extreme environmental conditions that characterize these altitudes, this species has to rely on distinct mechanisms to survive stressful temperatures. The purpose of this study was to determine low‐temperature resistance mechanisms in P. tarapacana. Tissue was sampled for carbohydrate and proline contents and micro‐climatic measurements were made at two altitudes, 4300 and 4850 m, during both the dry cold and wet warm seasons. Supercooling capacity (?3 to ?6 °C for the cold dry and ?7 to ?9 °C for the wet warm season) and injury temperatures (?18 to ?23 °C for both seasons), determined in the laboratory, indicate that P. tarapacana is a frost‐tolerant species. On the other hand, an increase in supercooling capacity, as the result of significant increase in total soluble sugar and proline contents, occurs during the wet warm season as a consequence of higher metabolic activity. Hence, P. tarapacana, a frost‐tolerant species during the colder unfavourable season, is able to avoid freezing during the more favourable season when minimum night‐time temperatures are not as extreme.