Factors contributing to seasonal increases in inoculative freezing resistance in overwintering fire-colored beetle larvae dendroides canadensis

The insects and microarthropods that vary seasonally in susceptibility to cross-cuticular inoculation by external ice (inoculative freezing) represent a phylogenetically diverse group; however, few studies have explored possible mechanisms experimentally. This study documents seasonally variable inoculative freezing resistance in Dendroides canadensis beetle larvae and combines immunofluorescence, in vivo removal of epicuticular lipids and in vitro chamber studies to explore the roles of seasonal modification in the cuticle and in epidermal and hemolymph antifreeze proteins (AFPs). Seasonal cuticular modifications contribute to the inhibition of inoculative freezing since more cold-hardy larvae froze inoculatively when epicuticular waxes were removed with hexane and, in in vitro chamber experiments, cuticle patches (with the underlying epidermis removed) from winter larvae provided greater protection from inoculative freezing than did cuticle patches from summer larvae. The results indicate that seasonal modifications in epidermal and hemolymph AFPs contribute most strongly to the inhibition of inoculative freezing. Subcuticular epidermal AFPs were present in immunocytochemically labeled transverse sections of winter larvae but were absent in summer ones. Winter integument patches (cuticle with epidermis) were more resistant to inoculative freezing than were summer integument patches. Integument patches resisted inoculative freezing as well as live winter-collected larvae only when hemolymph AFP was added. The results also suggest that some integumentary ice nucleators are removed in cold-hardy larvae and that AFP promotes supercooling by inhibiting the activity of these nucleators.