Feature

Suspended Animation

What happens to an insect when it freezes and comes alive again?

Jennifer Bedford and John Leader

When Moses led the Israelites through the wilderness, he quelled discord and saved their lives with a "miracle" food: manna.

Manna was almost certainly the sap of a desert plant, passed through the gut of an insect and excreted, before being sun-dried. Its major constituent was the sugar trehalose. Having long ago saved the Chosen People, several thousand years later trehalose has once again achieved the potential to become a significant player in the international economy. Trehalose may well prove to be a highly effective agent in the long-term preservation of biochemicals, pharmaceuticals, cells and even whole organs.

Modern interest in the effects of extreme stress on living things dates from the 17th century. In 1675, the first microscopist, Anthony van Leeuwenhoek, discovered, to his surprise, that when he added water to the dried scrapings from a gutter, many organisms appeared and swam about.

The "animalcules" that he saw were a variety of small animals. Besides a number of protozoans, he also found tardigrades, rotifers and nematodes. Nowadays we know that all these animals possess a significant feature in common in addition to their tolerance of dehydration -- a high concentration of the sugar trehalose in their bodies.

Trehalose is a disaccharide, comprising two molecules of glucose joined together. Possession of high concentrations of trehalose is common in a wide range of organisms which are exposed to water stress, either as a result of dehydration or low temperature.

In New Zealand, the pinkish tinge of the shores round the evaporation ponds of the salt factory near Blenheim is due to the presence of a small shrimp-like organism, Artemia salina. The eggs of this organism, found in salt lakes throughout the world, can be totally dehydrated. Even though no trace of life, as judged by normal criteria, can be detected, the eggs can remain in this state of suspended animation for many years. Once water is provided, the eggs swell up and immediately continue their development.

An even more spectacular example is that of the larva of the midge Polypedilum vanderplanki. This little insect lives in the mud at the bottom of small ponds throughout central Africa. When the ponds disappear in the dry season, the larva desiccates as well. All its vital activities cease, and it can remain for at least 50 years in this non-living state of "suspended animation" or cryptobiosis.

As soon as it rains again, it takes up water and resumes its life. The clear red granule swells rapidly as water diffuses into it. In about 10 minutes the pharynx, or foregut, can be seen to begin rhythmic contractions. Shortly afterwards, the heart starts beating and the rest of the larva begins to swell. In about 20 minutes, the larva is fully hydrated and actively feeding. More remarkably, since in the dry state no living processes occur, damage that would destroy the active larva in seconds can be inflicted in the dry state without any immediate consequence. The insect cannot die until it is once again rehydrated.

Practical Uses

Interest in this process of suspended animation has always been widespread, for reasons ranging from the bizarre desire of some people to live for ever, to a variety of more practical objectives, such as preservation of tissues and organs and embryos of humans for transplantation or the treatment of infertility, maintenance of animal material for stock improvement, or even the storage of rare and endangered species. Additionally there are strong economic pressures to find better and cheaper ways of preserving unstable biochemicals and pharmaceutical products.

A major step in achieving this was reached 50 years ago when Dr Audrey Smith first demonstrated the role of the antifreeze agent, glycerol, in tissue preservation. She added glycerol to suspensions of cells before cooling them to the temperature of liquid nitrogen (-186^oC), a temperature at which all living processes are slowed to immeasurable levels. When the cells were thawed again she found they showed a remarkable degree of recovery.

This discovery set in train a vast programme of agricultural and medical research. Successful protocols have been developed for the long-term preservation of a wide range of tissues and cells at low temperature. Indeed this longevity has led to some intriguing court battles as lawyers argue over the ownership of human eggs, sperm and embryos.

More mundanely, the ability to store semen of agriculturally important animals has led to rapid and sustained improvement in bloodstock throughout the world. A prize bull can become the sire of thousands of progeny. Desirable genetic features of sheep or pigs can be rapidly disseminated throughout the world.

There are however limitations. Not only is the freezing process only suitable for single cells, or small groups of cells, in suspension, but storage at liquid nitrogen temperatures is expensive and requires continuous monitoring. Furthermore not all cell types can be treated in this way.

It is for those reasons that two recent reports in the journal Nature Biotechnology have attracted attention. In February, Professor Levine reported that human cells, transfected with an adenovirus containing two genes encoding the enzymes for synthesising trehalose, could be totally dehydrated for up to five days. When rehydrated, the cells grew normally.

In a different approach, Professor Toner, from the Shriner Hospital of Children in Boston, used an engineered bacterial protein inserted in cell membranes, to enable human cells to take up and incorporate trehalose. Such cells could readily tolerate freezing to low temperatures. While these experiments are only a beginning, they point to new directions for the indefinite preservation of living material. Preservation in a dried state is not only inexpensive, but allows storage in, or transport to, regions of the world where access to liquid nitrogen is difficult or impossible.

We have approached this problem from a different direction. Together with Dr Helen Palmer, a Post-Doctoral Fellow funded by a Marsden grant, we have been studying the effect of dehydration and freezing stress on living cells and their constituent molecules.

In many parts of New Zealand, the air temperature can fall in winter to well below zero degrees Celsius for long periods. While this can be a source of pleasure to those humans who enjoy winter sports, it poses severe problems for cold-blooded animals.

The salty solution in which animal cells are bathed is liable to freeze at temperatures only a few degrees below the freezing point of water, and for many animals this is a lethal event, as the growing ice crystals disrupt cells and tissues, tearing cells apart, rupturing their membranes, and crushing organelles. In addition, when the body fluids freeze, pure water separates as ice, leaving behind a salty solution which becomes progressively more concentrated as freezing proceeds, destroying proteins and inhibiting the chemical reactions which sustain life.

These consequences are so damaging for most animals that they go to extreme lengths to avoid ice formation inside their bodies. Perhaps the commonest strategy is to hibernate, by seeking out an insulated spot where the temperature rarely if ever falls below zero. Burrowing only a few centimeters into the soil can often achieve this, and in places subject to snowfall, the snow itself can serve as an insulating layer beneath which life can continue normally.

Animal Antifreeze

Many insects protect themselves from the lethal effects of ice formation in much the same way as we protect our cars in winter, by additing a chemical antifreeze to the body fluids. The antifreeze found most commonly in insects is the polyhydric alcohol glycerol, demonstrating once again how science imitates nature.

Glycerol has several desirable features which make it useful for mitigation of freezing damage. It is non-toxic in high concentrations, mixes freely with water, and because of the hydrophilic properties of glycerol, mixtures of glycerol and water only begin to freeze at temperatures well below 0^oC.

This has several benefits. First, as it does in car radiators, glycerol can lower the freezing temperature to values which are rarely reached in nature, so that the insect never has to face the adverse consequences of ice formation. Secondly, the higher the concentration of glycerol, the lower is the temperature at which ice formation begins. As a result, the damaging consequences of ice formation are diminished, because both chemical and physical effects are slowed down at lower temperatures. It was these properties of glycerol which first made possible the preservation of mammalian tissues and organs at low temperatures.

Freezing is a stochastic process, meaning that a solution cooled below its freezing point will not necessarily freeze immediately. Such a supercooled liquid has a probability of freezing that increases both time and the degree of supercooling.

Some insects, for example may become supercooled by several degrees for long periods, and this may enable them to survive temperatures several degrees below freezing. This property may be enhanced by the ability of the insect to manufacture antifreeze proteins, which appear to act to prevent the growth of ice crystals, by becoming aligned on their growing facets, and thus impeding the addition of further water molecules.

However, when a supercooled liquid does freeze, it does so suddenly, causing sharp-fronted ice crystals to grow rapidly through the liquid. In an animal, these crystals tear apart cell membranes and organelles.

Supercooled insects thus live dangerously in winter, since if freezing does occur, the consequent disruption is generally fatal. If, therefore, there is a likelihood that the insect will be exposed to freezing temperatures, a better survival strategy is to encourage freezing to occur at a temperature close to the freezing point, so that ice crystal growth is slow and less disruptive. This is the tactical solution adopted by the montane weta, Hemideina maori.

Weta Ices

The montane weta is found throughout the South Island, where it lives in a variety of habitats from near sea level to high altitude. A spectacularly large variety is found under the large schist slabs above the 4,000-ft contour on the Rock and Pillar range. In this environment temperatures may drop below freezing at any time of the year, and in winter may fall to as low as -6 to -7^oC. Furthermore, the high winds experienced at this altitude means that most of the snow is blown away, so that the insects are exposed to the full effects of such low temperatures.

In the laboratory, H.maori can be frozen to temperatures of at least -10^oC for several hours. At this temperature about 90% of the body water is ice, and the tissues are then bathed in a solution more than 5x as concentrated as seawater. In this state the insects are rigid and brittle.

Nevertheless, when allowed to warm up, they soon thaw and become active again. Hemideina is easily the largest insect, and probably the largest animal, able to tolerate such low temperatures.

Climate records from the Rock and Pillar range suggest that this cycle of freezing and thawing may occur daily during the depths of winter, and that sometimes the insects may have to survive in the frozen state for several days at a time. Understanding how the insect may overcome such stresses may have important implications for the long-term preservation of human organs for transplantation and storage of agricultural products.

One important feature of the freezing tolerant ability of this insect lies in the composition of its blood. Studies have shown that the blood contains proteins which encourage the formation of ice at temperatures close to the freezing point. In winter, it also contains high concentrations of the sugar trehalose and an amino acid, proline. Trehalose concentrations may reach as high as 100 gm/l blood, about 100 times the concentration of blood sugar in humans.

Both of these substances seem to protect cells against the damage caused by freezing. Unlike mammalian cells, insect cells have transporting enzymes in their cell membranes, which can take trehalose from the blood and transport it into cells. Here the high sugar levels probably prevent the destructive formation of intracellular ice.

Nature in the Lab

It is possible to mimic the processes which go on during freezing by using isolated tissues of the weta. The "kidney" of insects is a good tissue to use for this purpose. It is formed by a large number of blindly ending tubules, the tubules of Malpighi, which float free in the body cavity. These tubules, formed by a single layer of cells, generate a secretion which drains into the gut, and which forms the primary urine.

It is a simple matter to anaesthetise the weta, make a small cut in its side, and pull out a few of the hundreds of tubules. The hole rapidly seals and the insect quickly recovers. Tubules can be transferred to an artificial solution, where they are contained within a small drop of a salty solution under paraffin, and the cut end is drawn out into the paraffin. The secretion from the tubules, the primary urine, appears as a droplet at the cut end, from where it can be collected in a fine pipette and its amount and composition analysed.

The viability of the tubules can be tested by adding small amounts of a bright red dye, phenol red, to the bathing solution. Functional tubules will avidly secrete the dye, so that the lumen of the tubules and the drop of secretion becomes bright red. The tubules are very hardy and remain functioning for up to a week if they are stored, in a suitable solution, in the refrigerator. This offers a valuable method of testing the effect of freezing on the cells.

Trehalose is clearly important for the preservation of function after freezing. Tubules frozen without trehalose in the bathing solution disintegrate when thawed. In the presence of concentrations of trehalose similar to those found in the blood, the tubules can be frozen and thawed several times without ill effects.

A number of recent experiments suggest that trehalose is acting in several ways. It happens to be the right molecular shape to fit onto the heads of the lipid bilayers which make up the cell membranes, helping to stabilise them. It is very hydrophilic and so imposes a structure on the water molecules in its immediate vicinity. This helps, by competing for the water shell which surrounds all proteins inside the cell, to pull protein structures more tightly together, and so prevent them from being distorted and damaged by the high salt concentrations which arise during freezing.

By acting as a physiologically neutral osmotic "filler" within cells, it also helps to retain water around the cellular machinery, enabling cell organelles to retain structure. Finally, trehalose solutions when frozen tend to form non-crystalline glass instead of ice crystals, minimising physical damage.

At present, it is not known which of these many functions of trehalose plays a key role in enabling H. maori to survive freezing. Our research is mainly aimed at clarifying the ways in which proteins are protected by trehalose during stress, since this is likely to lead to the most useful data.

It is important to remember that, although in recent years rapid strides have been made in analysis of macromolecular composition and structure, those phenomena which characterise the living system are additionally a function of supramolecular spatial arrangements, disturbance of which can lead rapidly to disruption and death.

Where it is protective of freezing and drying stresses, trehalose appears to function as a water substitute, forming a protective shield round macromolecules and thus preserving not only their structure, but also their physical relationships. Removal of water from the living cells, by freezing or drying, in the presence of high concentrations of trehalose, might then be merely a process of extracting water molecules from the interstices, imposing minimal physical stress. Rehydration would merely reverse the process, enabling restoration of living reactions.

Such a process could be universal. To achieve this however is to advance from what is still empirical interpretation of observed data towards a clearer theoretical understanding of the nature of the interactions between macromolecules and the aqueous environment within which all living processes occur. Such an understanding might bring us to a new promised land.

Jennifer Bedford is from the University of Otago
John Leader is from the University of Otago.