Wood Frog Freezing Survival
The wood frog (Rana sylvatica) inhabits forests ranging from the Appalachians to the Maritime provinces and west to northern Alaska, even to the Arctic Circle. Its overwintering site is a shallow burrow in the forest floor, well within the frost zone, that is overlain by leaves and other organic detritus. Our studies in southern Ohio suggest that wood frogs are subjected to several freezing episodes that typically last several days and expose the frogs to temperatures that fall as lows as -2° to -4°C; however, in more northerly regions they probably experience much lower temperatures and longer periods of frost.
Initiation of Freezing
Several mechanisms ensure that wood frogs freeze without supercooling extensively. First, owing to the highly permeable nature of amphibian skin, ice surrounding the frog can instantly trigger the freezing of the body fluids. Also, the frog’s winter refuge hosts an abundance of ice nucleating agents, such as various mineral particulates, organic acids, and certain microbes, that may cause the frog to freeze. Laboratory experiments suggest that ingestion of these agents promote ice formation in freeze-tolerant frogs. In fact, several strains of bacteria expressing potent ice nucleating activity have been cultured from the intestines of winter-collected wood frogs, indicating that such bacteria are retained throughout hibernation (Lee et al. 1995). Inoculation by ice or ice-nucleating agents in the winter environment probably is the primary mechanism initiating freezing in amphibians; there is no need for ice-nucleation proteins or other endogenous ice nuclei, as are found in some invertebrates (Costanzo et al. 1999).
Extensive freezing solidifies tissues, arrests vascular circulation, and deprives cells of oxygen. Because ice forms only in extracellular spaces, water inside cells is osmotically drawn externally where it joins the growing ice lattice. During this process cells may shrink substantially, potentially with damage to membranes and structural support systems. Macromolecules and solutes become crowded in a diminishing solvent volume, perhaps with adverse consequences. Ice formation within body fluids also poses the threat of mechanical injury by the growing ice lattice, particularly in compact and highly structured tissues and organs. Ice fronts may shear and separate tissues, disrupting intercellular communication systems. Upon thawing, large pools of dilute fluid form in extracellular spaces. Cell volume, hydroosmotic balance, and energy status must be restored.
Freeze Tolerance Capacity
Laboratory studies have shown that wood frogs can survive: (a) the freezing of up to 65-70% of their body water; (b) a minimum body temperature of -6°C; and (c) uninterrupted freezing for ≥ 4 wk. Freeze tolerance varies seasonally as frogs are most hardy during winter. Such seasonal variation in freeze tolerance capacity may partly reflect changes in the quantity of cryoprotectant that can be produced. Survival depends on slow freezing so that cryoprotective mechanisms can be more fully expressed.
Recovery is remarkably rapid, with basic physiological and behavioral functions usually returning within several hours of thawing
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In collaboration with Jack R. Layne, Jr. (Slippery Rock University), our work has shown that recovery dynamics are characterized by sequential restoration of fundamental to progressively more complex functions. For example, the heart resumes beating even before ice in the body has completely melted, and pulmonary respiration and blood circulation are restored soon thereafter. Contractility in hindlimb muscles returns 1-2 h after thawing, whereas function of the innervating sciatic nerve is restored within approximately 5 h. Hindlimb retraction and righting reflexes return several hours later and the frogs usually exhibit normal body postures and coordinated motor functions within 14-24 h. Higher order behaviors, such as mating drive and courting behavior, are not restored until at least several days later
et al. 1997).
Freeze Tolerance Adaptations
One response promoting freeze tolerance in freeze-tolerant frogs is the redistribution of up to 60% of the water normally found in tissues. Dissecting a frozen wood frog reveals that much of the ice is sequestered within the lymph system and in the coelom, where it may form without damaging delicate tissues and organs (Lee et al. 1992).
Freeze tolerance is also promoted by the rapid synthesis of glucose from liver glycogen and the distribution of this cryoprotective agent to cells throughout the body. The accumulated glucose apparently enhances the survival of cells, tissues, and organs because experimentally administering additional glucose to the frog increases its tolerance to freezing (Costanzo et al. 1993). One of the primary functions of glucose is to raise the osmotic pressure of the body fluids, which in turn reduces the amount of ice that forms at any given temperature. Glucose transported into cells acts as an osmolyte, decreasing the degree of cell shrinkage during freezing, and also serves as a fermentable fuel that can be metabolized in the absence of oxygen. The wood frog also uses urea as a cryoprotectant. Unlike glucose, urea is accumulated during autumn and early winter, and is already localized within cells when freezing begins. Some evidence suggests that urea is more efficacious than glucose in preventing cryoinjury (Costanzo and Lee 2005).
Aquaporins (AQPs) and facilitative urea transporters (UTs) are two transporter proteins that have been implicated in a wide range of physiological roles in various organisms. Recently, these proteins have been found in a variety of anurans; however, their physiological significance is not yet fully understood. In order to elucidate the importance of AQPs and UTs in osmolyte balance in hibernating frogs, we are examining expression of these proteins in frogs with varying degrees of terrestrialism. In addition we are measuring seasonal variations in expression, as well as changes in expression levels in response to winter-related stresses, in the wood frog.