Lichen species across Alaska produce highly active and stable ice nucleators.

Forty years ago, lichens were identified as extraordinary biological ice nucleators (INs) that enable ice formation at temperatures close to 0°C. By employing INs, lichens thrive in freezing environments that surpass the physiological limits of other vegetation, thus making them the majority of vegetative biomass in northern ecosystems. Aerosolized lichen INs might further impact cloud glaciation and have the potential to alter atmospheric processes in a warming Arctic. Despite the ecological importance and formidable ice nucleation activities, the abundance, diversity, sources, and role of ice nucleation in lichens remain poorly understood. Here, we investigate the ice nucleation capabilities of lichens collected from various ecosystems across Alaska. We find ice-nucleating activity in lichen to be widespread, particularly in the coastal rainforest of Southeast Alaska. Across 29 investigated lichen, all species show ice nucleation temperatures above -15 °C and ~30% initiate freezing at temperatures above -6 °C. Concentration series of lichen ice nucleation assays in combination with statistical analysis reveal that the lichens contain two subpopulations of INs, similar to previous observations in bacteria. However, unlike the bacterial INs, the lichen INs appear as independent subpopulations resistant to freeze-thaw cycles and against temperature treatment. The ubiquity and high stability of the lichen INs suggest that they can impact local atmospheric processes and that ice nucleation activity is an essential trait for their survival in cold environments.

Interspecies Differences in Proteome Turnover Kinetics Are Correlated With Life Spans and Energetic Demands.

Cells continually degrade and replace damaged proteins. However, the high energetic demand of protein turnover generates reactive oxygen species that compromise the long-term health of the proteome. Thus, the relationship between aging, protein turnover, and energetic demand remains unclear. Here, we used a proteomic approach to measure rates of protein turnover within primary fibroblasts isolated from a number of species with diverse life spans including the longest-lived mammal, the bowhead whale. We show that organismal life span is negatively correlated with turnover rates of highly abundant proteins. In comparison with mice, cells from long-lived naked mole rats have slower rates of protein turnover, lower levels of ATP production, and reduced reactive oxygen species levels. Despite having slower rates of protein turnover, naked mole rat cells tolerate protein misfolding stress more effectively than mouse cells. We suggest that in lieu of a rapid constitutive turnover, long-lived species may have evolved more energetically efficient mechanisms for selective detection and clearance of damaged proteins.

An ice-binding protein from an Arctic population of American dunegrass, Leymus mollis.

Several cold-hardy grasses have been shown to have ice-binding proteins (IBPs) that protect against freeze-thaw injury. Here, we looked for IBP activity in an Alaskan coastal grass, Leymus mollis (Pooidae), that had not previously been examined. Rhizome tissue had strong ice-structuring and ice recrystallization inhibiting (IRI) activities, indicating the probable presence of IBPs. The gene sequence of an IBP was obtained. The sequence encoded a 118-amino acid IRI domain composed of eight repeats and that was 80% identical to the IRI domain of the IBP of perennial ryegrass Lolium perenne. The predicted 3D structure of the IRI domain had eight beta-roll coils like those in L. perenne IBP.

Multi-year assessment (2006-2015) of persistent organic pollutant concentrations in blubber and muscle from Western Arctic bowhead whales (Balaena mysticetus), North Slope, Alaska.

Blubber and muscle were collected from male bowhead whales (n = 71) landed near Utqiagvik (Barrow), Alaska, between 2006 and 2015 and analyzed for lipid content and concentrations of persistent organic pollutants (POPs) in order to determine levels and trends over the collection period. Collection year was a significant predictor of blubber concentrations for most classes of POPs, while for a few classes, animal length (proxy for age) was also a significant predictor. This is the first report on levels of PBDEs in bowhead whales; concentrations of these compounds are low (≤55 ng/g wet weight). Blubber concentrations were lower than those reported in samples collected between 1992 and 2000, and many POP classes in blubber declined significantly between 2006 and 2015. Concentrations of POPs in bowhead whale tissues, which are subsistence foods for Native Alaskan communities, appear to be declining at rates comparable with previously reported temporal trends in Arctic biota.

How do baleen whales stow their filter? A comparative biomechanical analysis of baleen bending.

Bowhead and right whale (balaenid) baleen filtering plates, longer in vertical dimension (≥3-4 m) than the closed mouth, presumably bend during gape closure. This has not been observed in live whales, even with scrutiny of video-recorded feeding sequences. To determine what happens to the baleen during gape closure, we conducted an integrative, multifactorial study including materials testing, functional (flow tank and kinematic) testing and histological examination. We measured baleen bending properties along the dorsoventral length of plates and anteroposterior location within a rack of plates via mechanical (axial bending, composite flexure, compression and tension) tests of hydrated and air-dried tissue samples from balaenid and other whale baleen. Balaenid baleen is remarkably strong yet pliable, with ductile fringes, and low stiffness and high elasticity when wet; it likely bends in the closed mouth when not used for filtration. Calculation of flexural modulus from stress/strain experiments shows that the balaenid baleen is slightly more flexible where it emerges from the gums and at its ventral terminus, but kinematic analysis indicates plates bend evenly along their whole length. Fin and humpback whale baleen has similar material properties but less flexibility, with no dorsoventral variation. The internal horn tubes have greater external and hollow luminal diameter but lower density in the lateral relative to medial baleen of bowhead and fin whales, suggesting a greater capacity for lateral bending. Baleen bending has major consequences not only for feeding morphology and energetics but also for conservation given that entanglement in fishing gear is a leading cause of whale mortality.

Hydration affects the physical and mechanical properties of baleen tissue.

Baleen, an anisotropic oral filtering tissue found only in the mouth of mysticete whales and made solely of alpha-keratin, exhibits markedly differing physical and mechanical properties between dried or (as in life) hydrated states. On average baleen is 32.35% water by weight in North Atlantic right whales (Eubalaena glacialis) and 34.37% in bowhead whales (Balaena mysticetus). Baleen's wettability measured by water droplet contact angles shows that dried baleen is hydrophobic whereas hydrated baleen is highly hydrophilic. Three-point flexural bending tests of mechanical strength reveal that baleen is strong yet ductile. Dried baleen is brittle and shatters at about 20-30 N mm-2 but hydrated baleen is less stiff; it bends with little force and absorbed water is squeezed out when force is applied. Maximum recorded stress was 4× higher in dried (mean 14.29 N mm-2) versus hydrated (mean 3.69 N mm-2) baleen, and the flexural stiffness was >10× higher in dried (mean 633N mm-2) versus hydrated (mean 58 N mm-2) baleen. In addition to documenting hydration's powerful effects on baleen, this study indicates that baleen is far more pliant and malleable than commonly supposed, with implications for studies of baleen's structure and function as well as its susceptibility to oil or other hydrophobic pollutants.

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.

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.

Overwintering physiology and microhabitat use of Phyllocnistis populiella (Lepidoptera: Gracilliariidae) in interior Alaska.

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.

Investigating the deep supercooling ability of an Alaskan beetle, Cucujus clavipes puniceus, via high throughput proteomics.

Cucujus clavipes puniceus is a freeze avoiding beetle capable of surviving the long, extremely cold winters of the Interior of Alaska. Previous studies showed that some individuals typically supercool to mean values of approximately -40 °C, with some individuals supercooling to as low as -58 °C, but these non-deep supercooling (NDSC) individuals eventually freeze if temperatures drop below this. However, other larvae, especially if exposed to very cold temperatures, supercool even further. These deep supercooling (DSC) individuals do not freeze even if cooled to -100 °C. In addition, the body water of the DSC larvae vitrifies (turns to a glass) at glass transition temperatures of -58 to -70 °C. This study examines the proteomes of DSC and NDSC larvae to assess proteins that may contribute to or inhibit the DSC trait. Using high throughput proteomics, we identified 138 proteins and 513 Gene Ontology categories in the DSC group and 104 proteins and 573 GO categories in the NDSC group. GO categories enriched in DSC include alcohol metabolic process, cellular component morphogenesis, monosaccharide metabolic process, regulation of biological quality, extracellular region, structural molecule activity, and antioxidant activity. Proteins unique to DSC include alpha casein precursor, alpha-actinin, vimentin, tropomyosin, beta-lactoglobulin, immunoglobulins, tubulin, cuticle proteins and endothelins.

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.

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. (1)H 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.

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 degrees 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 beta-mannopyranosyl-(1-->4) beta-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-beta-(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.

Simultaneous freeze tolerance and avoidance in individual fungus gnats, Exechia nugatoria.

Freeze tolerance and freeze avoidance are typically described as mutually exclusive strategies for overwintering in animals. Here we show an insect species that combines both strategies. Individual fungus gnats, collected in Fairbanks, Alaska, display two freezing events when experimentally cooled and different rates of survival after each event (mean +/- SEM: -31.5 +/- 0.2 degrees C, 70% survival and -50.7 +/- 0.4 degrees C, 0% survival). To determine which body compartments froze at each event, we dissected the abdomen from the head/thorax and cooled each part separately. There was a significant difference between temperature levels of abdominal freezing (-30.1 +/- 1.1 degrees C) and head/thorax freezing (-48.7 +/- 1.3 degrees C). We suggest that freezing is initially restricted to one body compartment by regional dehydration in the head/thorax that prevents inoculative freezing between the freeze-tolerant abdomen (71.0 +/- 0.8% water) and the supercooled, freeze-sensitive head/thorax (46.6 +/- 0.8% water).

Freeze tolerance in an arctic Alaska stonefly.

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 degrees C in midwinter. When in contact with ice, autumn-collected N. arctica cool to -1.5+/-0.4 degrees C before freezing, but individuals survived temperatures as low as -15 degrees 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 degrees 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 degrees C to -8 degrees 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.

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

The beetle Cucujus clavipes is found in North America over a broad latitudinal range from North Carolina (latitude approximately 35 degrees N) to near tree line in the Brooks Range in Alaska (latitude, approximately 67 degrees 30' N). The cold adaptations of populations from northern Indiana (approximately 41 degrees 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 (approximately 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 degrees 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 degrees C, those of Alaska larvae were -35 to -42 degrees C, and at certain times Alaska C. clavipes did not freeze when cooled to -80 degrees C.