Climatic variability and the evolution of insect freeze tolerance

Insects may survive subzero temperatures by two general strategies: Freeze-tolerant insects withstand the formation of internal ice, while freeze-avoiding insects die upon freezing. While it is widely recognized that these represent alternative strategies to survive low temperatures, and mechanistic understanding of the physical and molecular process of cold tolerance are becoming well elucidated, the reasons why one strategy or the other is adopted remain unclear. Freeze avoidance is clearly basal within the arthropod lineages, and it seems that freeze tolerance has evolved convergently at least six times among the insects (in the Blattaria, Orthoptera, Coleoptera, Hymenoptera, Diptera and Lepidoptera). Of the pterygote insect species whose cold-tolerance strategy has been reported in the literature, 29% (69 of 241 species studied) of those in the Northern Hemisphere, whereas 85 % (11 of 13 species) in the Southern Hemisphere exhibit freeze tolerance. A randomization test indicates that this predominance of freeze tolerance in the Southern Hemisphere is too great to be due to chance, and there is no evidence of a recent publication bias in favour of new reports of freeze-tolerant species. We conclude from this that the specific nature of cold insect habitats in the Southern Hemisphere, which are characterized by oceanic influence and climate variability must lead to strong selection in favour of freeze tolerance in this hemisphere. We envisage two main scenarios where it would prove advantageous for insects to be freeze tolerant. In the first, characteristic of cold continental habitats of the Northern Hemisphere, freeze tolerance allows insects to survive very low temperatures for long periods of time, and to avoid desiccation. These responses tend to be strongly seasonal, and insects in these habitats are only freeze tolerant for the overwintering period. By contrast, in mild and unpredictable environments, characteristic of habitats influenced by the Southern Ocean, freeze tolerance allows insects which habitually have ice nucleators in their guts to survive summer cold snaps, and to take advantage of mild winter periods without the need for extensive seasonal cold hardening. Thus, we conclude that the climates of the two hemispheres have led to the parallel evolution of freeze tolerance for very different reasons, and that this hemispheric difference is symptomatic of many wide-scale disparities in Northern and Southern ecological processes.     

Heterologous aquaporin (AQY2-1) expression strongly enhances freeze tolerance of Schizosaccharomyces pombe

Aquaporin membrane proteins enable the transport of water across membranes in various organisms. In yeast their expression has been shown to correlate strongly with freeze tolerance. When we analyzed the freeze tolerance of Schizosaccharomyces pombe, an organism whose genome sequence has revealed no genes encoding a bona fide water channel, we found very low intrinsic freeze tolerance compared to other yeast species with aquaporin-encoding genes. Deletion of Spac977.17, which encodes a putative glycerol facilitator, resulted in no significant differences in freeze tolerance with its corresponding wild-type strain in all growth conditions tested. However, when we expressed the Saccharomyces cerevisiae aquaporin-encoding gene AQY2-1 in S. pombe cells, we found that the relatively low freeze tolerance of S. pombe could be significantly enhanced. Therefore, (i) the absence of a bona fide water channel in S. pombe might provide in part an explanation for its overall low freeze tolerance compared to other yeast species, and (ii) aquaporin overexpression might be a tool to improve cryopreservation of many other cell types as well, as has recently been shown for mouse oocytes and fish embryos.     

Disruption of the CAR1 Gene Encoding Arginase Enhances Freeze Tolerance of the Commercial Baker's Yeast Saccharomyces cerevisiae

The effect of intracellular charged amino acids on freeze tolerance in doughs was determined by constructing homozygous diploid arginase-deficient mutants of commercial baker's yeast. An arginase mutant accumulated higher levels of arginine and/or glutamate and showed increased leavening ability during the frozen-dough baking process, suggesting that disruption of the CAR1 gene enhances freeze tolerance.     

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.     

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.     

Freeze tolerance evolution among anurans: Frequency and timing of appearance

Despite numerous mechanistic studies on physiological responses supporting freeze tolerance in anurans, few have addressed the evolutionary significance of this trait. We thus investigated the phylogenetic relationships among anuran species whose freeze tolerance has been assessed and in combination with new data on freezing tolerance of two closely related species of the European brown frogs (Rana temporaria and Rana dalmatina). The species we studied exhibited short survival times in frozen state (around 8 h for both species). Phylogenetic analysis suggests that freeze tolerance evolved at least two times among Ranidae and one or two times among Hylidae and never in Bufonidae. Furthermore, in order to assess the timing of divergence of this character we used a relaxed molecular clock created, and found that the most recent separation between a freeze tolerant species and a freeze intolerant species dates from 15.9 ± 7.6 Myr (Rana arvalis and R. temporaria). The comparison between these two species thus represents the best current model to understand freeze tolerance evolution. Addressing the evolution of this trait with such large-scale approaches will not only improve our understanding of cold hardiness strategies, but might also create a framework guiding future comparative studies.     

Tolerance of freezing in caterpillars of the New Zealand Magpie moth (Nyctemera annulata)

In most insects known to tolerate freezing, the adaptation has been completely canalized and permanently incorporated into the genotype, either as a perennial or seasonal phenotypic switch. The exceptions to this (i.e. insects for which the adaptation is, in some manner, incomplete) represent examples of considerable evolutionary interest. To date, the few examples known of incomplete adaptation are readily identified by survival metrics. Caterpillars of the New Zealand Magpie moth (Nyctemera annulata Boisduval) represent a previously undescribed stage in the adaptive continuum of freeze tolerant insects from freeze avoidance to tolerance: a form of freeze tolerance that is intermediate between partial and complete freeze tolerance, the relative ‘incompleteness’ of which, is only apparent using indices of extended fitness (successful metamorphosis). This intermediate form is characterized by: the capacity to mechanistically tolerate equilibrium freezing (>75% survival); a narrow survival envelope below equilibrium freezing temperatures (3–4 °C); and a limited ability to complete metamorphosis after freezing (approximately 27% emergence). The low temperature capabilities of these caterpillars provide support for the hypothesis that the capacity to mechanistically tolerate internal extracellular ice formation by freeze tolerant holometabolous insects is acquired prior to the metabolic adaptations necessary to enable continuation of the life cycle.     

Proline: A Novel Cryoprotectant for the Freeze Preservation of Cultured Cells of Zea mays L

Proline is an effective cryoprotectant for the storage of cultured cells of Zea mays L. in liquid N₂. Increased freeze tolerance can be achieved by pregrowth for 3 to 4 days in medium containing proline. Cells cryoprotected with proline have an increased recovery potential when compared with cells cryoprotected with dimethylsulfoxide and glycerol. They also show a reduced postthaw viability loss and greater tolerance of a range of postthaw culture conditions. It is suggested that the mechanism of action of proline may be similar to that in its putative role of conferring protection against natural stresses. It may be protecting the cell against solution effects caused by dehydration during freezing. These findings are discussed in relation to other freeze tolerance enhancing treatments.     

Reptile freeze tolerance: metabolism and gene expression

Terrestrially hibernating reptiles that live in seasonally cold climates need effective strategies of cold hardiness to survive the winter. Use of thermally buffered hibernacula is very important but when exposure to temperatures below 0 degrees C cannot be avoided, either freeze avoidance (supercooling) or freeze tolerance strategies can be employed, sometimes by the same species depending on environmental conditions. Several reptile species display ecologically relevant freeze tolerance, surviving for extended times with 50% or more of their total body water frozen. The use of colligative cryoprotectants by reptiles is poorly developed but metabolic and enzymatic adaptations providing anoxia tolerance and antioxidant defense are important aids to freezing survival. New studies using DNA array screening are examining the role of freeze-responsive gene expression. Three categories of freeze responsive genes have been identified from recent screenings of liver and heart from freeze-exposed (5h post-nucleation at -2.5 degrees C) hatchling painted turtles, Chrysemys picta marginata. These genes encode (a) proteins involved in iron binding, (b) enzymes of antioxidant defense, and (c) serine protease inhibitors. The same genes were up-regulated by anoxia exposure (4 h of N2 gas exposure at 5 degrees C) of the hatchlings which suggests that these defenses for freeze tolerance are aimed at counteracting the injurious effects of the ischemia imposed by plasma freezing.     

Identification of chaperones in freeze tolerance in Saccharomyces cerevisiae

Exposure to low temperatures reduces protein folding rates and induces the cold denaturation of proteins. Considering the roles played by chaperones in facilitating protein folding and preventing protein aggregation, chaperones must exist that confer tolerance to cold stress. Here, yeast strains lacking individual chaperones were screened for reduced freezing tolerance. In total, 19 of 82 chaperone-deleted strains tested were more sensitive to freeze-thaw treatment than wild-type cells. The reintroduction of the respective chaperone genes into the deletion mutants recovered the freeze tolerance. The freeze sensitivity of the chaperone-knockout strains was also retained in the presence of 20% glycerol.     

Biochemistry of natural freeze tolerance in animals: molecular adaptations and applications to cryopreservation

For a wide variety of animals, winter survival in cold climates includes the ability to tolerate ice formation in extracellular body fluids. Among terrestrially hibernating vertebrates, freeze tolerance has been documented for five amphibian and two reptile species. These species may survive for days or weeks in a frozen state with no breathing and no heart beat, and with up to 65% of total body water as extracellular ice. The biochemical mechanisms involved in natural freeze tolerance include (i) the regulation of extracellular ice formation by proteinaceous ice nucleators in body fluids, (ii) the accumulation of high concentrations of low molecular weight carbohydrates as cryoprotectants to regulate cell volume reduction during freezing and stabilize macromolecular structure, and (iii) a well-developed ischemia tolerance that supports the survival of individual organs while frozen. The present article focuses on recent advances in our understanding of the biochemistry of natural freeze tolerance in lower vertebrates and the application of these studies to the improvement of cryopreservation technology for transplantable mammalian organs.     

Freezing survival, body ice content and blood composition of the freeze-tolerant European common lizard, Lacerta vivipara

To investigate the freeze tolerance of the European common lizard, Lacerta vivipara, we froze 17 individuals to body temperatures as low as -4 degrees C under controlled laboratory conditions. The data show that this species tolerates the freezing of 50% of total body water and can survive freezing exposures of at least 24-h duration. Currently, this represents the best known development of freeze tolerance among squamate reptiles. Freezing stimulated a significant increase in blood glucose levels (16.15+/- 1.73 micromol x ml(-1) for controls versus 25.06 +/- 2.92 micromol x ml(-1) after thawing) but this increase had no significant effect on serum osmolality which was unchanged between control and freeze-exposed lizards (506.0 +/- 23.8 mosmol x l(-1) versus 501.0 +/- 25.3 mosmol x l(-1), respectively). Tests that assessed the possible presence of antifreeze proteins in lizard blood were negative. Recovery at 5 degrees C after freezing was assessed by measurements of the mean time for the return of breathing (5.9 +/- 0.5 h) and of the righting reflex (44.8 +/- 4.5 h). Because this species hibernates in wet substrates inoculative freezing may frequently occur in nature and the substantial freeze tolerance of this lizard should play a key role in its winter survival.     

Roles of carbohydrate reserves for local adaptation to low temperatures in the freeze tolerant oligochaete Enchytraeus albidus

Geographic variation in cold tolerance and associated physiological adaptations were investigated in the freeze tolerant enchytraeid Enchytraeus albidus (Oligochaeta). Specimens from Svalbard, Greenland (Nuuk), Iceland (Hólar and Mossfellsbær) and continental Europe [Norway (Bergen), Sweden (Kullen) and Germany] were reared in the laboratory in a common-garden experiment. The aim was to test for variations in minimum lethal temperature, freeze duration tolerance, carbohydrate reserves and metabolic rate among the populations. Cold tolerance was related to environmental temperature of the respective location. Populations from the coldest climatic regions were able to tolerate freezing down to at least ?15 °C and endured being frozen at ?5 °C for 27–48 days, respectively. Populations from milder climates had a lower freeze duration tolerance (about ?9 °C) and endured ?5 °C for a shorter period (between 9 and 16 days). Glucose accumulation and glycogen reserves varied significantly between populations, but was not related directly to cold tolerance. Metabolic rate varied significantly between populations, but was not significantly related to cold tolerance. The metabolic rates at ?2 °C of frozen and unfrozen worms from Germany and Svalbard were tested. The metabolic depression due to freezing of these populations was relatively small (<50 %), suggesting that the large carbohydrate accumulations may also be important as fuel during long-term freezing at moderately low temperatures. Differences in metabolic depression may partly explain the difference in cold tolerance of these two populations, however, the mechanisms behind local adaptation to low winter temperatures in these enchytraeid populations seem more complex than earlier studies have indicated.     

A simple moder for estimating the ice content of freezing ectotherms

1. 1.Although body ice content is an important variable affecting freeze tolerance, present calorimetric methods for its measurement necessarily require the termination of a freezing protocol.

2. 2.A simple iterative model, based on the colligative properties of solutions and requiring precise measurements of only equilibrium freezing point (of the unfrozen organism) and of core body temperature, allows estimation of the percentage of body water frozen at any time during a freezing episode.

3. 3.This model can also predict the lethal temperature for a freezing ectotherm, assuming that death occurs due to osmotic dehydration when 67% (of any other known lethal fraction) of the body water is frozen.

4. 4.The basic model is easily extended to evaluate the effects of variables such as: body mass, initial body water content, initial osmotic concentration, and test chamber microenvironment.

5. 5.This model is not intended to supplant existing more exact biophysical models of freezing kinetics. Rather it is proposed as a first approximation which is generally supported by published data and which should be of significant practical value for investigators of freeze tolerant organisms.

Freeze/thaw-induced destabilization of the plasma membrane and the effects of cold acclimation

Disruption of the plasma membrane is a primary cause of freezing injury. In this review, the mechanisms of injury resulting from freeze-induced cell dehydration are presented, including destabilization of the plasma membrane resulting from (a) freeze/thaw-induced osmotic excursions and (b) lyotropic phase transitions in the plasma membrane lipids. Cold acclimation dramatically alters the behavior of the plasma membrane during a freeze/thaw cycle—increasing the tolerance to osmotic excursions and decreasing the propensity for dehydration-induced lamellar to hexagonal-II phase transitions. Evidence for a casual relationship between the increased cryostability of the plasma membrane and alterations in the lipid composition is reviewed.     

Aquaporin-mediated improvement of freeze tolerance of Saccharomyces cerevisiae is restricted to rapid freezing conditions

Previous observations that aquaporin overexpression increases the freeze tolerance of baker's yeast (Saccharomyces cerevisiae) without negatively affecting the growth or fermentation characteristics held promise for the development of commercial baker's yeast strains used in frozen dough applications. In this study we found that overexpression of the aquaporin-encoding genes AQY1-1 and AQY2-1 improves the freeze tolerance of industrial strain AT25, but only in small doughs under laboratory conditions and not in large doughs under industrial conditions. We found that the difference in the freezing rate is apparently responsible for the difference in the results. We tested six different cooling rates and found that at high cooling rates aquaporin overexpression significantly improved the survival of yeast cells, while at low cooling rates there was no significant effect. Differences in the cultivation conditions and in the thawing rate did not influence the freeze tolerance under the conditions tested. Survival after freezing is determined mainly by two factors, cellular dehydration and intracellular ice crystal formation, which depend in an inverse manner on the cooling velocity. In accordance with this so-called two-factor hypothesis of freezing injury, we suggest that water permeability is limiting, and therefore that aquaporin function is advantageous, only under rapid freezing conditions. If this hypothesis is correct, then aquaporin overexpression is not expected to affect the leavening capacity of yeast cells in large, industrial frozen doughs, which do not freeze rapidly. Our results imply that aquaporin-overexpressing strains have less potential for use in frozen doughs than originally thought.     

Physiological ecology of overwintering in hatchling turtles

Temperate species of turtles hatch from eggs in late summer. The hatchlings of some species leave their natal nest to hibernate elsewhere on land or under water, whereas others usually remain inside the nest until spring; thus, post-hatching behavior strongly influences the hibernation ecology and physiology of this age class. Little is known about the habitats of and environmental conditions affecting aquatic hibernators, although laboratory studies suggest that chronically hypoxic sites are inhospitable to hatchlings. Field biologists have long been intrigued by the environmental conditions survived by hatchlings using terrestrial hibernacula, especially nests that ultimately serve as winter refugia. Hatchlings are unable to feed, although as metabolism is greatly reduced in hibernation, they are not at risk of starvation. Dehydration and injury from cold are more formidable challenges. Differential tolerances to these stressors may explain variation in hatchling overwintering habits among turtle taxa. Much study has been devoted to the cold-hardiness adaptations exhibited by terrestrial hibernators. All tolerate a degree of chilling, but survival of frost exposure depends on either freeze avoidance through supercooling or freeze tolerance. Freeze avoidance is promoted by behavioral, anatomical, and physiological features that minimize risk of inoculation by ice and ice-nucleating agents. Freeze tolerance is promoted by a complex suite of molecular, biochemical, and physiological responses enabling certain organisms to survive the freezing and thawing of extracellular fluids. Some species apparently can switch between freeze avoidance or freeze tolerance, the mode utilized in a particular instance of chilling depending on prevailing physiological and environmental conditions.