Avoiding freezing

If a small quantity of pure water is cooled, it does not freeze at 0 °C and the temperature may fall to as low as —39 °C before the formation of ice crystals commences. This phenomenon is known as supercooling, which refers to the water remaining liquid at temperatures below its melting point. The temperature at which freezing eventually occurs is known as the temperature of crystallisation (or supercooling point). For freezing to occur, the water molecules need to come together to form an ice crystal that, once formed, results in the freezing of the whole body of water. This process is called nucleation and substances that cause the freezing of the water (such as an ice crystal or some other sort of particle) are called ice nucleators. The chances of the water molecules coming together spontaneously to form an ice crystal (which then acts as an ice nucleator) depends on the volume of water present, its temperature and the period of time it has been at that temperature. Substances other than ice (such as a dust particle) can act as ice nucleators and are usually responsible for initiating freezing in most natural situations.

You will rarely see supercooled bodies of water (lakes, ponds and puddles) in nature since there are usually plenty of nucleating agents (in the soil, sediment or dust) that will initiate the formation of ice and prevent supercooling. However, a small organism, which contains a small volume of water, can supercool by a substantial amount if it can prevent nucleation. This enables it to survive exposure to low temperatures by avoiding freezing.

Freeze-avoiding insects

There have been far more studies on the cold tolerance of insects than of any other group of organisms. This is due to the economic importance of insects as agricultural pests and the desire to study the survival of overwintering populations of insects, the numbers of which determine the likely size of the pest problem in the following growing season. The main pioneer of the study of insect cold tolerance was Reginald Salt, a Canadian entomologist who was inspired by the cold Canadian winters and the possibility that insect pests could be simply and cheaply controlled by exposing them to low temperatures over the winter. Salt was also impressed by being shown a technique that would allow the freezing of an insect to be measured. As he said:

A simple laboratory demonstration . . . of the heat of crystallisation given off by a freezing insect made a deep and lasting impression on me.

Insects are enclosed by a tough and opaque cuticle. It is difficult to tell whether they are frozen or not by touching or looking at them. The technique that made such an impression on Salt solved this problem and made possible the studies which he pursued for much of his career and the studies of investigators who followed him.

The technique used by Salt employed a thermocouple to monitor the temperature of an insect. A thermocouple consists of wires of two different metals that are fused together at both ends to form a circuit, the voltage of which varies as a function of temperature. The measuring junction of a thermocouple can be made quite small (less than 1 millimetre in diameter), which allows the temperature at the surface of even a tiny insect to be measured. When water is cooled, it often does not freeze at 0 °C but cools further (supercooling) until the formation of ice crystals commences. If there is sufficient water, freezing elevates the water temperature to 0 °C, since the change in state of the water from a liquid to a solid causes it to release energy in the form of latent heat of crystallisation. The temperature of the water will remain at 0 °C until the freezing process is completed and the heat generated dissipated and the temperature can then fall to that of the surroundings. If the temperature of an insect is monitored continuously using a thermocouple, the production of latent heat during freezing can be detected. For a small insect which supercools several degrees below

0 °C, and which contains only a small amount of water, the freezing event is detected as a blip on the thermocouple trace (Figure 5.2). This allows the temperature at which the insect froze to be determined.

The supercooling point of an insect is usually measured during cooling at a constant rate of 1 °C per minute and this is often taken to be the lower lethal temperature - the temperature below which the animal will not survive. This cooling rate is, however, much faster than those occurring in nature. Slower cooling rates are likely to produce freezing at higher temperatures, since the animal spends longer times at subzero temperatures. The longer an insect is exposed to low temperatures the greater is its risk of freezing. There may also be lethal effects of low temperatures that occur before the onset of freezing. The measurement of the supercooling point of an insect cooled at

1 °C per minute may thus give an over-optimistic estimate of its cold tolerance.

Potential ice nucleators for an insect include ice in the external environment (via inoculative freezing), the molecules which make up its own structure, food in the gut and microorganisms associated with its surface or intestine. Preventing nucleation by these sources allows

Time (mins)

figure 5.2 Freezing exotherm detected by a thermocouple attached to an Antarctic springtail (Gomphiocephalus hodgsoni) during cooling to —40 °C. The insect froze at — 37.1 °C. Data and photo: Brent Sinclair.

the insect to increase its ability to supercool and hence to avoid freezing and survive. Inoculative freezing is avoided by overwintering in dry sites, by having a cuticle which repels water or by producing a cocoon, or some other structure, which prevents nucleation from external ice. Nucleators in the gut could perhaps be avoided by simply ceasing to feed and emptying the gut at the onset of winter, although it may be dif ficult to remove all nucleators by this means. By removing sources of ice nucleation, an insect could supercool to perhaps — 20 °C. To survive to lower temperatures, further adaptations are required.

Freeze-avoiding insects produce a variety of small molecules, mainly sugars or sugar alcohols (polyols), which act as antifreezes. These substances include trehalose, glucose, fructose, glycerol, sorbitol, manni-tol and even ethylene glycol, which is the antifreeze that you add to your car radiator. These reduce the melting point and supercooling point of the insect by their colligative (water-binding) properties, which depend on the number of ions or molecules in solution - so that their effect is directly related to their concentration. By taking up space in the solution, the relative amount of water is reduced, which has the effect of lowering the melting and supercooling points. Some insects accumulate these antifreezes in high concentrations, so that they make up as much as 25 per cent of their body weight. An arctic fly, Rhabdophaga strobiloides, whose larvae overwinter in willow cone galls, produces high concentrations of glycerol during winter, depressing its supercooling point to —56.1 °C. Summer larvae, which contain low concentrations of glycerol, freeze at —26.5 °C. Seasonal patterns in the production of these antifreezes have been demonstrated in many species of insects, with levels increasing during autumn and declining during spring.

As well as these low molecular weight antifreezes, freeze-avoiding insects also produce proteins which act as antifreezes. Called thermal hysteresis proteins or antifreeze proteins, they have similar properties to the proteins discovered in the blood of Antarctic fish. They prevent the growth of small ice crystals by attaching to their surface and hindering the attachment of water molecules to the ice crystal lattice. The properties of these proteins will be considered further later in this chapter when we look at the freeze-avoidance mechanisms of polar fish. The supercooled state of a freeze-avoiding insect is highly unstable and the insect may freeze and die if there is a drop in temperature below its supercooling point or if it comes into contact with an ice nucleator. Both antifreeze proteins and antifreezes such as glycerol may help to stabilise the situation by reducing the chances of ice

FREEZE-AVOIDING INSECTS

prevent ice forming in their bodies

ICE NUCLEATION prevented by:

• emptying the gut

• avoiding surface moisture

• masking/removing ice-nucleating agents

ANTIFREEZE MEASURES

• small molecule antifreezes (e.g. glycerol)

• antifreeze proteins inhibit ice crystal growth

• partial desiccation figure 5.3 Some of the adaptations involved in the survival of subzero temperatures by a freeze-avoiding insect. Insert drawing of a springtail by Jo Ogier.

nucleation. During entry into winter, many insects partly desiccate. The resulting loss of water will increase the concentration of antifreeze components and thus improve the cold tolerance of the insect. There are thus a suite of adaptations involved in freeze avoidance by insects, and these are summarised in Figure 5.3.

Terrestrial Antarctic arthropods consist mainly of mites (arachnids) and springtails (collembola). There are just two species of higher insects, both of them chironomids (Belgioa antarotioa, found on the west coast of the Antarctic Peninsula and its associated islands, and Eretmoptera murphyi, an accidental introduction to Signy Island from South Georgia). The larval chironomids are freezing tolerant, but all the mites and springtails so far examined avoid freezing by supercooling and provide good examples of this method of surviving low temperatures. Professor Bill Block and his coworkers from the British Antarctic Survey, working on the mite Alaskozetes antarotious and the springtail Cryptopygus antarotious at Signy Island off the coast of the Antarctic Peninsula, have investigated their adaptations. They have accumulated data from eight years of studies on the cold tolerance of these species - forming a valuable long-term database.

Both of these animals die when they freeze but can supercool and use a freeze-avoiding strategy to survive the subzero temperatures they experience in the Antarctic (which go as low as — 26 °C in extreme winters). The supercooling points show distinct seasonal patterns, with average values ranging from — 6°C in summer to — 30 °C in winter. The seasonal change is due in part to the production of sugars and polyols as antifreezes. The springtail produces mainly sorbitol and mannitol, while the mite produces predominantly glycerol. The animals cease feeding at the onset of winter, thus emptying the gut of potential ice nucleators from gut-associated microorganisms and food. A. antarcticus produces an antifreeze protein that aids supercooling by inhibiting the growth of ice crystals. This may be important in preventing inoculative freezing since the mites can survive being encased in ice. The cuticle of C. antarcticus repels water, which prevents it from being trapped in water and ice. Both Alaskozetes and Cryptopygus can tolerate some desiccation with the former tolerating water loss that produces a 60 per cent reduction in its fresh weight. Desiccation may enhance cold tolerance by triggering the production of antifreezes, by reducing the amount of water and by increasing the concentration of protective compounds.

Freeze avoidance in other invertebrates

Many insects are fully terrestrial animals which, in most cases, can overwinter out of contact with water. They can largely avoid the problem of inoculative freezing that results from contact with the ice that forms in their environment. They can thus supercool and use a freeze-avoiding strategy. Invertebrates that live in the soil, or in small streams and ponds, which may partly or completely freeze during the winter, are more likely to be exposed to subzero temperatures in contact with water and to have a greater risk of inoculative freezing. Although some nematodes, for example, can survive periods of desiccation (by anhydrobiosis), they are essentially aquatic organisms and require at least a film of water surrounding soil particles for them to be active, grow and reproduce. If they are to use a freeze-avoiding strategy, they need a structure that prevents inoculative freezing from ice crystals forming in their environment.

The potato cyst nematode (Globodera rostochiensis) is a plant-parasitic nematode that evolved, along with its potato host, in the high Andes of South America. It has evolved cold tolerance abilities in response to the subzero temperatures it experiences in its native mountain environment. The nematode has been accidentally spread by humans, along with the introduction of potatoes to many parts of the world. After mating, the female potato cyst nematode becomes full of eggs. She then dies and her body wall becomes hardened to form a cyst, which protects the enclosed eggs - she truly lays down her life for her children. The eggs develop into infective larvae that do not hatch until they are stimulated to do so by chemicals released by the roots of a potato plant growing nearby.

The cysts contain up to 500 eggs and can contain viable larvae after many years within the soil. The eggs are resistant to desiccation and low temperatures. This is why this nematode is so hard to control. If it infests a commercial crop, potatoes cannot be grown on that land for many years. The eggshell, which encloses the infective larva, prevents inoculative freezing, enabling the larva to supercool to temperatures as low as —38°C, even though the cyst and eggs are encased in ice. The eggs supercool to such a large extent because they contain a small volume of water with no nucleators and because the egg fluid contains trehalose that acts as an antifreeze. Other nematodes that are enclosed as an embryo or larva within an eggshell or by a sheath (derived from a partly moulted cuticle) can also prevent inoculative freezing and use a freeze-avoiding strategy.

Invertebrates that can survive anhydrobiotically (some nematodes, rotifers, tardigrades, arthropod larvae) can avoid freezing since, in a state of anhydrobiosis, there is no water present to freeze. When a soil animal prevents inoculative freezing or freezes close to, but not in contact with, ice, it will undergo dehydration. This occurs because the vapour pressure (which is a measure of the tendency of a liquid or solid to change into the gaseous state and thus evaporate) of the supercooled water within the animal is higher than that of the surrounding ice. This happens in earthworm cocoons. Earthworms are restricted to soils that contain sufficient moisture and organic material. The cocoon contains one to 20 eggs (depending on the species), which are surrounded by a tough capsule. The wall of the cocoon prevents inoculative freezing, but is surrounded by ice, and the cocoon's contents thus lose water and it effectively starts to freeze dry. This process was first described by Martin Holmstrup of the National Environmental Research Institute at Silkeborg, Denmark, who considers it to be a separate mechanism of cold hardiness and calls it the 'protective dehydration mechanism' of cold hardiness. This also occurs in some springtails (a group of insects) and enchytraeids (annelids, related to earthworms). It may also occur in other soil or aquatic invertebrates (such as nematodes) which have structures that prevent inoculative freezing.

Freeze-avoiding strategies thus represent a group of mechanisms including: supercooling in the absence of external ice (fully terrestrial species), supercooling in contact with ice outside the body (by preventing inoculative freezing), freeze drying (the 'protective dehydration mechanism') and anhydrobiosis (no freezable water present).

Polar fishes

The cold waters of the oceans of the Arctic and those surrounding Antarctica have sea ice present for much of the year. The salts dissolved in seawater result in it freezing at —1.8 °C to —1.9 °C. The blood of teleost (bony) fish, however, is more dilute than seawater, reflecting their evolution in freshwater and brackish waters. The lower concentrations of salts in their blood means that teleosts freeze at —0.6°C to —1.0 °C, and we might expect those that live in polar waters to do the same. How, then, can they survive without freezing in polar waters, where the temperature is often below the freezing point of their blood? In order to do so, they need to supercool by —1.3 °C to —0.9 °C, which seems a rather modest amount in comparison with the supercooling capacities of some terrestrial insects. Ice crystals are, however, ubiquitous in coastal and surface polar waters. Apart from solid masses of ice in the form of pack ice, pancake ice and icebergs, the surface waters glitter with ice crystals in suspension - frazil ice consisting of floating needles and platelets. Fish will come into frequent contact with ice crystals and, as they are supercooled, are at constant risk of freezing by being nucleated by ice crystals coming into contact with the surface of their skin, their gills and even ingested along with their food.

Fish that inhabit deep polar waters can avoid freezing by supercooling. They are able to supercool because deep waters contain no ice crystals. Ice has a lower density than seawater and hence ice crystals tend to float to the surface. Ice crystals also tend to melt in the slightly warmer deeper waters. However, the temperature is often still below the freezing point of the fish, as was demonstrated in an experiment by Scholander in the 1950s. A fjord cod (Boreogadus saida) can be kept alive in a tank of seawater at —1.5 °C for a long time. If a cube of sea ice is added to the water, it melts and breaks up since the temperature is above the melting point of the seawater. The ice crystals coming into contact with the fish cause it to freeze, by inoculation, and the fish dies. Deep-sea fish are only able to survive in a supercooled state since they do not come into contact with ice crystals, which would cause them to freeze.

The capelin (Mallotus villosus) is a small fish that is one of the most important of commercial fish, caught in large quantities for the production of fish meal and oil. It is found in Arctic waters around the world. It lays its eggs in gravel, stones and seaweed close to the shore and, in some places, even on wave-washed beaches. Here, the fish are exposed to low air temperatures and, since they only survive in moist sites, to freezing in contact with external ice. The egg can supercool to temperatures as low as — 11 °C even when surrounded by ice, since its eggshell (chorion) prevents inoculative freezing. John Davenport, Director of the University of London's Marine Biological Station on the Isle of Cumbrae, showed that even a minute hole in the chorion breaches the barrier and allows the nucleation of the egg contents by ice crystals. He even observed a larval fish hatching from its egg as it was being cooled, with the tip of the fish's tail protruding through a

figure 5.4 The Antarctic cod(Dissosticusmawsoni), a member of the superfamily Notothenioidea. This fish is about 1.2-1.5 metres in length. Drawing by Jo Ogier.

small hole in the chorion. The fish froze, tail first, when it came into contact with ice.

Polar fishes that live in coastal waters cannot avoid contact with ice. It is present at the surface, on the bottom in shallow waters and as floating crystals in the water column. How can these fish survive in waters which are colder than the melting point of their blood and where they are at constant risk of freezing as a result of contact with ice crystals? Although there are nearly 20000 species of teleost fish, only 274 are found south of the Antarctic Polar Front. Nearly 95 per cent of fish caught in Antarctic coastal waters belong to the superfamily Notothenioidea, the Antarctic perches (Figure 5.4). The low diversity of Antarctic fish may be partly a reflection of their isolation but may also be due to the limited number of species that are able to solve the problems of living in Antarctic waters. Professor Arthur DeVries, now at the University of Illinois-Urbana, first obtained a clue as to how the Notothenioid fish do so. DeVries has been visiting Antarctica for nearly 40 years - during the period of 1969-1998, he only missed two field seasons. The clue he found early in his career was from the results of freezing different components of the blood of Notothenioid fish.

The blood of Notothenioids melts at —0.8°C, but does not freeze until —2.0 °C. There is thus a difference between the melting and freezing points (a phenomenon known as 'thermal hysteresis'). Since the temperature of Antarctic waters does not fall below —1.9 °C, it remains above the freezing point of the blood and this means the fish can survive indefinitely without freezing. Since the blood of non-polar teleosts does not show this property of thermal hysteresis, there must be something special about the blood of Antarctic Notothenioids.

The molecules dissolved within the plasma of the blood of Notothenioids can be separated into small and large components, by passing them through a membrane that allows the passage of small but not large molecules. The resulting solution containing small molecular weight components, mainly salts such as sodium chloride, freezes and melts at the same temperature as the melting point of the whole blood, —0.8 °C. The thermal hysteresis is due to large molecules in the blood plasma known as glycoproteins (a protein linked to a carbohydrate). These antifreeze glycoproteins comprise as much as 4 per cent by weight of the blood of the fish.

The antifreeze glycoproteins of Antarctic Notothenioids act in a different way to the low molecular weight antifreezes, such as glycerol, which are found in freeze-avoiding insects. The antifreeze proteins of fish attach to the surface of ice crystals and inhibit their growth by preventing further water molecules from joining the ice crystal lattice. If an ice crystal is introduced into a purified solution of antifreeze protein, at a temperature between its melting and freezing points, it will remain but not grow even after a week. Fish antifreeze proteins do not just slow the growth of ice crystals, they stop it completely. The freezing of the supercooled fish is thus prevented since any ice crystals that find their way into their bodies are immediately coated with antifreeze protein. If the temperature is lowered sufficiently, the inhibition will be overcome and an ice crystal will grow. Antifreeze proteins lower the freezing point of the fish by just a couple of degrees but this is enough to enable them to survive in an environment where the temperature does not fall below —1.9 °C.

Antifreeze proteins are present not just in the blood of the fish but also in the fluids of the body cavity and those of the heart, liver and muscles. These proteins are produced in the liver and circulated around the body. The brain is the only organ in which antifreeze proteins have not been found but this is surrounded by an extradural fluid which does contain them and which presumably acts as a barrier to ice formation. The gut is a particularly important site of potential ice nucleation since ice crystals are ingested along with the food. Antifreeze proteins are secreted into the gut via bile and there is evidence that they may have evolved from gut enzymes (see Chapter 8). There are no antifreeze proteins in the urine since they are filtered out by the kidneys, a process which retains the proteins within the blood.

Just about all Antarctic teleosts contain antifreeze glycoproteins. Although the Antarctic fish fauna is dominated by Notothenioids, there are fish from other families such as eel pouts (Zoarcidae) and icefish (Channichthyidae) that contain them. Arctic fish from a number of families also contain antifreeze proteins. These include Arctic and Greenland cod (Gadidae) and winter flounder (Pleuronectidae). These proteins have evolved independently in a number of different groups of fish to solve the problems of survival in icy waters. This is a striking example of convergent evolution, which I will tell you more about in Chapter 8. The antifreeze proteins of many Arctic fish appear on a seasonal basis, only developing significant concentrations during the winter. The production of antifreeze proteins in these fish is triggered by both decreasing temperatures and shorter day lengths and is under hormonal control.

Since their discovery in Antarctic fish, antifreeze proteins have also been found in freeze-avoiding insects. Here, they play a similar role by preventing the growth of ice crystals and thus stabilising the supercooled state. Antifreeze proteins are also called thermal hysteresis proteins in insects. They often have a much greater hysteresis activity than the antifreeze proteins from fish, producing a difference between the melting and freezing point which may be as much as 11 °C. These proteins have been reported from a variety of insects (although beetles feature most prominently) and from spiders, centipedes and mites. Antifreeze proteins have also been reported from freezing-tolerant insects and from other freezing-tolerant invertebrates (nematodes and molluscs). Their role may be rather different in this situation, as I will describe later, and these may turn out to be a rather different group of proteins. Antifreeze proteins have also been discovered in plants.

Polar fish have had to solve other problems which result from exposure to low temperatures, apart from the risk of freezing. Their enzymes, membranes and physiological processes have to function at temperatures which are constantly at about — 1.9°C. Liquids tend to become more viscous or sticky at low temperatures; try keeping your bottle of golden syrup in the fridge, it makes it harder to pour. The fish's heart needs to pump blood around the body to distribute food and oxygen to the tissues. The low temperature tends to make the blood more viscous and harder to move. This is offset by the fact that more oxygen will dissolve in the blood at low temperatures than will at high temperatures. The fish can thus make their blood less viscous by reducing the numbers of red blood cells, and hence the amount of haemoglobin (which carries oxygen), and yet still supply the tissues with enough oxygen. Icefish (Channichthyidae) have carried this to its ultimate conclusion. They have no haemoglobin or red blood cells, earning them the name white-blooded fish, and all their oxygen is carried dissolved in the plasma or fluid of the blood. Their blood can carry only 10 per cent of the oxygen transported by an equivalent volume of blood from a red-blooded fish. They compensate for this by having larger hearts to circulate a greater volume of blood and by having more blood vessels in their gills and skin to extract oxygen from the seawater.

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