The freezing process in lower vertebrates

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Ecology and Physiology of freeze tolerance in the wood frog, Rana sylvatica.
The ability to tolerate freezing has been reported in five species of frogs, Rana sylvatica, Hyla versicolor, H. chrysoscelis, Pseudacris crucifer and P. triseriata (Schmid, 1982; MacArthur and Dandy, 1982; Storey, 1984; Costanzo et al., 1992a). The wood frog R. sylvatica is one of the most studied model systems for freeze tolerance. Most research into vertebrate freeze tolerance deals with the mechanisms employed by R. sylvatica. The mechanisms that enable this survival are not fully known.
This essay reviews freeze tolerance in R. sylvatica and studies this phenomenon through several approaches. The biophysics of freezing and tolerance are discussed in terms of what freezing means to frogs and freeze-tolerant organisms. Freeze tolerance is investigated from an ecological view such as the involvement of geography and distribution. This essay also reviews the basic role that physiology plays in freeze tolerance for R. sylvatica.

I The biophysics of freeze tolerance
To survive freezing temperatures commonly seen over winter several strategies are used, such as: (A) anhydrobiosis - an extreme desiccation whereby all free water that can freeze is removed, (B) vitrification - water solidifies into an amorphous glass-like state rather than changing into ice crystals, (C) avoidance - various strategies that lower the point at which body water freezes, (D) tolerance - regulation of ice formation into extracellular spaces while the liquid state of cells is preserved. Anydrobiosis is commonly seen in nematodes and other microorganisms (Crowe and Crowe, 1992). Vitrification is seen in plants (Hirsch, 1987) and is a technique used in artificial tissue and cell cryopreservation (Pegg, 2001). Freeze avoidance strategies are used by many insects such as Epiblema scudderiana, which synthesize antifreeze proteins and cryoprotectants to lower the limit at which the body water will freeze (Storey and Storey, 1992). The majority of animals that deal with freezing temperatures employ a freeze avoidance strategy (Storey and Storey, 2001). A small group of animals, which includes R. sylvatica, the object of my research, tolerates freezing to survive the cold winter.
Small volumes of water can remain liquid well past the freezing point of 0oC (Angell, 1982). Being small allows some species of insects to take advantage of this phenomenon of water and survive temperatures as low as -70oC (Ring, 1986). By preventing the formation of ice crystals, the water supercools, and does not freeze until the supercooling point is reached. The supercooling point is the temperature at which the liquid body water will form ice crystals (Lee, 1991). Frogs and other vertebrates being larger have difficulty in taking full advantage of this strategy. Frogs do, however, supercool to some extent, which allows some species to be freeze tolerant given the right conditions and factors.
All freeze tolerant frogs have roughly the same weight. The smallest of the freeze tolerant frogs is H. versicolor, which reaches a few grams in weight. Adult R. sylvatica will reach 5-8g for males and 8-13g for females (Storey, 1985). The box turtle (Terrapene carolina), weighing in at 0.5 kg, is the largest known freeze-tolerant vertebrate (Storey et al., 1993).
To tolerate freezing temperatures a frog has to contend with: A) physical damage caused by ice crystal formation, B) anoxia (O2 deprivation) and ischemia due to the freezing of blood and plasma, and C) intracellular dehydration stress caused by the conversion of body water to extracellular ice (Storey, 1999). The changes that we see at the genetic and protein levels help the freeze-tolerant frog overcome these obstacles.
There is a characteristic curve seen when a frog freezes (Fig. 1). As the ambient temperature slowly drops, as would occur under snow cover in nature, the temperature of the frog decreases over time. A slow cooling rate ensures that ice formation is exclusively extracellular. The freezing process starts by inoculative freezing from the skin and proceeds inwards towards the core organs (Fig. 2.; Rubinsky et al., 1994). In nature the inoculation of freezing probably occurs from the damp hibernation sites of terrestrial hibernators. In fact, 98% of frogs held in damp containers froze at -2oC compared to only 20% when the frogs were in dry containers (Costanzo et al., 1999). Up to -2.0oC, the frog supercools and there is no internal ice formation. At -2.0oC the frog cannot supercool further and the ice crystallization process begins. Once freezing starts we see a jump in temperature to about -0.5oC called the freezing exotherm. The crystallization process gives off heat, which is why we see this increase in temperature. The temperature of the frog is held at this value while intracellular ice slowly forms. Eventually the body temperature will decrease to ambient levels (see Storey and Storey, 1984; Storey and Storey, 1986a). Proton magnetic resonance imaging of the freezing process shows that by seven hours the frog is completely frozen (Fig, 2.; Rubinsky et al., 1994).
As mentioned, a freeze-tolerant animal has to contend with other factors besides the conversion of body water to ice. Frogs have the capacity to tolerate low levels of oxygen (Hermes-Lima and Storey, 1996; Storey, 1996). Amphibians as a group also have a general tolerance of extensive water loss (Shoemaker, 1992; Churchill and Storey, 1993). Dehydration is seen during freezing as a result of the extracellular water turning into ice. Therefore, we can think of freezing, and not just evaporative water loss, as a form of dehydration. The ability to survive freezing probably evolved from an ability to tolerate dehydration (Churchill and Storey, 1993; Costanzo et al, 1999; Storey et al., 1996). However, some frogs that are dehydration tolerant are not freeze-tolerant. R. pipiens is dehydration and anoxia tolerant, but cannot survive freezing. Therefore there are other factors apart from the adaptations towards dehydration tolerance to being freeze-tolerant.

II Freeze tolerance from an ecological view
R. ...