freezing and animals

Chapter 12.2

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).

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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).