By: Jon P. Costanzo (Deptartment of Zoology, Miami University) © 2012 Nature Education
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
Not surprisingly, a species' capacity for cold hardiness is well matched to the thermal regimen to which
it has adapted. It may exhibit a latitudinal or altitudinal cline such that populations inhabiting colder
regions are adequately protected. Within a given population, survival limits, and even the cold
hardiness mechanism (i.e., freeze avoidance or freeze tolerance), can change from year to year (Kukal &
Duman 1989). For ectotherms with complex life cycles, such as holometabolous insects, cold hardiness
commonly is most pronounced in the overwintering life stages (Salt 1961).
Thermal Environments of Cold-hardy Ectotherms
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).
Citation: Costanzo, J. P. (2012) Extreme Cold Hardiness in Ectotherms. Nature
Education Knowledge 3(10):3
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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.
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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
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
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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).
Class Examples Known From
polyhydric alcohols (glycerol,
sorbitol, ethylene glycol);
sugars (glucose, trehalose);
Amino acids &
taurine, glycine, proline,
alanine, asparagine, glutamic
Table 1: Cryoprotectants used in animal freeze-avoidance and
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 wate