SUMMARYTo avoid freezing while overwintering beneath the bark of fallen trees, Dendroides canadensis (Coleoptera: Pyrochroidae) larvae produce a family of antifreeze proteins (DAFPs) that are transcribed in specific tissues and have specific compartmental fates. DAFPs and associated thermal hysteresis activity (THA) have been shown previously in hemolymph and midgut fluid, but the presence of DAFPs has not been explored in primary urine, a potentially important site that can contain endogenous icenucleating compounds that could induce freezing. A maximum mean THA of 2.65±0.33°C was observed in primary urine of wintercollected D. canadensis larvae. THA in primary urine increased significantly through autumn, peaked in the winter and decreased through spring to levels of 0.2-0.3°C in summer, in a pattern similar to that of hemolymph and midgut fluid. THA was also found in hindgut fluid and excreted rectal fluid, suggesting that these larvae not only concentrate AFPs in the hindgut, but also excrete AFPs from the rectal cavity. Based on dafp transcripts isolated from Malpighian tubule epithelia, cDNAs were cloned and sequenced, identifying the presence of transcripts encoding 24 DAFP isoforms. Six of these Malpighian tubule DAFPs were known previously, but 18 are new. We also provide functional evidence that DAFPs can inhibit ice nucleators present in insect primary urine. This is potentially critical because D. canadensis larvae die if frozen, and therefore ice formation in any body fluid, including the urine, would be lethal.

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.