Freeze Tolerance in Wood Frogs (Rana sylvantica)

Photograph of a wood frog. Used with permission of Michael Dorcas.

Box turtles, garter snakes, wood frogs, and many insects are able to survive freezing. Wood frogs must have this ability because they hibernate in shallow depressions in the forest where they are exposed to freezing temperatures. The skin of the frog cannot keep out ice from the environment, so freezing starts in the body by inoculation. In dry environments, a bacterium is used as an ice nucleating agent. Both of these strategies ensure that freezing starts at relatively high temperatures (around -0.5*C) which slows the rate of freezing. Also, the body temperature of the frog rises when water first freezes (the heat of fusion), which further helps slow the rate of freezing. The slow rate of freezing helps prevent ice from forming in the cells, allowing survival of the frogs. The increase in temperature after the release of the heat of fusion also leads to an increase in heart rate, which allows further distribution of the cytoprotectant (Costanzo and Lee 1994). As explained on the Old Hypothesis page, most freeze tolerant animals use sugar compounds called cryoprotectants which bind to free water prevent it from freezing. This prevents dehydration of the cells since it limits the increase in the concentration gradient (Storey and Storey 1992). In wood frogs, glucose is this cryoprotectant, shown by the higher survival rates with increased glucose in Figure 4.

Figure 4: Survival rates, ice content, and hemoglobin concentrations of wood frogs with different concentrations of glucose. From Costanzo and Lee 1994. Used with permission of Michael Dorcas.

Many freeze tolerant invertebrates also use glucose as a cryotprotectant, but they increase concentrations throughout the fall. The wood frog only releases it when freezing begins. This means the increased heart rate and continued distribution after nucleation is very important for survival (Costanzo and Lee, 1994).

After the initial crystallization, ice continues to propagate for hours or even days. When freezing is complete, about 65-70% of the frog's body water is frozen. There is no breathing or heart beat. The frog can survive these conditions until its body temperature reaches -6*C. When thawing begins,  the cardiovascular function is the first thing to resume, which helps the recovery of other systems (Costanzo and Lee 1994). The core organs of the wood frog thaw sooner than the peripheral ones due to a higher concentration of glucose. This thawing "from the inside out" was not found in painted turtles, meaning if they did freeze, the internal organs would not have a chance to prepare for resumption of their function (Rubinsky et. al. 1994).

Animals That Supercool

Many fish and some insects remain in a supercooled state when exposed to temperatures below the freezing point of their body fluids. They are able to remain supercooled as long as there is not nucleation in their body. To prevent this nucleation, many ectothermic animals use an antifreeze. Many insects use increased concentrations of glycerol as this antifreeze. For example, the willow gallfly (Rhabdophaga strobiloides) contains as much as 50% glycerol, and can supercool even in temperatures of -60*C. Eleven different families of fish use glycoproteins to supercool. These glycoproteins prevent the addition of water to ice, meaning ice cannot grow (Schmidt-Nielsen 2002).

Anthropods that supercool do not use an antifreeze. Instead, they clear their body of most ice-nucleatizing agents by purging their guts. This means that anthropods have a low tolerence for supercooling in the autum, but an increased tolerence after their guts are cleared in the winter. This is much like the painted turtles (Packard et. al. 2001).