Antifreeze and ice nucleator proteins in terrestrial arthropods

Terrestrial arthropods survive subzero temperatures by becoming either free ze tolerant (survive body fluid freezing) or freeze avoiding (prevent body fluid freezing). Protein ice nucleators (PINs), which limit supercooling an d induce freezing, and antifreeze proteins (AFPs), which function to preven t freezing, can have roles in both freeze tolerance and avoidance. Many fre eze-tolerant insects produce hemolymph PINs, which induce freezing at high subzero temperatures thereby inhibiting lethal intracellular freezing. Some freeze-tolerant species have AFPs that function as cryoprotectants to prev ent freeze damage. Although the mechanism of this cryoprotection is not kno wn, it may involve recrystallization inhibition and perhaps stabilization o f the cell membrane. Freeze-avoiding species must prevent inoculative freez ing initiated by external ice across the cuticle and extend supercooling ab ilities. Some insects remove PINs in the winter to promote supercooling. wh ereas others have selected against surfaces with ice-nucleating abilities o n an evolutionary time scale. However, many freeze-avoiding species do have proteins with ice-nucleating activity, and these proteins must be masked i n winter. In the beetle Dendroides canadensis, AFPs in the hemolymph and gu t inhibit ice nucleators. Also, hemolymph AFPs and those associated with th e layer of epidermal cells under the cuticle inhibit inoculative freezing. Two different insect AFPs have been characterized. One type from the beetle s D. canadensis and Tenebrio molitor consists of 12- and 13-mer repeating u nits with disulfide bridges occurring at least every six residues. The spru ce budworm AFP lacks regular repeat units. Both have much higher activities than any known AFPs.