Ability to survive freezing

A significant number of insects are known to be able to withstand freezing of body fluids, but a few species of anurans are the only vertebrates in which a similar ability has been observed. These anurans provide a valuable resource to those interested in the possibility of generating effective cyroprotectants in mammals.

Wood frogs utilize glucose as a cyroprotectant ("anti-freeze") agent. Glucose is synthesized and sent to body tissues in response to ice crystals forming in the body. Wood frogs don't act in anticipation of freezing; they can be exposed to long periods of near-freezing temperatures without beginning to produce glucose, but once the temperature drops a critical amount their bodies will quickly begin to synthesize the cyroprotectant (Storey 1984). This phenomenon is probably caused by the fact that wood frogs often find it unnecessary to withstand freezing temperatures during the course of a given winter. They usually hibernate within a foot of the surface of the ground, and with a heavy snow cover may never be exposed to freezing temperatures. On the other hand, an individual may find it necessary to freeze and re-thaw several times during the winter. By waiting to synthesize glucose until ice crystals actually begin to form, wood frogs avoid generating their cryoprotectant unnecessarily. Glucose is an ideal cryoprotectant for such short-notice synthesis, because it need pass through only three enzymatic steps to be created from glycogen (Storey 1984). The glucose used as a cryoprotectant is synthesized from glucose found in the liver, and the ability of an individual to withstand freezing varies dramatically between fall and spring as the level of available glycogen fluctuates. In the spring, when wood frogs have little glycogen in reserve, they are only able to accumulate 8umol/mL of glucose in their blood. In contrast, in the fall, with lots of glycogen at hand, they are able to increase the level of glucose in their blood to 300-500 umol/mL (Costanzo 1998). One measure of an individual frog's ability to withstand freezing is the minimum body temperature the individual can survive without suffering permanent damage. This temperature is known as the Lowest Lethal Temperature (LLT). One study found that frogs collected in the fall had a LLT of -5C to -6C, whereas individuals collected in the spring demonstrated an LLT of only -3C (Costanzo 1998). It was found that injecting ecogenous glucose into the spring-collected individuals augmented the natural cryoprotectant, and decreased their LLT.

Freezing generally occurs at -2C to -3C, although the LLT varies among populations found at different latitudes. Frogs in Ohio, for example, have been shown to suffer lethal damage when subjected to temperatures of -5.5C, while populations 600km to the north, in Canada, survive at even lower temperatures (Costanzo 1998). When wood frogs freeze, almost 50% of their body water may be converted into ice crystals (Storey 1984). In most organisms, such freezing would be lethal. The main reason is that intracellular freezing disrupts osmotic balance, and causes cell dehydration. When an organism freezes, the pure water in its body freezes first. This increases the extracellular osmotic concentration, causing the cells to dehydrate, which disturbs cellular structure and can lead to cell death. Extracellular freezing interferes with the delivery of necessary oxygen and nutrients to cells. In most organisms, these disruptions cause death when the animal thaws (Zug 254). The cryoprotectant utilized by wood frogs prevents intracellular freezing and ameliorates the effects of extracelluar freezing: it reduces osmotic shrinkage, keeps cell membranes intact, and acts collagatively to reduce the amount of ice that forms in the body. Thus, the wood frog is able to withstand freezing that would be lethal to almost every other vertebrate. The external indications of freezing in the wood frog are remarkable: the body becomes stiff, the abdomen rigid, and the eyes opaque. Breathing and heartbeat are suspended, and internally, ice fills the abdominal cavity and surrounds the organs and muscles (Storey 1984).

Wood frogs have the ability to survive for a substantial period of time while frozen. Experiments have shown that virtually all individuals can survive for 2 weeks at -1.5C (Costanzo 1991); some can survive for significantly longer. Frogs collected in autumn have the ability to survive longer than those collected in spring, because of the difference in available glycogen reserves, as previously mentioned. The longer the period of freezing the less likely the frog will be able to fully recover (Costanzo 1991). Wood frogs can thaw within a space of 12 to 24 hours (Storey 1984). The first functions to return are the heartbeat, vascular circulation, and other basic physiological activities; then the frog regains its power of movement. Wood frogs can withstand multiple cycles of freezing and thawing within a given winter.