Probability of freezing in the freeze-avoiding beetle larvae Cucujus clavipes puniceus (Coleoptera: Cucujidae) from interior Alaska.

Freeze-avoiding insects must resist freezing or die. A suite of adaptations to low temperatures, including the production of antifreeze proteins, colligative antifreezes (polyols), and dehydration allows most individuals to prevent freezing below the lowest ambient temperatures experienced in situ; however, there can be a wide variance in the minimum temperatures that individuals of freeze-avoiding species reach before freezing. We used logistic regression to explore factors that affect this variance and to estimate the probability of freezing in larvae of the freeze-avoiding beetle Cucujus clavipes puniceus. We hypothesized that water content ≤0.5 mg mg(-1) dry mass would lead to deep supercooling (avoidance of freezing below -58°C). We found a significant interaction between water content and ambient below-snow temperature and a significant difference between individuals collected from two locations in Alaska: Wiseman and Fairbanks. Individuals collected in Wiseman deep supercooled with greater water content and to a greater range of ambient temperatures than individuals collected in Fairbanks, leading to significantly different lethal water contents associated with 50% probability of freezing.

Deep supercooling, vitrification and limited survival to -100{degrees}C in the Alaskan beetle Cucujus clavipes puniceus (Coleoptera: Cucujidae) larvae.

Larvae of the freeze-avoiding beetle Cucujus clavipes puniceus (Coleoptera: Cucujidae) in Alaska have mean supercooling points in winter of -35 to -42 degrees C, with the lowest supercooling point recorded for an individual of -58 degrees C. We previously noted that some larvae did not freeze when cooled to -80 degrees C, and we speculated that these larvae vitrified. Here we present evidence through differential scanning calorimetry that C. c. puniceus larvae transition into a glass-like state at temperatures<-58 degrees C and can avoid freezing to at least -150 degrees C. This novel finding adds vitrification to the list of insect overwintering strategies. While overwintering beneath the bark of fallen trees, C. c. puniceus larvae may experience low ambient temperatures of around -40 degrees C (and lower) when microhabitat is un-insulated because of low snow cover. Decreasing temperatures in winter are correlated with loss of body water from summer high levels near 2.0 to winter lows near 0.4 mg mg(-1) dry mass and concomitant increases in glycerol concentrations (4-6 mol l(-1)) and thermal hysteresis. Finally, we provide direct evidence that Cucujus from Wiseman, Alaska, survive temperatures to -100 degrees C.

Antifreeze proteins in Alaskan insects and spiders.

Prior to this study, antifreeze proteins (AFPs) had not been identified in terrestrial arthropods from the Arctic or anywhere in Alaska. The hemolymph of 75 species of insects and six spiders from interior and arctic Alaska were screened for thermal hysteresis (a difference between the freezing and melting points), characteristic of the presence of AFPs. Eighteen species of insects and three spiders were shown to have AFPs. Ten of the insects with AFPs were beetles including the first species from the families Chrysomelidae, Pythidae, Silphidae and Carabidae. In addition, the first Neuropteran to have AFPs was identified, the lacewing Hemerobius simulans together with the second and third Diptera (the first Tipulids) and the second and third Hemiptera, the stinkbug Elasmostethus interstinctus (the first Pentatomid), and the water strider Limnoporus dissortis (the first Gerrid). Prior to this study, 33 species of insects and three spiders had been reported to have AFPs. Most AFP-producing terrestrial arthropods are freeze avoiding, and the AFPs function to prevent freezing. However, some of the AFP- producing insects identified in this study are known to be freeze tolerant (able to survive freezing) to very low temperatures (-40 to -70 degrees C).