Freeze intolerance

Freeze-intolerant species have been known for a considerable period (see Salt 1961). In part, these species rely on the fact that small volumes of water can be cooled well below their melting point before spontaneous nucleation, or freezing, takes place (Lee 1989). Although this implies a relationship between volume and SCP (Lee and Costanzo 1998), in practice several other factors mean that this relationship is often not found. In preparation for winter, the SCPs decline in freeze-intolerant species, often over a period of several weeks (Rickards et al. 1987) (Fig. 5.18). This increase in supercooling capacity is partially the result of the removal of ice nucleating agents (INAs) from the gut and haemo-lymph and various tissues, and partly the result of the accumulation of either one or several low

Figure 5.18 Alterations in (a) SCP, (b) glycerol content, and (c) glycogen content in larvae of the freeze-intolerant moth Epiblema scudderiana over winter.

Source: Insects at Low Temperatures, 1991, pp. 64-93, Storey and Storey, with kind permission of Kluwer Academic Publishers.

Figure 5.18 Alterations in (a) SCP, (b) glycerol content, and (c) glycogen content in larvae of the freeze-intolerant moth Epiblema scudderiana over winter.

Source: Insects at Low Temperatures, 1991, pp. 64-93, Storey and Storey, with kind permission of Kluwer Academic Publishers.

molecular weight cryoprotectants, although dehydration (i.e. reduction in water content) may also be important (Zachariassen 1985; Block 1990; Ramlov 2000).

Cryoprotectants

The low molecular weight cryoprotectants include polyhydric alcohols (polyols), such as glycerol, sorbitol, mannitol, threitol (Somme 1982), sugars, such as trehalose and occasionally glucose and fructose, and amino acids, such as proline (Duman et al. 1991; Ramlov 2000). The polyols often increase to molar levels over winter, and in the beetle Ips acuminatus (Coleoptera, Scolytidae), molar levels of ethylene glycol, a compound toxic to humans, can be found (Gehrken 1984). These low molecular weight cryoprotectants either stabilize membranes and proteins, or act in a colligative manner to prevent ice formation, that is, they depress the freezing point in a way that is related to the number of molecules in solution (Ramlov 2000). In general, they decrease the melting point by 1.860Cmolal~1 and the SCP by approximately twice that amount (Zachariassen 1985). To effectively control ice formation, these compounds are soluble in aqueous solution, do not perturb protein structure, counteract the denaturing effects on proteins of high ionic concentrations, cold and dehydration, and are non-toxic and non-reactive even at high concentrations (Ramlov 2000). The synthesis and breakdown of these low molecular weight substances has been reviewed in detail by Storey (Storey and Storey 1991, 1996; Storey 1997).

Antifreeze proteins

Several freeze-intolerant species also produce antifreeze proteins (AFPs, or thermal hysteresis proteins) (Duman 2001; Walker et al. 2001). These proteins lower the non-equilibrium freezing point of water, but do not affect the melting point. In consequence, the melting and freezing points differ, and this is known as thermal hysteresis. Although AFPs have been described from about 40 species of insects, the sequences of AFPs from only three insect species have been published, and their structures are discussed in detail by Duman (2001). Antifreeze proteins increase the extent of supercooling in a non-colligative manner (see Duman 2001 for a description of the mechanism) and may also inactivate various ice nucleators (Ramlov 2000; Duman 2001). In addition, they are also important in preventing inoculative freezing (i.e. contact with external ice resulting in spontaneous nucleation). Although the wax-coated, hydrophobic cuticle is thought to provide an effective barrier to inoculative freezing, many instances are known where this barrier has been penetrated (Ramlov 2000; Duman 2001), and thus where external contact with moisture can increase the SCP (Fig. 5.19). Antifreeze proteins are accumulated in the haemolymph and in epidermal cells, and together with cuticular changes play a large role in preventing inoculative freezing (Duman 2001).

Inoculative freezing

While these changes may be sufficient to prevent inoculative freezing in some species, in others the

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