Extreme Cold Hardiness in Ectotherms

Ectotherms, animals whose body temperature closely tracks ambient temperature, occur in virtually every ecological niche on Earth. By virtue of some remarkable adaptations, they thrive even at high latitudes and altitudes in habitats characterized by seasonal or continuous cold (Addo-Bediako et al. 2000). Because aquatic habitats tend to be relatively warm and thermally stable, even in the Polar Regions, this article focuses on ectotherms that occupy terrestrial, arboreal, or intertidal habitats where temperatures may fall appreciably below the freezing point (FP) of body fluids.

For any ectotherm, even brief exposure to subzero temperatures carries the risk of irreparable injury or death. Cold impairs cellular functions by rigidifying membranes, slowing ion pumps, inducing oxidative damage, denaturing proteins, and altering energy balance. Freezing of tissues also provokes myriad stresses that are injurious and potentially lethal to most species. Even for cold-hardy ectotherms, survival depends on cooling rate, exposure temperature, and the duration and frequency of subzero chilling episodes.

Avoidance often is an animal's primary means to protection from extreme temperatures. If migration to warmer climes is not an option, survival may depend on finding an overwintering site, or hibernaculum, that insulates from damaging cold. For example, some toads and terrestrial turtles, being proficient excavators, descend into the soil column and overwinter below the reach of frost. Various snakes and woodland salamanders evade frost by following abandoned rodent burrows or root channels to underground lairs. Nevertheless, many species encounter subzero cold, either because their winter refuge lacks adequate insulation or because they are behaviorally active during cold weather (Figure 1).

Figure 1: Garter snakes (Thamnophis sirtalis) emerging from a subterranean hibernaculum in late February.

T. sirtalis is the most northerly distributed, and the earliest and latest active, of all North American snakes. Hibernation usually is below the frost line, but transient exposure to subzero temperatures can occur during the colder portions of the activity season; these it survives by virtue of its freeze tolerance (Costanzo et al. 1988).

To survive in winter, even cold-hardy ectotherms must seek thermally buffered sites. The ideal hibernaculum also conceals its occupant from potential predators, permits gas exchange, and prevents excessive desiccation. Some species prefer relatively exposed sites from which they can readily detect environmental cues stimulating spring emergence. For others, such as plant gall-inhabiting insects (Baust et al. 1979) and the hatchlings of some turtles (Costanzo et al. 2008), there is no choice in the matter: winter is passed in the very place where one hatches.

The thermal regimens to which overwintering ectotherms are exposed vary geographically, with the intensity and frequency of subzero exposures increasing with altitude and latitude. Even locally, temperatures can range from mild to severe, depending on site characteristics and the physical features of individual hibernacula, and, being subject to the vagaries of the weather, can vary markedly from year to year (Figure 2). Within hibernacula, prevailing temperatures follow a pronounced seasonal rhythm, usually attaining the lowest values at high winter, and also oscillate on a diel cycle.

Figure 2: Regional, local, and annual variation in winter temperatures encountered by hatchlings of the painted turtle (Chrysemys picta).

In North America, C. picta occurs farther north than any other turtle. Although their eggs hatch in late summer, neonates commonly remain inside the nest, in the company of siblings, until the following spring. Temperatures inside these nests, which are constructed in open areas near water bodies, commonly fall below zero and, in extreme cases, may approach -15°C (Costanzo et al. 2003). Winter minima within nests tend to be higher in locales where air temperature is temperate and snow cover is extensive and frequent, such as the upper Great Lakes region, and lower in colder locales where snow cover is scarce, such as the Great Plains region. Shown are minimum temperatures recorded during winter 2000-01 inside 13 nests in the Sandhills of west-central Nebraska and seven nests near a millpond in northern Indiana (adapted from Costanzo et al. 2004). Thermal minima within individual nests vary markedly due to differences in topography, aspect, and other environmental factors. Data collected at the Indiana site over five winters illustrate pronounced annual variation in thermal regimens owing to the vagaries of weather. The graph depicts the winter minimum temperature attained in the warmest and coldest nests, as well as the average for all nests studied in each winter (N as shown). Adapted from Baker et al. (2010).

Many biologists would be surprised to learn that a solution or organism does not necessarily freeze at its FP, but under certain conditions can cool much further whilst remaining unfrozen (liquid), or "supercooled." Indeed, a small volume of pure water can be chilled to nearly -40°C before it spontaneously freezes at its so-called "supercooling point," or, more accurately, temperature of crystallization (T_c).

To exploit the purely physical phenomenon of supercooling in a freeze-avoidance strategy, an organism must remain free of potent ice-nucleating agents (INAs), any of various inorganic particulates, microorganisms, proteins, and organic residues that can organize water molecules into a crystalline arrangement. Ubiquitous in nature, INAs of various potencies occur in diverse habitats, including the overwintering sites of molluscs (Ansart et al. 2010), insects (Zachariassen & Kristiansen 2000), amphibians (Costanzo et al. 1999), and reptiles (Costanzo et al. 2000), and may enter the body through orifices or be inadvertently ingested with food. Many freeze-avoiding ectotherms prepare for dormancy by eliminating ingested INAs, and also by masking or inhibiting endogenous ice-nucleating proteins (Costanzo et al. 2003, Duman 2001). It is crucial that they avoid physical contact with ice, which potentially can invade the body and initiate freezing. Species that rely on supercooling for winter survival can reduce the risk of such "inoculative freezing" by selecting hibernacula that limit their exposure to environmental ice. Some harbor in their tissues antifreeze proteins (AFPs) that effectively inhibit inoculation (Duman 2001).

To avoid ice nucleation, many cold-hardy ectotherms accumulate one or more cryoprotectants in advance of winter (Zachariassen & Kristiansen 2000). Representing several classes of compounds, these solutes vary by species, but all are of low molecular mass and benign in high concentrations (Table 1). They not only colligatively depress the organism's FP, much as automotive antifreeze (e.g., ethylene glycol) prevents radiator fluid from freezing, but also can enhance supercooling (Figure 3). Supercooling capacity is further increased by partial dehydration of the body, which occurs preparatory to winter in many insects (Lee 2010).

Permutations of the freeze-avoidance strategy include vitrification, a process in which body fluids form a glass when cooled to extreme temperatures (Sformo et al. 2010), and cryoprotective dehydration, a survival adaptation of various invertebrates that overwinter in frozen substrata (Holmstrup et al. 2002, Sørensen & Holmstrup 2011). Because the vapor pressure of supercooled water exceeds that of ice, water tends to leave the unfrozen body until the internal vapor pressure reaches that of its frozen environment. Concomitantly, tissue FP drops as solutes become concentrated in a reduced water volume (and, commonly, as cryoprotectants are synthesized), thereby totally eliminating the risk of freezing. For this mechanism to be effective-and for ice inoculation to be avoided-dehydration must proceed quickly enough that the organism's FP consistently remains near the temperature of its cooling environment. Consequently, this strategy can be useful only to small ectotherms with a highly-permeable integument and profound desiccation tolerance.

Excessively deep or prolonged chilling can result in transient or permanent neuromuscular dysfunction or cold narcosis ("chill coma"), and even death (Salt 1961). Underlying mechanisms of chilling injury are not well understood, but probably include disturbance of ion homeostasis and metabolic functions, oxidative stress, and adverse phase changes in membranes (Kostal et al. 2006, MacMillan & Sinclair 2011).

Figure 3: Effect of solute concentration on a solution’s freezing point (FP) and supercooling capacity, as represented by its temperature of crystallization (T_c)

Upper (red) line depicts the theoretical FP depression of -1.86°C per osmole. Lower lines are regressions fitted to empirically determined Tc values for 5-µl samples of solutions containing various quantities of solute, with or without hemolymph (5% v/v) from the cold-hardy beetle, Eleodes blanchardi. Note that a potent ice-nucleating agent in the hemolymph markedly raised the solution’s T_c but did not alter the effect of solute concentration on supercooling capacity. Modified from Lee et al. (1981).

Found in a small number of arthropods, molluscs, nematodes, annelids, amphibians, and reptiles, freeze tolerance is an adaptation for the survival of tissue freezing under ecologically-relevant thermal and temporal conditions. Freezing and thawing of the body, whilst potentially lethal, can be managed by mounting a diverse array of molecular and physiological responses that limit injury to cells and tissues.

For many species, freeze tolerance is expressed seasonally, usually developing in autumn in response to environmental cues and becoming most robust during the coldest months. In the laboratory, capacity for freezing survival can be enhanced by acclimation to high salinity and low environmental temperature, oxygen tension, and water potential (Lee 2010, Murphy 1983).

Control of the freezing process is key to survival in freeze-tolerant ectotherms (Lee & Costanzo 1998). In many invertebrates, which are prone to supercool owing to their small size, freezing is initiated by INAs in the hemolymph or other tissues. In other taxa, particularly species (e.g., amphibians) having a permeable integument, inoculation by external ice ensures that tissues freeze at relatively high temperatures (Costanzo & Lee 1996, Layne et al. 1990). Slow cooling enhances freezing survival, perhaps by allowing the organism adequate time to mount adaptive responses and by restricting ice to extracellular spaces; intracellular freezing is lethal for all but a few species (Lee 2010, Murphy 1983).

Ectotherms vary markedly in their limits of freeze tolerance. Known from laboratory studies, freeze endurance extends from perhaps a few days to several months of continuous freezing, although multiple freeze-thaw episodes are commonly experienced in nature (Layne et al. 1999). Depending on the taxon, the minimum survivable temperature ranges from a degree or two below 0°C to about -80°C (Table 2). Despite this variation, apparently no ectotherm can withstand the freezing of more than 50-80% of their body water.

Molecular and physiological adaptations to freezing and thawing help prevent cell death from dehydration, mechanical distortion, damage to macromolecules and membranes, metabolic perturbation, and ischemia/reoxygenation (Storey & Storey 1992). Dehydration is a particularly onerous problem. As ice accumulates in extracellular spaces, water is lost from the supercooled cells to the interstitium where solutes, rejected by the forming ice crystals, become increasingly concentrated in the as yet unfrozen solution. The rising osmotic potential of this solution withdraws water from inside the cells, progressively shrinking them and concentrating cytosolic solutes to potentially harmful levels (Mazur 1984). Oxidative damage, precipitated by the return of oxygen to thawed tissues, also contributes to cryoinjury (Storey & Storey 2010, Swartz 1972).

Freeze avoidance is an effective survival strategy in small ectotherms that either use dry hibernacula or possess an impermeable integument. Although exposure to extreme cold or prolonged chilling can be harmful, cryoinjury can be minimized if freezing is avoided. Accordingly, freeze-avoiding species generally tolerate a relatively broad range of subzero temperatures. On the other hand, overwintering in the supercooled state is a precarious proposition, as death is the likely consequence of an inadvertent nucleation event.

Freeze-tolerant ectotherms may be of any size and utilize myriad hibernacula, including ones that harbor ice. For those overwintering in an icy environment, tissue freezing eliminates the vapor pressure gradient across the integument and thereby prevents the organism from desiccating. On the downside, freezing/thawing stresses can be highly deleterious and, therefore, survival is possible over a relatively limited range of temperatures and exposure durations.