Large Molecular Weight Antifreezes and Related Adaptations in Freeze-Tolerant Alaskan Insects

The overwintering physiology of aquatic insects has not been well characterized. The stonefly, Nemoura arctica, was collected from the headwaters of the Chandalar River, where streambed temperatures measured as low as -12.7Ì܁ÁC in midwinter. When in contact with ice, N. arctica froze at -1.5Ìâå±0.4Ì܁ÁC, but individuals survived temperatures down to -15Ì܁ÁC, making this the first described species of freeze tolerant stonefly. Nemoura arctica produce both glycerol and ice thermal hysteresis factors in response to low temperature. Hemolymph glycerol concentrations increased from 3 mM to 930Ìâå±114 mM in response to acclimation at -8Ì܁ÁC and glycerol concentrations increased even after nymphs were frozen. Although, the hemolymph of individual cold acclimated nymphs occasionally exhibited more than a degree of thermal hysteresis, typically the hemolymph exhibited only hexagonal crystal growth and recrystallization inhibition. The freeze tolerant beetle Upis ceramboides is the only animal species known to accumulate high concentrations of threitol (250 mM) as a cryoprotectant; however the biosynthetic pathway has not been investigated. In vivo metabolism of 13C-labeled glucose isotopomers was monitored by 13C{1H} NMR spectroscopy, demonstrating that C3-C6 of glucose become C1-C4 of threitol, which is expected for four-carbon saccharides arising from the pentose phosphate pathway (PPP). In vitro experiments show that threitol is synthesized from erythrose 4-phosphate (E4P), a C4 intermediate in the PPP. Threitol 4-phosphate appears to be the preferred substrate of the sugar phosphatase(s), promoting threitol synthesis over that of erythritol. In addition, erythritol is catabolized under conditions that promote threitol synthesis, leading to the accumulation of high concentrations of threitol without concomitant accumulation of erythritol. Upis ceramboides also produces a novel, highly active antifreeze glycolipid. This glycolipid exhibits 3.7Ìâå±0.3Ì܁ÁC of thermal hysteresis at 5 mg/ml, whereas fish AF(G)Ps exhibit only 1.5Ì܁ÁC of thermal hysteresis at 20-40 mg/ml. Consistent with our structural determinations, thermal hysteresis activity is unaffected by protease, but is rapidly lost upon treatment with endo Ì_å_-(1?4) xylanase. This is the first high molecular weight antifreeze to be isolated from a freeze tolerant animal. We have subsequently isolated similar TH-active glycolipids from numerous organisms, including other insects, plants, fish and amphibians.