Physiological responses to supercooling and hypoxia in the hatchling painted turtle, Chrysemys picta

We investigated physiological responses to supercooling in hatchling painted turtles (Chrysemys picta) which remain in their natal nests over winter and therefore may become exposed to subzero temperatures. These turtles are freeze tolerant but also must rely on supercooling to survive exposure to the lower temperatures occurring in nests during winter. We compared whole-body concentrations of lactate, glucose, glycerol, and ATP in turtles chilled at 0 degrees C, -4 degrees C, or -6 degrees C for 5 days, or at 6 degrees C for 19 days. In a companion experiment, we measured metabolite concentrations in turtles exposed to a hypoxic environment for 1 day, 4 days, or 8 days. Supercooling and hypoxia exposure were both associated with an increase in concentrations of lactate and glucose and a decrease in glycerol concentrations (albeit no change in the ATP pool), suggesting that supercooling induces functional hypoxia. We conclude that hypoxia tolerance may be an important pre-adaptation for surviving exposure to subzero temperatures in hatchling C. picta.     

Characteristics of nest soil, but not geographic origin, influence cold hardiness of hatchling painted turtles

We investigated environmental factors influencing cold hardiness in hatchling painted turtles (Chrysemys picta) indigenous to northeastern Indiana and the Sandhills of west-central Nebraska. In both locations, hatchlings overwinter in their natal nests. Survival of hatchlings chilled to minimum temperatures between -2.5 and -6.0 degrees C inside explanted natal nests ranged from 30 to 100%. Mortality likely was caused by freezing of the turtles that was induced by contact with ice nuclei in the surrounding soil. Susceptibility to inoculative freezing was strongly influenced by moisture content (7.5-25%, w/w) of the frozen soil in which hatchlings were cooled. When chilled in soil containing 15% moisture, turtles from Indiana resisted inoculative freezing better than hatchlings from Nebraska, but this variation was due to physical characteristics of the soils indigenous to each locale rather than genetic differences between populations. Soil in which the Indiana turtles nested contained relatively higher amounts of clay and organic matter, and bound more moisture, than the loamy sand at the Nebraska site. Soil collected from both locales contained potent ice nuclei that may constrain supercooling of the hatchlings, even in the absence of soil moisture. In addition to temperature and precipitation, local and regional variation in soils is an important determinant of overwintering survival of hatchling C. picta.     

The relationship between gut contents and supercooling capacity in hatchling painted turtles (Chrysemys picta)

Painted turtles (Chrysemys picta) typically spend their first winter of life in a shallow, subterranean hibernaculum (the natal nest) where they seemingly withstand exposure to ice and cold by resisting freezing and becoming supercooled. However, turtles ingest soil and fragments of eggshell as they are hatching from their eggs, and the ingestate usually contains efficient nucleating agents that cause water to freeze at high subzero temperatures. Consequently, neonatal painted turtles have only a modest ability to undergo supercooling in the period immediately after hatching. We studied the limit for supercooling (SCP) in hatchlings that were acclimating to different thermal regimes and then related SCPs of the turtles to the amount of particulate matter in their gastrointestinal (GI) tract. Turtles that were transferred directly from 26 degrees C (the incubation temperature) to 2 degrees C did not purge soil from their gut, and SCPs for these animals remained near -4 degrees C for the 60 days of the study. Animals that were held at 26 degrees C for the duration of the experiment usually cleared soil from their GI tract within 24 days, but SCPs for these turtles were only slightly lower after 60 days than they were at the outset of the experiment. Hatchlings that were acclimating slowly to 2 degrees C cleared soil from their gut within 24 days and realized a modest reduction in their SCP. However, the limit of supercooling in the slowly acclimating animals continued to decline even after all particulate material had been removed from their GI tract, thereby indicating that factors intrinsic to the nucleating agents themselves also may have been involved in the acclimation of hatchlings to low temperature. The lowest SCPs for turtles that were acclimating slowly to 2 degrees C were similar to SCPs recorded in an earlier study of animals taken from natural nests in late autumn, so the current findings affirm the importance of seasonally declining temperatures in preparing animals in the field to withstand conditions that they will encounter during winter.     

Physiological ecology of overwintering in the hatchling painted turtle: multiple-scale variation in response to environmental stress

We integrated field and laboratory studies in an investigation of water balance, energy use, and mechanisms of cold-hardiness in hatchling painted turtles (Chrysemys picta) indigenous to west-central Nebraska (Chrysemys picta bellii) and northern Indiana (Chrysemys picta marginata) during the winters of 1999-2000 and 2000-2001. We examined 184 nests, 80 of which provided the hatchlings (n=580) and/or samples of soil used in laboratory analyses. Whereas winter 1999-2000 was relatively dry and mild, the following winter was wet and cold; serendipitously, the contrast illuminated a marked plasticity in physiological response to environmental stress. Physiological and cold-hardiness responses of turtles also varied between study locales, largely owing to differences in precipitation and edaphics and the lower prevailing and minimum nest temperatures (to -13.2 degrees C) encountered by Nebraska turtles. In Nebraska, winter mortality occurred within 12.5% (1999-2000) and 42.3% (2000-2001) of the sampled nests; no turtles died in the Indiana nests. Laboratory studies of the mechanisms of cold-hardiness used by hatchling C. picta showed that resistance to inoculative freezing and capacity for freeze tolerance increased as winter approached. However, the level of inoculation resistance strongly depended on the physical characteristics of nest soil, as well as its moisture content, which varied seasonally. Risk of inoculative freezing (and mortality) was greatest in midwinter when nest temperatures were lowest and soil moisture and activity of constituent organic ice nuclei were highest. Water balance in overwintering hatchlings was closely linked to dynamics of precipitation and soil moisture, whereas energy use and the size of the energy reserve available to hatchlings in spring depended on the winter thermal regime. Acute chilling resulted in hyperglycemia and hyperlactemia, which persisted throughout winter; this response may be cryoprotective. Some physiological characteristics and cold-hardiness attributes varied between years, between study sites, among nests at the same site, and among siblings sharing nests. Such variation may reflect adaptive phenotypic plasticity, maternal or paternal influence on an individual's response to environmental challenge, or a combination of these factors. Some evidence suggests that life-history traits, such as clutch size and body size, have been shaped by constraints imposed by the harsh winter environment.     

Intracellular ice formation in yeast cells vs. cooling rate: Predictions from modeling vs. experimental observations by differential scanning calorimetry

To survive freezing, cells must not undergo internal ice formation during cooling. One vital factor is the cooling rate. The faster cells are cooled, the more their contents supercool, and at some subzero temperature that supercooled cytoplasm will freeze. The question is at what temperature? The relation between cooling rate and cell supercooling can be computed. Two important parameters are the water permeability (Lp) and its temperature dependence. To avoid intracellular ice formation (IIF), the supercooling must be eliminated by dehydration before the cell cools to its ice nucleation temperature. With an observed nucleation temperature of −25 °C, the modeling predicts that IIF should not occur in yeast cooled at <20 °C/min and it should occur with near certainty in cells cooled at ?30 °C/min. Experiments with differential scanning calorimetry (DSC) confirmed these predictions closely. The premise with the DSC is that if there is no IIF, one should see only a single exotherm representing the freezing of the external water. If IIF occurs, one should see a second, lower temperature exotherm. A further test of whether this second exotherm is IIF is whether it disappears on repeated freezing. IIF disrupts the plasma membrane; consequently, in a subsequent freeze cycle, the cell can no longer supercool and will not exhibit a second exotherm. This proved to be the case at cooling rates >20 °C/min.     

Adaptations to terrestrial overwintering of hatchling northern map turtles,<Emphasis Type="Italic"> Graptemys geographica</Emphasis>

We conducted a 3-year field and laboratory study of winter biology in hatchlings of the northern map turtle (Graptemys geographica). At our study area in northern Indiana, hatchlings routinely overwintered in their natal nests, emerging after the weather warmed in spring. Winter survival was excellent despite the fact that hatchlings were exposed frequently to subfreezing temperatures (to –5.4 °C). In the laboratory, cold-acclimated hatchlings exhibited low rates of evaporative water loss (mean=2.0 mg g^–1 day^–1), which would enable them to conserve body water during winter. Laboratory-reared hatchlings were intolerant of freezing at –2.5 °C for 24 h, conditions that are readily survived by freeze-tolerant species of turtles. Winter survival of hatchling G. geographica probably depended on their extensive capacity for supercooling (to –14.8 °C) and their well-developed resistance to inoculative freezing, which may occur when hatchlings contact ice and ice-nucleating agents present in nesting soil. Supercooled hatchlings survived a brief exposure to –8 °C. Others, held at –6 °C for 5 days, maintained ATP concentrations at control levels, although they did accumulate lactate and glucose, probably in response to tissue hypoxia. Therefore, anoxia tolerance, as evidenced by the viability of hatchlings exposed to N₂ gas for 8 days, may promote survival during exposure to subfreezing temperatures.Abbreviations EWL evaporative water loss - FP_eq equilibrium freezing point - INA ice-nucleating agents - T_c temperature of crystallizationCommunicated by L.C.-H. Wang     

Accumulation of lactate by supercooled hatchlings of the painted turtle (Chrysemys picta): implications for overwinter survival

Hatchlings of the North American painted turtle (Chrysemys picta) typically spend their first winter of life inside the shallow, subterranean nest where they completed embryogenesis the preceding summer. Neonates at northern localities consequently may be exposed during winter to subzero temperatures and frozen soil. Hatchlings apparently survive exposure to such conditions by supercooling, but the physiological consequences of this adaptive strategy have not been examined. We measured lactate in hatchling painted turtles after exposure to each of three temperatures (0 °C, −4 °C, and −8 °C) for three time periods (5 days, 15 days, and 25 days) to determine the extent to which overwintering hatchlings might rely on anaerobic metabolism to regenerate ATP. Whole-body lactate increased with increasing duration of exposure and decreasing temperature, and the highest levels were associated with the group that experienced the highest mortality. These results indicate that animals may develop a considerable lactic acidosis during a winter in which temperatures fall below 0 °C for weeks or months and that accumulation of lactate may contribute to mortality of overwintering animals. Accepted: 20 October 1999     

Natural freeze-tolerance in hatchling painted turtles?

Hatchlings of the North American painted turtle (Family Emydidae: Chrysemys picta) typically spend their first winter of life inside a shallow, subterranean hibernaculum (the natal nest) where life-threatening conditions of ice and cold commonly occur. Although a popular opinion holds that neonates exploit a tolerance for freezing to survive the rigors of winter, hatchlings are more likely to withstand exposure to ice and cold by avoiding freezing altogether-and to do so without the benefit of an antifreeze. In the interval between hatching by turtles in late summer and the onset of wintery weather in November or December, the integument of the animals becomes highly resistant to the penetration of ice into body compartments from surrounding soil, and the turtles also purge their bodies of catalysts for the formation of ice. These two adjustments, taken together, enable the animals to supercool to temperatures below those that they routinely experience in nature. However, cardiac function in hatchlings is diminished at subzero temperatures, thereby compromising the delivery of oxygen to peripheral tissues and eliciting an increase in reliance by those tissues on anaerobic metabolism for the provision of ATP. The resulting increase in production of lactic acid may disrupt acid/base balance and lead to death even in animals that remain unfrozen. Although an ability to undergo supercooling may be key to survival by overwintering turtles in northerly populations, a similar capacity to resist inoculation and undergo supercooling characterizes animals from a population near the southern limit of distribution, where winters are relatively benign. Thus, the suite of characters enabling hatchlings to withstand exposure to ice and cold may have been acquired prior to the northward dispersal of the species at the end of the Pleistocene, and the characters may not have originated as adaptations specifically to the challenges of winter.     

Reptile freeze tolerance: metabolism and gene expression

Terrestrially hibernating reptiles that live in seasonally cold climates need effective strategies of cold hardiness to survive the winter. Use of thermally buffered hibernacula is very important but when exposure to temperatures below 0 degrees C cannot be avoided, either freeze avoidance (supercooling) or freeze tolerance strategies can be employed, sometimes by the same species depending on environmental conditions. Several reptile species display ecologically relevant freeze tolerance, surviving for extended times with 50% or more of their total body water frozen. The use of colligative cryoprotectants by reptiles is poorly developed but metabolic and enzymatic adaptations providing anoxia tolerance and antioxidant defense are important aids to freezing survival. New studies using DNA array screening are examining the role of freeze-responsive gene expression. Three categories of freeze responsive genes have been identified from recent screenings of liver and heart from freeze-exposed (5h post-nucleation at -2.5 degrees C) hatchling painted turtles, Chrysemys picta marginata. These genes encode (a) proteins involved in iron binding, (b) enzymes of antioxidant defense, and (c) serine protease inhibitors. The same genes were up-regulated by anoxia exposure (4 h of N2 gas exposure at 5 degrees C) of the hatchlings which suggests that these defenses for freeze tolerance are aimed at counteracting the injurious effects of the ischemia imposed by plasma freezing.     

The role of the integument as a barrier to penetration of ice into overwintering hatchlings of the painted turtle (Chrysemys picta)

Hatchlings of the North American painted turtle (Chrysemys picta) spend their first winter of life inside a shallow, subterranean hibernaculum (the natal nest) where they may be exposed for extended periods to ice and cold. Hatchlings seemingly survive exposure to such conditions by becoming supercooled (i.e., by remaining unfrozen at temperatures below the equilibrium freezing point for body fluids), so we investigated the role of their integument in preventing ice from penetrating into body compartments from surrounding soil. We first showed that hatchlings whose epidermis has been damaged are more likely to be penetrated by growing crystals of ice than are turtles whose cutaneous barrier is intact. We next studied integument from a forelimb by light microscopy and discovered that the basal part of the alpha-keratin layer of the epidermis contains a dense layer of lipid. Skin from the forelimb of other neonatal turtles lacks such a layer of lipid in the epidermis, and these other turtles also are highly susceptible to inoculative freezing. Moreover, epidermis from the neck of hatchling painted turtles lacks the lipid layer, and this region of the skin is readily penetrated by growing crystals of ice. We therefore conclude that the resistance to inoculation imposed by skin on the limbs of hatchling painted turtles results from the presence of lipids in the alpha-keratin layer of the epidermis. Neonates apparently are able to avoid freezing during winter by drawing much of the body inside the shell, leaving only the ice-resistant integument of the limbs exposed to ice in the environment. The combination of behavior and skin morphology enables overwintering hatchlings to exploit an adaptive strategy based on supercooling.     

Effect of age and body size on selected temperature by juvenile wood turtles (Glyptemys insculpta)

In an aquatic thermal gradient of 15–30 °C, 3-, 6-, and 12-month-old juvenile wood turtles (Glyptemys insculpta) acclimated to 20 °C selected the warmest temperature available (30 °C) and avoided the coldest temperatures available (15 and 18 °C). Mean selection of chambers differed between control and gradient tests across all temperatures except 27 °C. Turtles of all age classes relocated between chambers less often when the gradient was present than during control tests. Six- and 12-month-old turtles selected 30 °C more frequently, and selected colder temperatures less frequently, than 3-month-old turtles, suggesting that the ability to select preferred temperatures is better developed in older hatchlings.     

Oxidative stress and antioxidant capacity of a terrestrially hibernating hatchling turtle

Hatchlings of the painted turtle, Chrysemys picta, hibernate terrestrially and can survive subfreezing temperatures by supercooling or by tolerating the freezing of their tissues. Whether supercooled or frozen, an ischemic hypoxia develops because tissue perfusion is limited by low temperature and/or freezing. Oxidative stress can occur if hatchlings lack sufficient antioxidant defenses to minimize or prevent damage by reactive oxygen species. We examined the antioxidant capacity and indices of oxidative damage in hatchling C. picta following survivable, 48 h bouts of supercooling (−6°C), freezing (−2.5°C), or hypoxia (4°C). Samples of plasma, brain, and liver were collected after a 24 h period of recovery (4°C) and assayed for Trolox-equivalent antioxidant capacity (TEAC), thiobarbituric acid reactive substances (TBARS), and carbonyl proteins. Antioxidant capacity did not vary among treatments in any of the tissues studied. We found a significant increase in TBARS in plasma, but not in the brain or liver, of frozen/thawed hatchlings as compared to untreated controls. No changes were found in the concentration of TBARS or carbonyl proteins in supercooled or hypoxia-exposed hatchlings. Our results suggest that hatchling C. picta have a well-developed antioxidant defense system that minimizes oxidative damage during hibernation.     

Influence of freezing temperatures on a cactus,Coryphantha vivipara

Summary Coryphantha vivipara (Nutt.) Britton & Rose var. deserti (Engelm.) W.T. Marshall (Cactaceae) survived snow and tissue temperatures of-12°C in southern Nevada. However, the freezing point depression of the cell sap was only about 0.9°C. When the nocturnal air temperature in the laboratory was reduced from 10°C to-10°C for one night, the optimum temperature for CO₂ uptake shifted from 10°C to 6°C and uptake was reduced 70%, but full recovery to the original values occurred in 4 days. Nocturnal temperatures of-15°C killed 2 out of 5 plants and-20°C killed 5 out of 5, as judged by lack of net CO₂ uptake at night over a 2-month observation period. when the stems were cooled at 2° C/h, supercooling to about-6°C occurred followed by an exothermic reaction that presumably represented the freezing of extracellular water. When the subzero temperature was lowered further, no other exothermic reaction was observed and the cells became progressively dehydrated. Freezing-induced tissue death was ascribed to this cellular dehydration, which led to about 94% loss of intracellular water at-15°C. when the tissue temperature was lowered, the ability of chlorenchyma cells to plasmolyze and to take up a stain decreased, both being nearly 70% inhibited at-15°C and completely abolished at-20°C. Some cold-bardening occurred, since lowering the air temperature from 30° to-10°C in 10°C increments at weekly intervals caused the subzero temperature for 50% inhibition of staining to decrease from-10°C to-17°C. Extension of the range of C. vivipara to regions with wintertime freezing apparently reflects the tolerance of considerable freeze dehydration by its protoplasts.     

Supercooling and freeze-tolerance in the European wall lizard,Podarcis muralis,with a revisional history of the discovery of freeze-tolerance in vertebrates

Summary Wall lizards were collected in the fall of 1988 from a population introduced in 1951 into Cincinnati, OH. They were acclimated to 5 °C for several weeks prior to testing at sub-zero temperatures. Eleven super-cooled lizards were removed from the cooling chamber prior to crystallization after between 15 min and 26 h at body temperatures ranging from -2.2 to -5.9 °C. With the exception of one individual supercooled to-5.0 °C, all lizards recovered fully. The crystallization temperatures of 15 lizards which froze ranged from -0.6 to -6.4 °C. Frozen lizards were stiff with a distinct blue color, which faded upon thawing at 3 °C. The ice contents of frozen lizards were determined calorimetrically and/or estimated from a theoretical model, the two methods being generally in close agreement. Remarkably, five individuals recovered fully from exposures as long as 2 h and with as much as 28% of their body water frozen. Although these animals are not as tolerant as certain other vertebrates they are clearly able to withstand freezing under some circumstances. Failure to survive freezing was attributed either to excessive ice accumulation during a prolonged freeze or to excessive supercooling prior to freezing, which induced a large initial surge of ice formation upon crystallization. Our results accord with those of Weigmann (1929). We accordingly recognize him as the first to demonstrate freeze-tolerance in vertebrates, and we further recognize P. muralis as the first vertebrate known to survive freezing.     

Low temperature survival in different life stages of the Iberian slug, Arion lusitanicus

The slug Arion lusitanicus Mabille (Gastropoda: Pulmonata: Arionidae) is an invasive species which has spread to most parts of Europe. The area of origin is unknown, but A. lusitanicus seems to cope well with the local conditions in the countries to which it has migrated. It spreads rapidly, occurs often in high densities and has become a serious pest in most European countries. Therefore there is an urgent need for better knowledge of the ecophysiology of A. lusitanicus, such as the influence of climatic conditions, in order to develop prognostic models and strategies for novel pest management practises.The aim of our study was to investigate the influence of subzero temperatures in relation to winter survival. A. lusitanicus is shown to be freeze-tolerant in some life stages. Most juveniles and some adult slugs survived being frozen at −1.3 °C for 3 days, but none of the slugs survived freezing at −3 °C. The eggs survived subzero temperatures (down to −2 °C) probably by supercooling. Juveniles and adults may also survive in a supercooled state (down to −3 °C) but are generally poor supercoolers. Therefore, the winter survival of A. lusitanicus depends to a high degree on migration to habitats protected from low winter temperatures, e.g. under plant litter, buried in the soil or in compost heaps.     

Physiological ecology of overwintering in hatchling turtles

Temperate species of turtles hatch from eggs in late summer. The hatchlings of some species leave their natal nest to hibernate elsewhere on land or under water, whereas others usually remain inside the nest until spring; thus, post-hatching behavior strongly influences the hibernation ecology and physiology of this age class. Little is known about the habitats of and environmental conditions affecting aquatic hibernators, although laboratory studies suggest that chronically hypoxic sites are inhospitable to hatchlings. Field biologists have long been intrigued by the environmental conditions survived by hatchlings using terrestrial hibernacula, especially nests that ultimately serve as winter refugia. Hatchlings are unable to feed, although as metabolism is greatly reduced in hibernation, they are not at risk of starvation. Dehydration and injury from cold are more formidable challenges. Differential tolerances to these stressors may explain variation in hatchling overwintering habits among turtle taxa. Much study has been devoted to the cold-hardiness adaptations exhibited by terrestrial hibernators. All tolerate a degree of chilling, but survival of frost exposure depends on either freeze avoidance through supercooling or freeze tolerance. Freeze avoidance is promoted by behavioral, anatomical, and physiological features that minimize risk of inoculation by ice and ice-nucleating agents. Freeze tolerance is promoted by a complex suite of molecular, biochemical, and physiological responses enabling certain organisms to survive the freezing and thawing of extracellular fluids. Some species apparently can switch between freeze avoidance or freeze tolerance, the mode utilized in a particular instance of chilling depending on prevailing physiological and environmental conditions.     

Effects of acclimation and egg-incubation temperature on selected temperature by hatchling western painted turtles (Chrysemys picta bellii)

The ability of hatchling turtles to detect environmental temperature differences and to effectively select preferred temperature is a function that critically impacts survival. In some turtle species, temperature preference may be influenced by embryonic and post-hatching conditions, such as egg-incubation and acclimation temperature. We tested for effects of embryonic incubation temperature (27.5 °C, 30 °C) and acclimation temperature (20 °C, 25 °C) on the selected temperature and movement patterns of 32 Chrysemys picta bellii (Reptilia: Emydidae) hatchlings in an aquatic thermal gradient of 14-34 °C and in single-temperature (20 °C, 25 °C) control tests. Among 10-11 month old hatchlings, acclimation temperature and egg-incubation temperature influenced temperature selection and movement patterns. Acclimation temperature affected activity and movement: in thermal gradient and single-temperature control tests, 25 °C-acclimated turtles relocated between chambers significantly more frequently than individuals acclimated to 20 °C. Acclimation temperature also affected temperature selection: 20 °C-acclimated turtles selected a specific temperature during gradient tests, but 25 °C-acclimated turtles did not. Among 20 °C-acclimated turtles, egg-incubation temperature was inversely related to selected temperature: hatchling turtles incubated at 27.5 °C selected the warmest temperature available (34 °C); individuals incubated at 30 °C selected the coldest temperature (14 °C). These results suggest that interactions of environmental conditions may influence post-hatching thermoregulatory behavior in C. picta bellii, a factor that ultimately affects fitness.     

A Leech Capable of Surviving Exposure to Extremely Low Temperatures

It is widely considered that most organisms cannot survive prolonged exposure to temperatures below 0°C, primarily because of the damage caused by the water in cells as it freezes. However, some organisms are capable of surviving extreme variations in environmental conditions. In the case of temperature, the ability to survive subzero temperatures is referred to as cryobiosis. We show that the ozobranchid leech, Ozobranchus jantseanus, a parasite of freshwater turtles, has a surprisingly high tolerance to freezing and thawing. This finding is particularly interesting because the leach can survive these temperatures without any acclimation period or pretreatment. Specifically, the leech survived exposure to super-low temperatures by storage in liquid nitrogen (−196°C) for 24 hours, as well as long-term storage at temperatures as low as −90°C for up to 32 months. The leech was also capable of enduring repeated freeze-thaw cycles in the temperature range 20°C to −100°C and then back to 20°C. The results demonstrated that the novel cryotolerance mechanisms employed by O. jantseanus enable the leech to withstand a wider range of temperatures than those reported previously for cryobiotic organisms. We anticipate that the mechanism for the observed tolerance to freezing and thawing in O. jantseanus will prove useful for future studies of cryopreservation.     

Cellular Damage to the Callus Cells of Potato Subjected to Freezing

Callus cells of potato (Solanum tuberosum L.) cv. Désirée were exposed to various subzero temperatures and examined for the freezing damage. In the cells subjected to –3 °C, plasma membranes appeared to be intact, while tonoplast seemed to be damaged and organelles to be swollen. After freezing at –6 °C, the damage became severe and plasma membranes were ruptured. After exposure to –10 °C, the damage was so severe that the cell organelles could not be recognised and cytoplasm became fragmented.     

Supercooling,ice inoculation and freeze tolerance in the European common lizard,Lacerta vivipara

The European common lizard (Lacerta vivipara) is widely distributed throughout Eurasia and is one of the few Palaearctic reptiles occurring above the Arctic Circle. We investigated the cold-hardiness of L. vivipara from France which routinely encounter subzero temperatures within their shallow hibernation burrows. In the laboratory, cold-acclimated lizards exposed to subfreezing temperatures as low as -3.5°C could remain unfrozen (supercooled) for at least 3 weeks so long as their microenvironment was dry. In contrast, specimens cooled in contact with ambient ice crystals began to freeze within several hours. However, such susceptibility to inoculative freezing was not necessarily deleterious since L. vivipara readily tolerated the freezing of its tissues, with body surface temperatures as low as -3.0°C during trials lasting up to 3 days. Freezing survival was promoted by relatively low post-nucleation cooling rates (0.1°C·h^-1) and apparently was associated with an accumulation of the putative cryoprotectant, glucose. The cold-hardiness strategy of L. vivipara may depend on both supercooling and freeze tolerance capacities, since this combination would afford the greatest likelihood of surviving winter in its dynamic thermal and hydric microenvironment.Abbreviations bm body mass - SVL snout-vent length - T_b body surface temperature - T _c crystallization temperature