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Internal ice formation leads to wholesale changes in ionic, osmotic and pH homeostasis, energy metabolism, and mechanical damage, across a small range of temperatures, and is thus an abiotic stressor that acts at a distinct, physiologically relevant, threshold. Insects that experience repeated freeze-thaw cycles over winter will cross this stressor threshold many times over their lifespan. Here, we examined the effect of repeatedly crossing the freezing threshold on short-term physiological parameters (metabolic reserves and cryoprotectant concentration) as well as long-term fitness-related performance (survival and egg production) in the freeze-tolerant goldenrod gall fly, Eurosta solidaginis We exposed overwintering prepupae to a series of low temperatures (-10, -15 or -20°C) with increasing numbers of freezing events (3, 6 or 10) with differing recovery periods between events (1, 5 or 10 days). Repeated freezing increased sorbitol concentration by about 50% relative to a single freezing episode, and prompted prepupae to modify long-chain triacylglycerols to acetylated triacylglycerols. Long-term, repeated freezing did not significantly reduce survival but did reduce egg production by 9.8% relative to a single freezing event. Exposure temperature did not affect any of these measures, suggesting that threshold crossing events may be more important to fitness than the intensity of stress in overwintering E. solidaginis ., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Rainbow smelt, Osmerus mordax, experience a wide range of temperatures in their native habitat. In response to cold, smelt express anti-freeze proteins and the osmolytes glycerol, trimethylamine N-oxide (TMAO) and urea to avoid freezing. The physiological influences of these osmolytes are not well understood. Urea destabilizes proteins, while TMAO counteracts the protein-destabilizing forces of urea. The influence of glycerol on muscle function has not been explored. We examined the effects of urea, glycerol and TMAO through muscle mechanics experiments with treatments of the three osmolytes at physiological concentrations. Experiments were carried out at 10°C. The contractile properties of fast-twitch muscle bundles were determined in physiological saline and in the presence of 50 mmol l(-1)urea, 50 mmol l(-1)TMAO and/or 200 mmol l(-1)glycerol in saline. Muscle exposed to urea and glycerol produced less force and displayed slower contractile properties. However, treatment with TMAO led to higher force and faster relaxation by muscle bundles. TMAO increased power production during cyclical activity, while urea and glycerol led to reduced oscillatory power output. When muscle bundles were exposed to a combination of the three osmolytes, they displayed little change in contraction kinetics relative to control, although power output under lower oscillatory conditions was enhanced while maximum power output was reduced. The results suggest that maintenance of muscle function in winter smelt requires a balanced combination of urea, glycerol and TMAO., (© 2016. Published by The Company of Biologists Ltd.)

Ice-binding proteins (IBPs) assist in subzero tolerance of multiple cold-tolerant organisms: animals, plants, fungi, bacteria etc. IBPs include: (1) antifreeze proteins (AFPs) with high thermal hysteresis antifreeze activity; (2) low thermal hysteresis IBPs; and (3) ice-nucleating proteins (INPs). Several structurally different IBPs have evolved, even within related taxa. Proteins that produce thermal hysteresis inhibit freezing by a non-colligative mechanism, whereby they adsorb onto ice crystals or ice-nucleating surfaces and prevent further growth. This lowers the so-called hysteretic freezing point below the normal equilibrium freezing/melting point, producing a difference between the two, termed thermal hysteresis. True AFPs with high thermal hysteresis are found in freeze-avoiding animals (those that must prevent freezing, as they die if frozen) especially marine fish, insects and other terrestrial arthropods where they function to prevent freezing at temperatures below those commonly experienced by the organism. Low thermal hysteresis IBPs are found in freeze-tolerant organisms (those able to survive extracellular freezing), and function to inhibit recrystallization - a potentially damaging process whereby larger ice crystals grow at the expense of smaller ones - and in some cases, prevent lethal propagation of extracellular ice into the cytoplasm. Ice-nucleator proteins inhibit supercooling and induce freezing in the extracellular fluid at high subzero temperatures in many freeze-tolerant species, thereby allowing them to control the location and temperature of ice nucleation, and the rate of ice growth. Numerous nuances to these functions have evolved. Antifreeze glycolipids with significant thermal hysteresis activity were recently identified in insects, frogs and plants., (© 2015. Published by The Company of Biologists Ltd.)

We investigated the ecological physiology and behavior of free-living wood frogs [Lithobates (Rana) sylvaticus] overwintering in Interior Alaska by tracking animals into natural hibernacula, recording microclimate, and determining frog survival in spring. We measured cryoprotectant (glucose) concentrations and identified the presence of antifreeze glycolipids in tissues from subsamples of naturally freezing frogs. We also recorded the behavior of wood frogs preparing to freeze in artificial hibernacula, and tissue glucose concentrations in captive wood frogs frozen in the laboratory to -2.5°C. Wood frogs in natural hibernacula remained frozen for 193 ± 11 consecutive days and experienced average (October-May) temperatures of -6.3°C and average minimum temperatures of -14.6 ± 2.8°C (range -8.9 to -18.1°C) with 100% survival (N=18). Mean glucose concentrations were 13-fold higher in muscle, 10-fold higher in heart and 3.3-fold higher in liver in naturally freezing compared with laboratory frozen frogs. Antifreeze glycolipid was present in extracts from muscle and internal organs, but not skin, of frozen frogs. Wood frogs in Interior Alaska survive freezing to extreme limits and durations compared with those described in animals collected in southern Canada or the Midwestern United States. We hypothesize that this enhancement of freeze tolerance in Alaskan wood frogs is due to higher cryoprotectant levels that are produced by repeated freezing and thawing cycles experienced under natural conditions during early autumn., (© 2014. Published by The Company of Biologists Ltd.)

During the austral summer, larvae of the terrestrial midge Belgica antarctica (Diptera: Chironomidae) experience highly variable and often unpredictable thermal conditions. In addition to remaining freeze tolerant year-round, larvae are capable of swiftly increasing their cold tolerance through the rapid cold-hardening (RCH) response. The present study compared the induction of RCH in frozen versus supercooled larvae. At the same induction temperature, RCH occurred more rapidly and conferred a greater level of cryoprotection in frozen versus supercooled larvae. Furthermore, RCH in frozen larvae could be induced at temperatures as low as -12°C, which is the lowest temperature reported to induce RCH. Remarkably, as little as 15 min at -5°C significantly enhanced larval cold tolerance. Not only is protection from RCH acquired swiftly, but it is also quickly lost after thawing for 2 h at 2°C. Because the primary difference between frozen and supercooled larvae is cellular dehydration caused by freeze concentration of body fluids, we also compared the effects of acclimation in dehydrated versus frozen larvae. Because slow dehydration without chilling significantly increased larval survival to a subsequent cold exposure, we hypothesize that cellular dehydration caused by freeze concentration promotes the rapid acquisition of cold tolerance in frozen larvae.

During cold periods ectotherms may improve low temperature tolerance via rapid cold hardening (RCH) over a period of hours and/or long-term cold acclimation (LTCA) during days, weeks or months. However, the effect of duration and the major underlying mechanisms of these processes are still not fully understood. In the present study, the molecular and biochemical responses to RCH (1-3 h) and LTCA (1-3 days) and the corresponding benefits to survival were investigated using the chill-sensitive collembolan Folsomia candida. We investigated osmolyte accumulation, membrane restructuring and transcription of candidate genes as well as survival benefits in response to RCH and LTCA. RCH induced significant upregulation of targeted genes encoding enzymes related to carbohydrate metabolic pathways and genes encoding small and constitutively expressed heat shock proteins (Hsps), indicating that the animals rely on protein protection from a subset of Hsps during RCH and probably also LTCA. The upregulation of genes involved in carbohydrate metabolic processes initiated during RCH was likely responsible for a transient accumulation of myoinositol during LTCA, which may support the protection of protein and membrane function and structure. Membrane restructuring, composed especially of a significantly increased ratio of unsaturated to saturated phospholipid fatty acids seems to be a mechanism supplementary to activation of Hsps and myoinositol accumulation in LTCA. Thus, the moderate increase in cold shock tolerance conferred by RCH seems to be dominated by effects of Hsps, whereas the substantially better cold tolerance achieved after LTCA is dominated by post-transcriptional processes increasing membrane fluidity and cryoprotectant concentration.

Ectothermic animals inhabiting the subarctic and temperate regions have evolved strategies to deal with periods of continuous frost during winter. The earthworm Dendrobaena octaedra is freeze tolerant and accumulates large concentrations of glucose upon freezing. The present study investigates the roles of glucose accumulation for long-term freeze tolerance in worms kept frozen at -2 degrees C for 47 days. During this period, worms were sampled periodically for determination of survival and for measurements of glucose, glycogen, lactate, alanine and succinate. In addition we performed calorimetric measurements to assess metabolic rate of frozen and unfrozen worms. Long-term freezing was associated with a gradual depletion of glucose and worms that succumbed during this period were always characterised by low glucose and glycogen levels. The anaerobic waste products lactate and alanine increased slightly whereas succinate levels remained constant. However, it is argued that other waste products (particularly propionate) could be the primary end product of a continued anaerobic metabolism. Calorimetric measures of the metabolic rate of frozen worms were in accord with values calculated from the reduction in glucose assuming that most ( approximately 90%) glucose was metabolised anaerobically. Both estimates of metabolic rate demonstrated a 10-fold metabolic depression associated with freezing. Thus, in addition to the suspected role of glucose as cryoprotectant, the present study demonstrates that glucose accumulation is vital to ensure substrate for long-term anaerobic metabolism in frozen worms. On the basis of the estimated metabolite levels, we calculate that the combined effect of metabolic depression and large glucose stores enables a projected 3 months survival of freezing at -2 degrees C of the ;average' D. octaedra. Such conditions are very likely to occur in the northern distribution ranges of this stress-tolerant earthworm.

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.

The physiological responses supporting freeze tolerance in anurans are well known, but the evolution of this trait remains little studied. This is the first common-garden study of geographic variation in cryoprotective responses to freezing and the degree of freeze tolerance. We studied the gray treefrogs (Hyla versicolor and H. chrysoscelis) from sympatric sites in Minnesota, Indiana and Missouri. Patterns in the literature suggest that northern frogs produce more cryoprotectants upon freezing, but we found no geographic variation in cryoprotective responses or degree of freeze tolerance. The concentration of glucose produced upon freezing was higher than previously reported for this species (liver: 475 micro mol g(-1) dry mass). Unfrozen frogs had high levels of glycerol (liver: approx. 150 micro mol g(-1) dry mass), and did not produce more upon freezing. Liver glycogen content (concentration multiplied by liver mass) was highest in frogs from Minnesota and Missouri, and was stored in preference to lipids in Minnesota frogs, possibly to provide energy for the longer northern winters. Minnesota frogs accumulated more ice (53.4+/-1.8%) after freezing to-2.5 degrees C than Indiana frogs (45.5+/-3.3%). The two species differed in body size but not in any of the physiological parameters measured. We conclude that these populations show no adaptive variation in freeze tolerance and that comparing published studies may be misleading because of different acclimation and feeding regimes.

In contrast to most studies of rapid cold-hardening, in which abrupt transfers to low temperatures are used to induce an acclimatory response, the primary objectives of this study were to determine (i) whether rapid cold-hardening was induced during the cooling phase of an ecologically based thermoperiod, (ii) whether the protection afforded was lost during warming or contributed to increased cold-tolerance during subsequent cycles and (iii) whether the major thermally inducible stress protein (Hsp70) or carbohydrate cryoprotectants contributed to the protection afforded by rapid cold-hardening. During the cooling phase of a single ecologically based thermoperiod, the tolerance of Drosophila melanogaster to 1 h at -7 degrees C increased from 5 +/- 5% survival to 62.5 +/- 7.3% (means +/- S.E.M., N=40-60), while their critical thermal minima (CTmin) decreased by 1.9 degrees C. Cold hardiness increased with the number of thermoperiods to which flies were exposed; i.e. flies exposed to six thermoperiods were more cold-tolerant than those exposed to two. Endogenous levels of Hsp70 and carbohydrate cryoprotectants were unchanged in rapidly cold-hardened adults compared with controls held at a constant 23 degrees C. In nature, rapid cold-hardening probably affords subtle benefits during short-term cooling, such as allowing D. melanogaster to remain active at lower temperatures than they otherwise could.