Some insects respond to seasonally low temperatures by the production of thermal hysteresis proteins (THP), at times known as antifreeze proteins (AFP). These proteins lower the freezing temperature of the insect by binding to microscopic ice crystals and inhibiting ice growth. The spruce budworm moth (Choristoneura fumiferana) and the meal worm beetle (Tenebrio molitor) each produce a different, but extremely active, 8-9 kDa AFP in their overwintering stages. Expression studies show that the spruce budworm produces AFP transcripts in the 2mm caterpillar/hibernaculum stage and that these transcripts rapidly decrease about the time of molting to the third instar. In Tenebrio, AFP mRNA abundance increases throughout larval development, but declines throughout the pupal and adult stages, consistent again with the observation that it is the larvae that overwinter. In both insects there are multiple copies of these genes, which are distinct for each species. The isolation and sequencing of cDNAs encoding AFP isoforms for both these species' proteins, as well as in vitro mutagenesis, has allowed us to identify conserved amino acids. Conserved residues are candidates to confer structural stability as well as for ice binding. In both AFP types, cysteines appear to be important, suggesting that they have been conserved for proper folding of the proteins by disulfide bridge formation. Threonines, often as part of a repetitive motif, are also well conserved and suggest models which could interact with ice. This work, coupled with structural analysis, has lead to the development of two different protein models and their inhibition of ice growth. Thus, although these evolutionary distant species have evolved unique proteins, they share some common features that promise insight into the interaction of ice surfaces with antifreeze proteins, the developmental regulation of AFP genes and an appreciation of the strategies employed by insects to avoid freezing.

Index terms: Choristoneura fumiferana, Tenebrio molitor, thermal hysteresis proteins, gene expression in development.

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