Todd Sformo

A nonprotein thermal hysteresis-producing xylomannan antifreeze in the freeze-tolerant Alaskan beetle Upis ceramboides

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 β-mannopyranosyl-(1→4) β-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-β-(1→4)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.

Deep supercooling, vitrification and limited survival to –100°C in the Alaskan beetle Cucujus clavipes puniceus (Coleoptera: Cucujidae) larvae

SUMMARY Larvae 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–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°C.

Comparative overwintering physiology of Alaska and Indiana populations of the beetle Cucujus clavipes (Fabricius): roles of antifreeze proteins, polyols, dehydration and diapause.

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.

A thermal hysteresis-producing xylomannan glycolipid antifreeze associated with cold tolerance is found in diverse taxa

The presence of large-molecular-mass, thermal hysteresis (TH)-producing antifreezes (e.g., antifreeze proteins) has been reported in numerous and diverse taxa, including representative species of fish, arthropods, plants, fungi, and bacteria. However, relatively few of these antifreeze molecules have been chemically characterized. We screened diverse species by subjecting their homogenates to ice-affinity purification and discovered the presence of a newly identified class of antifreeze, a xylomannan-based TH-producing glycolipid that was previously reported in one species of freeze-tolerant Alaskan beetle. We isolated xylomannan-based antifreeze glycolipids from one plant species, six insect species, and the first frog species to be shown to produce a large-molecular-mass antifreeze. 1H NMR spectra of the ice-purified molecules isolated from these diverse freeze-tolerant and freeze-avoiding organisms were nearly identical, indicating that the chemical structures of the glycolipids were highly similar. Although the exact functions remain uncertain, it appears that antifreeze glycolipids play a role in cold tolerance.

Freeze tolerance in an arctic Alaska stonefly

SUMMARY Most aquatic insects do not survive subzero temperatures and, for those that do, the physiology has not been well characterized. Nemoura arctica is a species of stonefly widely distributed throughout arctic and subarctic Alaska. We collected nymphs from the headwaters of the Chandalar River, where we recorded streambed temperatures as low as –12.7°C in midwinter. When in contact with ice, autumn-collected N. arctica cool to –1.5±0.4°C before freezing, but individuals survived temperatures as low as –15°C, making this the first described species of freeze-tolerant stonefly. N. arctica clearly survive freezing in nature, as winter-collected nymphs encased in ice demonstrated high survivorship when thawed. In the laboratory, 87% of N. arctica nymphs frozen to –15°C for 2.5 weeks survived and, within one month of thawing, 95% of the last-instar nymphs emerged. N. arctica produce both glycerol and ice-binding factors (e.g. antifreeze protein) in response to low temperature. Hemolymph glycerol concentrations increased from 3 mmol l–1 to 930±114 mmol l–1 when temperatures were decreased from 4°C to –8°C, and N. arctica continued to produce glycerol even while 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, indicating a low concentration of ice-binding factor. Hemolymph of nymphs acclimated to subzero temperatures had recrystallization inhibition. These results demonstrate that, in the face of freezing conditions, N. arctica exhibit overwintering adaptations similar to those of terrestrial insects.

Elucidating the Biochemical Overwintering Adaptations of Larval Cucujus clavipes puniceus, a Nonmodel Organism, via High Throughput Proteomics

Cucujus clavipes puniceus (C.c.p.) is a nonmodel, freeze-avoiding beetle that overwinters as extremely cold-tolerant larvae in the interior boreal forests of Alaska to temperatures as low as -100 °C. Using a tandem MS-based approach, we compared the proteomes of winter- and summer-collected C.c.p. to identify proteins that may play functional roles in successful overwintering. Using Gene Ontology (GO) analysis and manual interpretation, we identified 104 proteins in winter and 128 proteins in summer samples. We found evidence to indicate a cytoskeletal rearrangement between seasons, with Winter NDSC possessing unique actin and myosin isoforms while summer larvae up-regulated α actinin, tubulin, and tropomyosin. We also detected a fortification of the cuticle in winter via unique cuticle proteins, specifically larval/pupal rigid cuticle protein 66 precursor and larval cuticle protein A2B. Also, of particular interest in the winter larvae was an up-regulation of proteins related to silencing of genes (bromodomain adjacent to zinc finger domain 2A and antisilencing protein 1), proteins involved with metabolism of amines (2-isopropylmalate synthase and dihydrofolate reductase), and immune system process (lysozyme C precursor), among others. This represents the first high throughput MS/MS analysis of a nonmodel, cold-tolerant organism without a concurrent microarray analysis.

Overwintering Physiology and Microhabitat Use of Phyllocnistis populiella (Lepidoptera: Gracilliariidae) in Interior Alaska

ABSTRACT We investigated the overwintering physiology and behavior of Phyllocnistis populiella Chambers, the aspen leaf miner, which has caused severe and widespread damage to aspen in Alaska over the past 10 yr. Active P. populiella moths caught in spring and summer supercooled to an average temperature of -16°C, whereas dormant moths excavated from hibernacula in the leaf litter during fall and winter supercooled to an average of -32°C. None of the moths survived freezing in the laboratory. Counts of overwintering moths in leaf litter across microhabitats in interior Alaska demonstrated that moths occurred at significantly higher density beneath white spruce trees than beneath the aspen host, several other hardwood species, or in open areas among trees. During winter, the temperature 1–2 cm below the surface of the leaf litter beneath white spruce trees was on average 7–9°C colder than beneath aspen trees, and we estimate that during at least one period of the winter the temperature under some white spruce trees may have been cold enough to cause mortality. However, the leaf litter under white spruce trees was significantly drier than the litter from other microhabitats, which may assist P. populiella moths to avoid inoculative freezing because of physical contact with ice. We conclude that in interior Alaska, P. populiella overwinter in a supercooled state within leaf litter mainly under nonhost trees, and may prefer relatively dry microhabitats over moister ones at the expense of lower environmental temperature.

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.

Cold tolerance in Arabidopsis kamchatica

UNLABELLED • PREMISE OF THE STUDY Cold tolerance is a critically important factor determining how plants will be influenced by climate change, including changes in snowcover and extreme weather events. Although a great deal is known about cold tolerance in Arabidopsis thaliana, it is not highly cold tolerant. This study examined cold tolerance and its genetic diversity in an herbaceous subarctic relative, Arabidopsis kamchatica, which generally occurs in much colder climates.• METHODS Thermal analysis and electrolyte leakage were used to estimate supercooling points and lethal temperatures (LT50) in cold-acclimated and nonacclimated families from three populations of A. kamchatica.• KEY RESULTS Arabidopsis kamchatica was highly cold tolerant, with a mean LT50 of -10.8°C when actively growing, and -21.8°C when cold acclimated. It also was able to supercool to very low temperatures. Surprisingly, actively growing plants supercooled more than acclimated plants (-14.7 vs. -12.7°C). There was significant genetic variation for cold tolerance both within and among populations. However, both cold tolerance and genetic diversity were highest in the midlatitude population rather than in the far north, indicating that adaptations to climate change are most likely to arise in the center of the species range rather than at the edges.• CONCLUSIONS Arabidopsis kamchatica is highly cold tolerant throughout its range. It is far more freeze tolerant than A. thaliana, and supercooled to lower temperatures, suggesting that A. kamchatica provides a valuable complement to A. thaliana for cold tolerance research.

Severe Bone Loss as Part of the Life History Strategy of Bowhead Whales

The evolution of baleen constituted a major evolutionary change that made it possible for baleen whales to reach enormous body sizes while filter feeding on tiny organisms and migrating over tremendous distances. Bowhead whales (Balaena mysticetus) live in the Arctic where the annual cycle of increasing and decreasing ice cover affects their habitat, prey, and migration. During the nursing period, bowheads grow rapidly; but between weaning and approximately year 5, bowhead whales display sustained baleen and head growth while limiting growth in the rest of their bodies. During this period, they withdraw resources from the skeleton, in particular the ribs, which may lose 40% of bone mass. Such dramatic changes in bones of immature mammals are rare, although fossil cetaceans between 40 and 50 million years ago show an array of rib specializations that include bone loss and are usually interpreted as related to buoyancy control.