Freezing tolerant species are capable of surviving extracellular ice formation, and apparently, in a few instances, intracellular ice formation in the fat body (Denlinger and Lee 1998). In consequence, they must be able to resist the injuries that are associated with this process. This is done, in part, by controlling ice formation at relatively high temperatures. This means that the site of ice formation, ice growth rate (and hence osmotic equilibration time), and the quantity of ice formed (i.e. the concentration of the non-frozen fluids and the extent of dehydration) (Ramlov 2000) can be controlled, so limiting injury.
As soon as the temperature of an organism has declined below its melting point there is a risk of ice formation. Crystallization may take place either by aggregation of water molecules into an ice nucleus (homogeneous nucleation) or via their aggregation around some substance or irregularity (heterogeneous nucleation) (Ramlov 2000). When freezing takes place, additional water is added to the nucleus or nuclei, and effectively the animal begins to desiccate. The removal of water from the solution causes an increase in solute concentration. The progressive concentration of the body fluids may lead to protein denaturation, changes in pH, and alterations of membrane potential and transport properties. In addition, cellular shrinkage may occur owing to removal of water from the cells and this may damage the cell membrane to such an extent that it cannot recover following thawing (the critical minimum cell volume hypothesis) (Denlinger and Lee 1998). The extent to which this damage is incurred depends on the rate of cooling (Ramlov 2000), which may account for higher order (whole-individual) effects of cooling rate (Section 5.1.1). Of course, there may also be injury due to mechanical damage of cells by sharp ice crystals and their growth during the freezing process.
Inoculation at high subzero temperatures Although many freezing tolerant species can survive very low temperatures, virtually all of them freeze at relatively high subzero temperatures (Section 5.3.1). These high SCPs are generally the result of inoculation of freezing by extracellular
INAs (Ramlev 2000; Duman 2001). By initiating freezing at relatively high subzero temperatures, INAs, which function as sites of heterogeneous nucleation, ensure that ice formation is controlled and restricted to the extracellular spaces (Duman et al. 1991), thus protecting cells from freezing damage. The best known of these INAs are the haemolymph protein (PIN) and lipoprotein (LPIN) ice nucleators. Knowledge of the relationship between PIN and LPIN structure and function is limited, although there is some information on the size and structure of both PINs and LPINs (Duman 2001).
Not all freeze-tolerant species make use of haemo-lymph PINs and LPINs to initiate inoculative freezing. In Eurosta solidaginis it appears that crystals of calcium carbonate, uric acid and potassium phosphate may ensure crystallization at relatively high subzero temperatures (Lee and Costanzo 1998). In some species, such as the highly freeze-tolerant Chymomyza costsata, external inoculation by ice crystals at relatively high subzero temperatures (—2°C) is essential for the development of freeze tolerance, and this is true also of several other species (Duman et al. 1991). In some instances, cooling in the absence of ice results in a depression of the SCP, but complete mortality on freezing. In E. solidaginis it appears that external inoculation via frozen gall tissue is essential for induction of freezing early on in the winter season, whereas endogenous nucleators become more important as the galls they inhabit dry out (Lee and Costanzo 1998).
Cryoprotection and recrystallization inhibition Freezing tolerant species are also characterized by high levels of low molecular weight cryoprotec-tants. The colligative cryoprotectants, such as glycerol and sorbitol, reduce the percentage of ice that can be formed, thus preventing the intracel-lular volume from declining below a critical minimum, and lower the rate of ice formation (Storey 1997). In addition, they may also protect proteins against denaturation (Duman et al. 1991). The non-colligative cryoprotectants, such as trehalose and proline, interact directly with the polar head groups of lipids to stabilize the bilayer structure, thus preventing an irreversible transition to the gel state (Storey and Storey 1996; Storey 1997). The metabolism of cryoprotectants, in the context of freezing tolerance, has been reviewed by Storey (1997).
Although AFPs are usually more characteristic of freeze intolerant insect species, several freeze tolerant species also have AFPs. While the presence of both INAs and AFPs in a single species appears somewhat paradoxical, Duman et al. (1991) point out that AFPs may function not to depress the freezing point, but rather to prevent recrystalliza-tion. Essentially recrystallization proceeds by crystal growth within the frozen matrix (Duman 2001), and can cause damage to tissues either during thawing or temperature changes (Ramlov 2000). The danger of this is particularly severe at relatively high subzero temperatures. AFPs inhibit recrys-tallization by preventing ice crystal growth and this function may be particularly important in species that spend long periods frozen, or in freezing tolerant species from more temperate areas, where the risk of recrystallization is high.
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