Freeze Tolerant Animals
For many animals that live in climates with extreme winter temperatures,
the ability to survive freezing of body fluids is a necessary part of
their existance. Natural freeze tolerance occurs in aquatic animals such
as polar fishes and intertidal invertebrates, terrestrial amphibians and
reptiles, and various polar and temperature insects. Various strategies
have been worked out by these disparate animals to withstand the rigours
associated with ice formation at low temperatures.
Freeze Tolerance in Amphibians and Reptiles
There are many examples of lower vertebrates that hibernate in
temperature regions where ice growth in the extracellular fluid is
tolerated within certain limits. For example, the wood frog, Rana
sylvatica, is capable of withstanding temperatures as low as -8°C,
with 65% of its body water converted to ice, or at temperatures of
-2.5°C for periods of up to 2 weeks. Ice formation of this magnitude
causes the cessation of all muscle movements (heart, breathing,
vasoconstriction, skeletal), the onset of ischemia, and large changes in
the volume of cells and organs. Other terrestrial frogs and some turtles
display similarly advanced freeze tolerance while there are also many
other reptiles and amphibians that are able to withstand short, mild
freezing exposures typical of overnight frosts.
There are several factors that influence the ability of a vertebrate to
survive extracellular ice formation (there are no examples of
vertebrates that can withstand intracellular ice formation).
Assessment and Control of Ice Formation
It is essential that freeze tolerant animals initiate freezing within
their body fluids at high sub-freezing temperatures, and that they can
detect the presence of ice in their bodies. When ice forms in
supercooled water, ice growth is rapid and the osmotic stresses that the
cells face is severe. With no cryoprotectants present, the cells will be
subjected to a high salt concentration and frozen into channels where no
cryoprotectant is likely to appear, in order to mitigate this salt. If,
on the other hand, ice growth is initiated near the freezing point, then
the animal can take steps to minimize the physical damage by reacting to
this ice growth.
Typically, wood frogs only supercool to -2°C or -3°C before ice growth
begins. Spontaneous nucleation at such low degrees of supercooling is
unlikely, thus there are probably ice nucleating proteins or bacteria on
the surface of the frog that catalyze ice formation. Alternatively,
contact with external ice will lead to ice growth inside the body cavity
at the freezing temperature (-0.5°C). In addition to external ice
nucleators, all freeze tolerant species generate ice nucleating proteins
within their blood plasma during the hibernation season. These proteins
are less efficient than the external ones, requiring supercooling to
about -5°C to ensure ice nucleation. Because the plasma will freeze well
before this temperature is reached, the function of these ice nucleators
is uncertain. They may be involved in facilitating ice growth within
capillaries, where the high curvature of the ice crystal would be
inhibitory.
Ice growth through the organism is carefully controlled. Ice usually
starts in the hind limbs and begins spreading throughout the body from
there, taking several hours to grow throughout the body. Ice grows
around the vital organs long before freezing occurs
within the organs. Ice forms in the brain last, with the fluid portion
freezing before the neural tissue. Melting does not occur with this same
directional rigidity, but instead begins in the vital organs
simultaneously, and then spreads outwards. Since the organs are the last
to freeze, the cryoprotectant concentration is highest there, causing
these regions to have the lowest melting point. The amount of ice that forms
within the organs is limited by dehydration and ice formation in the fluid
regions that surround the organs. At a temperature of -2.5°C, where 50% of
the frog's body water is frozen, the eyes lose 3% of their water, the brain
loses 9%, skeletal muscle loses 13%, the liver loses 20%, and the heart loses
24% of its water content.
Ice formation in the skin is detected virtually immediately by freeze
tolerant frogs and the biological response, beginning in the liver, is
fully active within two minutes.
Cryoprotection
The biological action that freeze tolerant species initiate, upon
finding ice growing within their body, is the production and
dissemination of enormous quantities of cryoprotectant. The
cryoprotectants used are colligative in action, glucose and glycerol are
two of the most common cryoprotectants (a particular species confines
itself to the use of a single cryoprotectant).
When freezing is detected, a signal is transmitted to the liver where
glycogenolysis, the conversion of glycogen to glucose, begins in
earnest. Glucose levels within the liver will have risen by over six
times within the first 4 minutes, and remain elevated for several hours.
Blood flow distributes glucose throughout the body (there is no
supplemental glucose production from other locations within the body)
until freezing brings a halt to circulation. Thus the lowest
concentration of glucose is in the skin and skeletal muscle (which
freeze first) and the highest concentrations are in the vital organs
(thereby depressing their freezing points the most).
The liver of freeze tolerant frogs is specialized for this task. It
contains much higher levels of glycogen than is found in comparable non-
freeze tolerant species. Likewise, the frogs' cells have much higher
numbers of glucose transporters within the membranes to support
cryoprotectant entry into the cells, the increase being seasonal as
well. Animals that use glycerol as a cryoprotectant do not need to add
transport proteins as cell membranes are naturally permeable to
glycerol. It is not yet known whether aquaporins play a role in
accelerating the large cellular water losses that must accompany
freezing for colligative cryoprotection to be effective.
Lowered Metabolism and Limiting of Ischemia
The lowest temperature that freeze tolerant species are able to
withstand is about -8°C. This is much too high to bring an effective
halt to biochemical reactions; and indeed, the freeze concentration that
occurs with ice formation may even accelerate some reactions. Thus these
species must minimize ischemic damage while in the frozen state. There
appears to be an active metabolic homeostasis that is maintained in the
frozen state, along with significant production of antioxidant enzymes
to combat reactive oxygen species.
Reanimation
All organ systems shut down during freezing with the heart being the
last to stop (at about 12 hours after the onset of freezing). During
thawing, the heartbeat is also the first physiological activity that is
restored. Blood flow to the periphery is restored soon after, followed
by breathing and then skeletal muscle activity. There are undoubtedly
many biochemical event that must occur to reverse the process of
cryoprotectant release and repair ischemic damage, but shortly after the
ice melts, the freeze tolerant animal is able to hop (or walk or
slither, as the case may be) away.
Aquatic Animals at Low Temperatures
There are many intertidal invertebrates that survive brief periods of
extracellular ice formation. This occurs primarily by the mechanism of
colligative cryoprotection, as in terrestrial animals. The real action
here is the strategy that polar fishes have evolved for dealing with the
freezing environment.
Seawater freezes at -1.9°C, a temperature that is reached during the
winter in polar and temperature ocean regions. This is well below the
melting point of the body fluids of marine fishes (-0.8°C). Although it
is conceivable for an organism to exist with 1 degree of supercooling,
the marine environment is one in which there are small ice crystals
suspended throughout the seawater when it reaches the freezing point.
Since it is necessary for fishes to live in this environment, and pass
this water (with its ice crystals) through their gills, it is impossible
for these organisms to avoid ice growth within their bodies. Yet they
appear not to freeze until the temperature drops below -2°C, at which
point they will freeze and die.
The melting point of the fluids within these polar fishes is -0.8°C, but
the apparent freezing point (the temperature where ice begins to grow)
is significantly lower. This freezing point depression is not
colligative (although the depression of the melting point is) and is
lost when the fluid is dialyzed through a molecular sieve with a cutoff
of about 2500. The agents responsible for this freezing point depression
are either glycoproteins (Antarctic and North-temperate fish) or
proteins (Arctic fish). These compounds are generically referred to as
antifreeze proteins.
Antifreeze Proteins
Antifreeze glycopeptides have a molecular weight between 2600 and 34000
while the antifreeze peptides range between 3200 and 14000. In solution
these compounds form extended structures, usually helical rods. They are
amphipathic molecules, with one side of the rod being composed of
hydrophobic residues and the other side being composed of hydrophilic
residues. On the hydrophilic side, there is a 4.5 Å separation between
repeating threonine and aspartate residues that bind the protein to an
ice lattice.
The thermal hysteresis, or the difference between the freezing point
(crystal growth point) and the melting point is shown on the following
graph for several of the antifreeze compounds.
Fig. 12.2.1 Freezing and Melting Points of Antifreeze Compounds.
Ice that grows in the presence of antifreeze proteins (AFP's) is
spicular, forming long, thin structures with a hexagonal cross-section.
The AFP's bind to ice steps along the a-axes of an ice crystal, so that
spicular growth is growth that is restricted to the c-axis. The
following diagram illustrates the structure of a hexagonal ice
crystal.
Fig. 12.2.2 Hexagonal Ice Crystal.
When AFP binds to the a-axis steps, then water molecules cannot join the
crystal between the AFP binding sites without introducing significant
curvature to the growth along the a-axis.
Fig. 12.2.3 AFP binding to Hexagonal Ice Crystal.
At low degrees of supercooling, this increased curvature will completely
impede ice growth along the a-axes. The basal plane will then get
smaller with each step and the crystal will form a microscopic bi-
pyramidal shape that is thermodynamically stable at temperatures below
its melting point. Further cooling will lead to more rapid accumulations
along the c-axis compared to the a-axes, leading to spicular crystals.
Growth in the a-axes requires that the temperature be low enough to
support the curvature between AFP molecules bound to the crystal. When
this happens, the AFP's will be incorporated into the crystal as
defects, rather than excluded as colligative solutes.
One consequence of the amphipathic nature of AFP's is that an ice
crystal that has a surface coated with these molecules will present a
hydrophobic exterior. This allows such ice crystals to grow
through lipid bilayers without any thermodynamic exclusion. Once through
the membrane and into the cytoplasm (that does not contain AFP's), the
ice growth quickly spreads throughout the cell and leads to cell
death.
Insect Adaptations to Low Temperatures
The ability of insects to adapt to diverse ecological conditions is
legendary. This tremendous diversity is justly illustrated by their
ability to withstand the intense cold of arctic and alpine environments.
Indeed, the Arctic spring is accompanied by a veritable deluge of biting
insects; a grim but unmistakable testament to their overwintering
capabilities.
The adaptations that have evolved to allow insects to survive low
temperatures are legion, but they can be classified along two general
lines, freeze tolerance (the ability to survive following ice
formation within the body cavity) and freeze avoidance (the
prevention of ice formation within the body cavity at temperatures where
such freezing would normall occur).
In addition to the problems posed by ice formation, there are also
significant problems that must be solved for normal metabolism to occur
at low temperatures. The maintenance of neural function, fluidity of
cell membranes, pH control, activity of enzymes, adaptation to hypoxia,
dehydration of body fluids, etc. all present obstacles to low
temperature survival. Although these difficulties are formidable, they
will not be discussed further here, as the adaptations to freezing
temperatures are the focus of this chapter.
Very few insect species are actually exposed to the full rigors of
winter temperatures as most choose an overwintering microhabitat that
provides a buffered temperature. The habitats provided inside vegetation
(logs, stumps, etc.) or under the soil provide thermal buffering,
especially when covered with snow. In many climates, however, the
organisms are still exposed to potentially lethal conditions throughout
the winter. The particular adaptations associated with freeze tolerance
and freeze avoidance allow these organisms to survive in such harsh
environments.
Freeze Tolerance
Many species of insects have developed a tolerance for ice formation
within their body fluids. The degree to which these species withstand
freezing varies widely, from just a few degrees below freezing to -87°C
for an Alaskan beetle. The strategies employed are legion, although the
principle means for minimizing injury from ice formation is the use of
cryoprotectants to reduce the amount of ice formed and the salt
concentration at a given temperature.
Cryoprotectants
The cryoprotectants used by freeze tolerant species are similar (almost
exactly so) to the compounds used by freeze avoidant insects to
colligatively depress the freezing point. Glycerol is the most common
cryoprotectant, followed by sorbitol and erythritol, ribitol, threitol,
and sucrose. A multicomponent cryoprotection scheme is common, to reduce
the concentrations of any given cryoprotectant to sub-toxic levels. The
mechanism of cryoprotection appears to be simple colligative action, as
in cryopreservation, with the additional benefit of stabilization of
protein structure against low temperature denaturation.
Ice Nucleating Proteins
One strategy for mitigating the damaging effects of ice formation is to
nucleate ice at a high sub-freezing temperature to avoid the high
osmotic stresses associated with rapid freezing. Ice nucleators that
have been found in insects are generally not too efficient, initiating
ice growth at supercooling points between -7°C and -10°C, thus they are
probably involved in avoiding intracellular ice growth rather than the
directed ice growth seen in freeze-tolerant amphibians.
Some freeze tolerant insects have neither specialized ice nucleating
proteins, nor an absence of ice nucleation sites (as in some freeze-
avoidant insects). In a dry environment, they will supercool to near
-20°C and then freeze and die. If they are inoculated with environmental
ice at higher temperatures, however, they can survive. It seems, though,
that most freeze tolerant insects produce ice nucleating proteins as
part of their strategy for survival.
Stabilization of Bound Water
The water associated with macromolecules (bound water) is important for
maintaining the tertiary structure of some molecules as well as the
structure of membranes. It has been found that freeze tolerant organisms
increase the amount of bound water in their systems during cold
acclimation. That this confers additional tolerance to freezing is still
a point of speculation.
Antifreeze Proteins
In many cases, damage from freezing has been linked to the rate of
thawing, implicating the degree to which the ice undergoes
recrystallization as a damaging mechanism. Since a few freeze tolerant
insects produce antifreeze proteins, it has been speculated that these
proteins minimize the injury associated with recrystallization of
extracellular ice (AFP's are potent inhibitors of recrystallization). In
fact, many of the freeze tolerant species that produce AFP's contain
them in too low a concentration to produce thermal hysteresis; they are
concentrated enough to inhibit recrystallization, however, since the
thermodynamic driving force is much lower than for crystal growth in
supercooled water.
Vitrification
It has been found that the hemolymph of a particular insect can be
partially vitrified at cooling rates that are likely to occur in nature.
Furthermore, the very low temperatures that some insects survive in the
absence of ice indicate that vitrification could easily be achieved if
the temperature went below the glass transition temperature. Thus it has
been speculated that vitrification, or at least partial vitrification,
may well be a strategy for freeze tolerance although direct evidence is
thus far lacking.
Freeze Avoidance
Insects that are freeze-susceptible (ice formation in their body fluids
is lethal) need to avoid freezing during the winter months. There are
three basic strategies that insects employ to avoid ice formation within
their body cavity: 1. Colligative depression of the freezing point
through the concentration of a low molecular weight solute; 2.
Production of an antifreeze protein (AFP) to lower the crystal-growth
temperature non-colligatively; 3. Lowering of the nucleation temperature
by removal of ice nucleation sites.
Colligative Freezing Point Depression
Colligative freezing point depression is simply the addition of low
molecular weight solutes to the body fluids, exactly as occurs in normal
cryoprotectant use. The solutes must be non-toxic in the concentrations
required (molar), excluding many salts and small organic molecules. The
polyhydroxy alcohols are the most common antifreeze solutes. Glycerol is
undoubtedly the most prevalent polyol found in insects, but other
compounds, such as ethylene glycol, sorbitol, and mannitol are also
found in some species. There are other polyols that are found in
elevated concentrations during the winter, but not in the molar
quantities of an antifreeze solute (the combined effect, however, is to
further reduce the freezing point of the solution); these include
inositol, fucitol, arabitol, zylitol, rhamnitol, and ribitol. In
conjunction with the polyols, elevated levels of the sugars trehalose,
glucose and fructose are often found during the winter, as are elevated
levels of the amino acid alanine. Such a multicomponent approach to
freezing point depression allows a significant colligative action
without bringing any one solute to the point of chemical toxicity.
Non-Colligative Freezing Point Depression
Thermal hysteresis producing antifreeze proteins (AFP's) have been found
in many species of insects. The AFP's found in insects have, in some
cases, been found to have a much higher activity than the AFP's isolated
from polar fishes, primarily due to the increased concentration found in
insects. Insect AFP activity has been found with a depression of the
crystal growth temperature by as much as 6°C to 9°C below the melting
point. None of the insect AFP's have been found to have carbohydrate
moieties, in contrast to the antifreeze glycoproteins commonly found in
antarctic fish.
The advantage of AFP's over colligative freezing point depression is in
the much lower concentrations required and the ability to concentrate
these molecules in the gut, where ice contamination is likely.
Supercooling
Pure water has a homogeneous nucleation temperature of -40°C, thus in
the absence of any nucleation sites, it should be possible for an insect
to survive very cold temperatures in a supercooled state if it could rid
itself of all ice nucleators and prevent external ice from contacting
its body fluids. It has been shown that some insects become much more
susceptible to freeze injury if they are fed ice nucleating bacteria
before exposure to cold temperatures, indicating that supercooling is a
naturally occuring strategy for freeze-avoidant insects. In addition,
insects have been found to have significantly lower supercooling points
in winter, as compared with summer, without any lowering of their
melting points or crystal growth temperatures. Some species remove ice
nucleation sites seasonally whereas others have removed them permanently,
over evolutionary time (a strategy that is evidently not compatible
with all lifestyles).
There are some insect species found in the Canadian Rockies that have
supercooling points of -60°C, combining colligative freezing point
depression with an absence of ice nucleation sites to avoid ice
formation at any terrestrial temperature (although these species are
also freeze tolerant, there is simply no chance for them to experience
temperatures of -60°C except in the laboratory).
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Document last updated Mar. 1, 1999.
Copyright © 1999, Ken Muldrew.