This section of our course is devoted to adaptations to environment. We can regard the material in this section in several terms, including physiological ecology, enviornmental physiology or comparative physiology. As mentioned earlier, all these terms are meant to convey an interest in how insects are adapted to live in the habitats they occupy. Now let us move away from our interest in the importance and mechanisms of maintaining a homeostasis of water and salt balance. In this chapter, we consider some of the adaptations to life in northern temperate and polar regions of our planet. We begin with the idea that insects inhabiting these regions are subject to freezing temperatures for a greater or lesser proportion of each year. Our main interest will lie in understanding how insects are adapted to very cold environmental temperatures.

One adaptation for life in freezing temperatures is to protect the body from freezing conditions. Many insects, and some fishes, protect themselves from freezing by biosynthesis of anti-freeze compounds. These compounds convey some degree of frost resistance, that is, an avoidance of freezing. The hemolymph concentrations of anti-freeze compounds increase and decrease in a seasonal pattern. The slug caterpiller Monema flavescens, is a good example. In this insect, concentrations of the polyol glycerol increase in hemolymph as winter approaches. As outdoor temperatures decrease in September there is a reduction in body sugar levels, which remain low throughout the winter. Attending this drop in body sugar is an increase in glycogen. Glycogen is nothing more than a long polymer of linked glucose molecules. When ambient temperatures drop to about 10 oC, glycogen rapidly decreases in this species while glycerol increases to about 25 mg/g body weight. After winter, as environmental temperatures increase to 20 oC, glycogen and glycerol concentrations are inverted again. In this example, increasing hemolymph glycerol to about 5% of fresh body weight is associated with tolerance of freezing temperatures as low as -20 oC. It appears that some insects can avoid freezing by generating anti-freeze.

It is fair to ask something like, well, OK, how do we know the gylcerol protects the insect body from freezing? The outcome of a well-designed experiment shows glycerol does, in fact, convey frost resistance to pupae of M. flavescens. Two groups of insects were treated. In one situation pupae were kept in chambers at 20 oC. Insects maintained at this high temperature express no increase in hemolymph glycerol during the winter period. The low glycerol concentrations correspond to a relatively small increase in frost resistance, which reaches a peak in early November. The other group of pupae were reared outdoors through the fall. Glycerol rapidly increased during October. The increased glycerol concentration was accompanied by a dramatic increase in frost resistance.

Rates of increase in body glycerol concentration are often related to the ambient temperatures insects experience. We can consider the glycerol concentrations in several groups of overwintering Papilio pupae. Each group of pupae were maintained at a separate ambient temperatures. Pupae kept at 20 oC experienced a small, baseline increase in body glycerol. Glycerol levels increase at higher rates when the pupae were kept at lower temperatures. Of course, glycerol is biosynthesized by enzyme actions, and at very low temperatures glycerol synthesis capability is reduced. For example, glycerol synthesis at 0 oC is faster than at -5 oC.

The next point we want to make is that glycerol is a common cellular metabolite, which we appreciate by regarding glycerol biosynthesis. The glycolytic pathways were presented in the treatment of energy metabolism. The aldolase reaction is a key step in this pathway. Aldolase splits one 6-carbon compound, fructose-1,6-diphosphate, into two 3-carbon compounds, dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate. The step from DHAP to glycerol-3-phosphate (G-3-P) is very common because this compound provides the backbone structure for biosynthesis of triacylglycerol, with which we are comfortably familiar. The phosphate is removed from G-3-P by phosphatase, yielding glycerol. My speculation is that glycerol synthesis is regulated at the phosphatase step. Overall, glycerol is just two metabolic steps away from DHAP, a common intermediate in the glycolytic pathway.

Please recall that in spring months glycerol levels are reduced, with a concomitant increase in glycogen. This is arranged by simply reversing the steps just covered in glycerol biosynthesis. The aldolase reaction is commonly reversible, as are most of the steps in glycolysis. The flow of carbon from glycogen >>> glucose >>> glycerol and back again glycerol >>> glucose >>> glycogen is a straight forward metabolic pathway. The key point is recognizing the carbon atoms are not lost, they are simply moved around some.

It seems appropriate to ask: How does glycerol increase the cold hardiness of an insect? The answer is glycerol probably works in three ways. One is freezing point depression, due simply to colligative properties of molecules. A second is supercooling. Finally, glycerol provides protection from ice damage to tissues and cells. First, we recollect from basic chemistry courses what are known as colligative properties of solutes. By this we mean those effects of a solute that express their influence by the number of particles, rather than by the nature of particles. For example, a 1.0 molal solution (1 mole of solute in 1 liter of water) lowers the freezing point of water by 1.86 oC; it also raises the boiling point and creates other physical sorts of events. The influence of all colligative properties can be calculated from the influence of any other colligative property. The idea for our purposes is that one effect of increasing hemolymph glycerol concentrations is to lower the freezing point of body fluids due to the colligative properties of solutes. Putting this together with our information on regulation of osmotic balance, it should be clear that colligative properties alone would not be a very effective overall strategy to avoid freezing.

The second mechanism allows lowering temperatures of body fluids below freezing points without formation of ice crystals. This is called supercooling. Let us consider a single example. First, animal body fluids are not pure water, and animal freezing points are typically a bit lower than 0 oC. Second, with supercooling, body fluids can decline considerably below zero without freezing. Finally, a supercooling point obtains. This is the point at which supercooled fluids freeze. We usually see a small temperature rise at this point, due to heat of fusion.

We can consider a graph illustrating supercooling in pupae of Papilo xuthus. When these pupae are cold hardened they can be cooled to about 20 oC below the usual freezing point without ice crystal formation. The temperature at which the fluids finally do freeze is called the supercooling point. This event is associated with a sudden rise in body temperature due to heat of fusion. On a larger scale, the temperature increase due to heat of fusion is why very large lakes exert a moderating effect on environmental temperatures in some climates. Many insects from northern latitudes can be supercooled to 20 to 30 oC below 0 oC. In a more extreme case, larvae of the braconid wasp Bracon cephi can be supercooled to -47 oC.

Some insects appear to actually freeze because environmental extremes far exceed supercooling capacities. In these cases glycerol also seems to protect tissues and cells from injuries that otherwise obtain due to ice crystal formation. This non-specific protection from ice crystal formation is well known. Glycerol has been used for decades in laboratories to protect vertebrate tissues during freezing. For example, human sperm can be kept frozen, and viable, for years if glycerol is added before freezing. Freezing is lethal to sperm that are not protected with glycerol.

Glycerol exerts three actions in cold hardiness. Glycerol acts as a cryoprotectant by lowering freezing points of body fluids, by enhancing supercooling capacities and by protection from ice-crystal formation. Glycerol is not the only substance seen in studies of cold hardiness. The other most commonly found compound is the 6-carbon polyol sorbitol. Sorbitol is formed by a single step reduction of glucose-6-phosphate, followed by removal of the phosphate group. The sortibol mode of action within a biological matrix appears to be much like the action of glycerol. Beyond glycerol and sorbitol, a few insects increase trehalose concentrations for onset of winter. Still other species biosynthesize mannitol, another polyol.

Although it is certain that glycerol can exert an important cryoprotectant influence on many insects, there still exists a certain ambiguity as to its precise role. Looking at some data on the point, we find examples of three cases: In one case, for example the lepidopteran mentioned earlier, Monema flavescens, the prepupae accumulate glycerol (to about 5% of fresh weight) and gain protection from freezing. In a second case, insects accumulate glycerol, but they do not gain protection. This occurs in extreme cases in some species. For example, larvae of the beetle Dendroctanus manticolae accumulate up to 23% of fresh body weight in glycerol and gain no protection from freezing. In a third case, insects do not accumulate glycerol, but they are able to tolerate very cold ambient temperatures. Larvae of the lepidopteran Hestina japonica, for example, can tolerate freezing to -15 oC with no increases in body glycerol.

These data are not presented to suggest that the formation of glycerol does not play the frost resistance roles we just considered. Rather, we want to take the idea that other, yet unspecified, biochemical activities appear to act in the physiology of cold hardiness.

Now, the physiological mechanisms we have looked at so far suggest that insects either build up anti-freeze compounds, or they develop a capacity to supercool, which may or may not involve glycerol. These strategies are known as freezing resistance. Another strategy is known as freezing tolerance. Freezing tolerance is the ability to survive formation of ice within an organism. Freezing tolerance is known in a number of insect species. The larvae of one species of arctic midge, for example, overwinters in solidly frozen pools. These larvae begin to freeze at -1 to -2 oC. They can be frozen to -25 oC and subsequently thawed without injury to the tissues. They can, in fact, tolerate many cycles of freezing and thawing. In the neighborhood of -15 oC, about 90% of this midge's body water is frozen. How can some insects tolerate freezing?

One explanation is called the site of freezing theory. The first point is that intracellular freezing is fatal, whereas extracellular freezing, to some extent, is not necessarily fatal. A second point is that freezing usually begins extracellularly, with any freezing within intracellular compartments occurring subsequent to extracellular freezing. This is because the formation of ice crystals depends entirely upon the chance of an aggregation of water molecules getting to the critical size of a crystal nucleus; the probability of such nucleation increases with the bulk volume of water, which is greater in extracellular compartments. As ice develops around the cells, an increase in concentration of extracellular osmotic particles attends the decrease in free water. This is followed by movement of water from the intracellular compartment into extracellular compartment, thereby increasing solute concentrations within cells. Loss of intracellular fluid has two effects. First, the increased solute concentrations makes cells more resistant to freezing. Second, if a cell does freeze, the resulting ice crystals are much smaller, and they do less physical damage to intracellular organelles.

One prediction that is consistent with the site of freezing theory is that the rate of cooling could effect outcomes of freezing experiences. Here are some data from the sawfly Trichiocampus. In the following experiments all insects were cooled to -20 oC, but at different rates of cooling.

These data are consistent with the idea that insect physiologists do some pretty weird experiments. The data also show that when cooled very rapidly, the intracellular and extracellular body fluid compartments can freeze at about the same time, without a crucial time lag between initial freezing in extracellular compartment and later freezing within cells. The time lag would allow for osmotic movement of water from intracellular to extracellular spaces. It is thought the deleterious effects of intracellular freezing in the fully hydrated state result from mechanical damage. Expansion of ice crystals within cells would disrupt cellular organization, injure organelles and possibly rupture membranes. Slower cooling rates allow time for water to move out of cells before they freeze, thereby lessening potential for physical damage.

We can infer a little bit more from this information. At least part of the ability to survive freezing is related to ability to withstand dehydration. The most extreme example of the ability to survive at very low temperatures comes from studies on larvae of the midge Polypedilum vanderplankii. These larvae are active in temporary pools in tropical Africa, an area little supposed to have much to do with freezing. The temporary pools are regularly subject to complete dehydration, and the larvae are adapted to survive loss of up to 92% of their body water. In studies incidental to this primary adaptation, it was shown these larvae can be cooled to -270 oC (essentially to the lowest temperature obtainable in our universe) and immediately resume activity upon being thawed and rehydrated. I imagine surviving water loss during naturally slow cooling processes is the primary adaptation to freezing.

Most insects living in high mountains or in cold temperate regions are well adapted to resist or to tolerate freezing. Nonetheless, the most extensive adaptations to cold environments are seen in the relatively few insects inhabiting the high arctic and a few islands in the southern polar regions. In these areas of relatively harsh circumstances, with low temperature, high winds and brief periods in which to affect mating and growth, freezing tolerance is but one of several adaptations to special situations.

The most extensive specializations appear to occur among members of the Simuliidae, commonly known as the black flies. In ordinary, off-the-shelf southern species, females fly in search of blood meals from avian or mammalian hosts. After feeding females settle down until blood is digested and eggs are matured. Eventually the females embark on oviposition flights, following which another cycle of feeding, egg maturation and oviposition may begin afresh. A female may complete several such cycles during her adult life. Flight is also important in mating, because males and females meet in swarms where males respond to females that are detected visually through enlarged facets in upper parts of their compound eyes.

Unlike their less extreme relatives, most the adult female of most arctic species is a modified non-feeding form. Mouthparts in these species are so reduced that it is not possible to penetrate host integuments. Their eggs are developed on nutrients that were accumulated in larval stages. In fact, eggs start to develop before adults emerge from pupal cases. In some species the eggs are mature at adult eclosion. These changes in developmental patterns have the effect of telescoping the life cycles. Moreover, mating occurs as a result of contacts made on the ground near sites of emergence, rather than in a swarm. Mating chances are enhanced by minimal dispersal of adults and by dense larval aggregation in immature stages. Often the enlarged eye facets of males are reduced. The main points are reduction of feeding and mating flights, early egg development and re-designed mate-finding systems. These adaptations reach even greater extremes in a few species: populations occur as male-less pathenogenic reproductive females.

Other dipterans inhabiting the tundra regions express similar adaptations. The biting midge Culicoides is autogenous, that is, it does not require a blood meal for egg maturation, and their mouthparts are slightly reduced. Also, in most arctic Chironomidae mating takes place on the ground without a mating flight. There are also examples of parthenogenic races. In arctic Tipulidae the wings are reduced to a greater or lesser extent; females of one species have only vestiges of wings.

Arctic mosquitoes tend to retain their normal appearances, but most species are facultatively autogenous. Two species of Culiseta hibernate as mated, but not blood-fed adults while some Aedes species hibernate as eggs. Even though most of these species can mature their eggs in the absence of a blood meal, there is reduced fecundity associated with this trait.

The most striking feature of arctic lepidopterans is the long length of larval lives. Larvae may develop over a period of five years or more before finally pupating. These larval stages are remarkably tolerant of extreme cold and exposed habitats in winter. Other insect groups are represented in arctic regions, including aphids, scales insects, lice, bumblebees, flightless beetles, mayflies and caddisflies. A few common ideas run through the adaptive complex of all insects found in arctic regions. Flight in unstable thermal conditions that are punctuated by high winds is a very hazardous business. Many of the adaptations noted here are well suited for reducing the importance of flight. Host and mate finding may be very difficult under these extreme conditions, and certain points tend to minimize these requirements.

A final point is that these insects, as well as many other temperate species, share the ability to survive in temperatures well below freezing.