Thermal hysteresis (TH), a difference between the melting and freezing points of a solution that is indicative of the presence of large-molecular-mass antifreezes (e.g., antifreeze proteins), has been described in animals, plants, bacteria, and fungi. Although all previously described TH-producing biomolecules are proteins, most thermal hysteresis factors (THFs) have not yet been structurally characterized, and none have been characterized from a freeze-tolerant animal. We isolated a highly active THF from the freeze-tolerant beetle, Upis ceramboides, by means of ice affinity. Amino acid chromatographic analysis, polyacrylamide gel electrophoresis, UV-Vis spectrophotometry, and NMR spectroscopy indicated that the THF contained little or no protein, yet it produced 3.7 ؎ 0.3°C of TH at 5 mg/ml, comparable to that of the most active insect antifreeze proteins. Compositional and structural analyses indicated that this antifreeze contains a ␤-mannopyranosyl-(134) ␤-xylopyranose backbone and a fatty acid component, although the lipid may not be covalently linked to the saccharide. Consistent with the proposed structure, treatment with endo-␤-(134)xylanase ablated TH activity. This xylomannan is the first TH-producing antifreeze isolated from a freeze-tolerant animal and the first in a new class of highly active THFs that contain little or no protein.antifreeze protein ͉ insect cold tolerance ͉ glycolipid ͉ membrane associated antifreeze

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SUMMARYLarvae of the freeze-avoiding beetle Cucujus clavipes puniceus (Coleoptera: Cucujidae) in Alaska have mean supercooling points in winter of -35 to -42°C, with the lowest supercooling point recorded for an individual of -58°C. We previously noted that some larvae did not freeze when cooled to -80°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°C and can avoid freezing to at least -150°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°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 ) and thermal hysteresis. Finally, we provide direct evidence that Cucujus from Wiseman, Alaska, survive temperatures to -100°C.

We analyzed scarring data for Bering-Chukchi-Beaufort (BCB) Seas bowhead whales (Balaena mysticetus) harvested by Alaska Native hunters to quantify the frequency of line entanglement, ship strikes, and killer whale-inflicted injuries. We had 904 records in our database for whales landed between 1990 and 2012, and after data quality screening, we found 521 records containing information on scarring. Logistic regression was used to evaluate different combinations of explanatory variables (i.e., body length, sex, year, year-group) to develop a prediction model for each scar type. We also list bowhead whales that were harvested, found dead, or observed alive entangled in commercial line/fishing gear. Our findings suggest that about 12% of harvested bowheads show entanglement scars. Their frequency is highly correlated with body length and sex: about 50% of very large bowheads (> 17 m) show such scars, while whales under 9 m rarely do, and males show a significantly higher rate than females. Scars associated with ship strikes are infrequent and occur on ~2% of all harvested whales; body length, sex, and year were not significant factors. Scarring from attempted killer whale predation was evident on ~8% of landed whales. As with entanglement injuries, the frequency of killer whale scars was much higher (> 40%) on whales more than 16 m in length and statistically more frequent in the second half of the study (2002 – 12). Increased killer whale injuries in the recent decade are consistent with studies conducted on bowheads of the Eastern Canada-West Greenland population. The findings presented here reflect the most thorough analysis of injury rates from entanglement, ships, and killer whales for the BCB bowheads conducted to date. They indicate that (1) entanglement rates primarily from pot fishing gear (crab or cod or both) are relatively high for very large and presumably older bowheads, (2) collisions with ships are infrequent at present, and (3) scarring from killer whales is frequent on very large adult whales (> 17 m). Considering that bowhead habitat is changing rapidly (e.g., sea ice reduction), industrial ship traffic in the Arctic is increasing, and commercial fishing operations are expanding to the north, we strongly recommend that monitoring of scarring and injuries on harvested bowheads continue into the future as a means of documenting change.

SUMMARY The beetle Cucujus clavipes is found in North America over a broad latitudinal range from North Carolina (latitude ∼35°N) to near tree line in the Brooks Range in Alaska (latitude, ∼67°30′ N). The cold adaptations of populations from northern Indiana (∼41°45′N) and Alaska were compared and, as expected, the supercooling points (the temperatures at which they froze) of these freeze-avoiding insects were significantly lower in Alaska insects. Both populations produce glycerol, but the concentrations in Alaska larvae were much higher than in Indiana insects(∼2.2 and 0.5 mol l–1, respectively). In addition, both populations produce antifreeze proteins. Interestingly, in the autumn both populations have the same approximate level of hemolymph thermal hysteresis,indicative of antifreeze protein activity, suggesting that they synthesize similar amounts of antifreeze protein. A major difference is that the Alaska larvae undergo extreme dehydration in winter wherein water content decreases from 63–65% body water (1.70–1.85 g H2O g–1 dry mass) in summer to 28–40% body water(0.40–0.68 g H2O g–1 dry mass) in winter. These 2.5–4.6-fold reductions in body water greatly increase the concentrations of antifreeze in the Alaska insects. Glycerol concentrations would increase to 7–10 mol l–1 while thermal hysteresis increased to nearly 13°C (the highest ever measured in any organism) in concentrated hemolymph. By contrast, Indiana larvae do not desiccate in winter. The Alaska population also undergoes a diapause while insects from Indiana do not. The result of these, and likely additional, adaptations is that while the mean winter supercooling points of Indiana larvae were approximately –23°C, those of Alaska larvae were –35 to–42°C, and at certain times Alaska C. clavipes did not freeze when cooled to –80°C.