Tissue Specific Transcription of Antifreeze Proteins and the Role of Malpighian Tubule Antifreeze Proteins in the Overwintering Strategy of Dendroides canadensis

To avoid freezing while overwintering beneath the bark of fallen trees, Dendroides canadensis (Coleoptera: Pyrochroideae) larvae produce a family of antifreeze proteins (DAFPs) that are transcribed in specific tissues and have specific compartmental fates. Thermal hysteresis activity (THA) and DAFPs have been conclusively shown in hemolymph and midgut fluid, but the presence of DAFPs or ice binding factors (IBFs) has not previously been explored in primary urine which has the potential to contain endogenous ice nucleating compounds that could induce freezing. A maximum mean thermal hysteresis activity of 2.65 + .33oC was observed in primary urine of winter acclimatized D. canadensis larvae. Thermal hysteresis activity in primary urine increased significantly through fall, peaked in the winter and decreased through spring to levels less than 1oC in summer, in a pattern similar to that described previously in hemolymph and midgut fluid. Thermal hysteresis activity 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. Addition of recombinant DAFPs to solutions containing ice nucleating crystals that have been shown in Malpighian tubules significantly decreased the supercooling points of the solutions suggesting a potential role for DAFPs in Malpighian tubule fluid (primary urine). Short photoperiod (10 hour critical PP) and low temperature (10oC) have been shown to induce thermal hysteresis in D. canadensis. However, late summer collected D. canadensis larvae, acclimated to varying temperature and photoperiods, responded primarily to temperature. The TH induction in response to short PP described 30 years ago could not be reproduced. Perhaps because of a changing global climate, D. canadensis larvae are relying on temperature cues rather than photoperiodic cues for induction of thermal hysteresis. Also, when D. canadensis larvae were treated with juvenile hormone III (JH-III) and held under non-inductive conditions, THA in hemolymph and midgut fluid increased significantly after seven days. Previous studies that used JH-I did not show significant induction of THA until 14 days.