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Winter life of painted turtle hatchlings
- Reptile freeze tolerance: Metabolism and gene expression

The ability to endure the freezing of extracellular body fluids is an integral part of winter cold hardiness for numerous vertebrate and invertebrate animals living in seasonally cold environments. Among vertebrates, natural freeze tolerance is well-developed in several species of frogs that hibernate on land and the biochemistry and physiology of vertebrate freeze tolerance has been most extensively studied using the wood frog, Rana sylvatica, as the model animal [25], [75] and [81]. A number of reptile species also survive freezing [22] and [80]. Whereas it is clear that freeze tolerance is an integral part of winter survival for various terrestrially hibernating frog species, there has been much more debate about whether freeze tolerance is an ecologically relevant part of reptile cold hardiness. A number of reptile species can endure brief freezing exposures at mild subzero temperatures with low amounts of body ice but die when time stretches beyond a few hours, temperature drops much past −2 °C, or equilibrium ice content is achieved. This probably means that some peripheral freezing of skin and skeletal musculature is tolerable but ice penetration through the body core, halting blood flow and the vital functions of internal organs, is not. Among the species that fall into this category are wall lizards (Podarcis muralis, P. sicula), garter snakes (Thamnophis sirtalis), boreal adders (Vipera berus) and hatchlings of some turtles including red-eared sliders (Trachemys scripta elegans), map turtles (Graptemys geographica) and snapping turtles (Chelydra serpentine) (Table 1) [1], [2], [5], [11], [13], [14], [16], [22] and [30]. Several other reptile species show much better freeze tolerance including survival for one or more days at subzero temperatures normally encountered in their hibernacula with ice penetration throughout the body cavity and ice contents that reach equilibrium values of over 50% of total body water. For these, freeze tolerance appears to be ecologically relevant to survival in their natural environment. Species in this category include box turtles (Terrapene carolina, T. ornata) [15], [19] and [83], the European common lizard (Lacerta vivipara) [17], [87] and [89] and hatchlings of several turtle species that spend their first winter within the natal nest including painted turtles (Midland and Western subspecies, Chrysemys picta marginata and C. p. belli, respectively), Blanding’s turtle (Emydoidea blandingii), and the diamondback terrapin (Malaclemys terrapin) [12], [22], [29], [30] and [85] (Table 1). Adult box turtles, at about 0.5 kg body mass, are the largest known freeze tolerant animals. Statistics on reptile freeze tolerance to date include hatchling C. picta that variously survived freezing for 3–11 days at −2 to −2.5 °C [12] and [66], E. blandingii hatchlings endured 3 days at −3.5 °C [29], L. vivipara endured 1–3 days frozen at −3 °C [17] and [88], and T. carolina endured 2–3 days frozen [15] and [83]. Survival at equilibrium ice contents of not, vert, similar50% of total body water has been reported in many cases [81].

However, despite significant freezing survival by these species, reptiles appear to use a mixture of strategies to get through the winter and the mixture can vary between geographic populations, between years, within different parts of the winter, and depend on the hibernation site, soil moisture characteristics, insulation/snowcover and weather. This confounds our anthrocentric need to put labels “freeze tolerant” versus “freeze intolerant” on species and a rigid categorization may not work for reptiles. Terrestrially hibernating frogs have the highly water-permeable skin of amphibians which dictates two consequences: (a) they must hibernate in moist sites to avoid death by desiccation, and (b) they are highly susceptible to inoculative freezing by contact with environmental ice [80] and [81]. Furthermore, they have little ability to dig and do not seek refuge deep underground. If ambient temperature at the hibernation site drops below the freezing point of frog blood (about −0.5 °C), freezing is virtually unavoidable and, hence, adaptations supporting freeze tolerance are well-developed and prominently expressed in anuran species living in seasonally cold environments [81]. The situation for reptiles is less clear-cut. Reptiles have a less water-impermeable skin that makes them less susceptible to inoculative freezing and may also allow them to winter in drier hibernation sites than could be used by frogs. Hence, a freeze avoidance (supercooling) strategy for dealing with subzero temperatures is realistic in some cases. Indeed, numerous laboratory studies have documented substantial supercooling capacity by reptiles with supercooling points of −10 to −15 °C measured in some cases [2], [23] and [59]. Furthermore, apart from hatchling turtles that are restricted to their natal nest over the winter, most other reptiles can modify their cold exposure by situating themselves in thermally buffered hibernation sites (e.g., underground caves, burrows, grass or moss hummocks) where they may not be subjected to microenvironment temperatures below 0 °C, despite low subzero ambient air temperatures [3], [39], [58] and [60].

However, a mechanism to deal with subzero exposure must always be present in case the choice of hibernaculum turns out to be poor. For example, Bernstein and Black [3] recorded body temperatures of box turtles (T. o. ornata) overwintering in sand dunes and found that 23 animals had body temperatures that never dipped below 0–0.5 °C over the whole winter but two individuals showed continuous subzero body temperatures (−2 to −8 °C) from late December to mid-February. Variation in weather conditions from year to year can also change the overwintering experience. Nagle et al. [61] reported 45% mortality of hatchling C. picta in a winter when soil temperatures fell to −7 to −9 °C without snow cover whereas mortality was E. blandingii could remain supercooled at −4 °C for 7 days or cool continuously to −14 °C in supercooling tests but when placed in moist soil, they cooled to only −1.3 °C before freezing [29]. European common lizards illustrate several types of variation that make it hard to assign a single cold hardiness strategy to the species: (a) highland populations show better freezing survival than do their lowland counterparts, (b) adults are more freeze tolerant than juveniles but juveniles are found at a greater vertical depth than adults in the bogs where they hibernate, and (c) viviparous strains show better long term freezing survival than do oviparous strains [17], [40], [88] and [89]. Hence, reptile species may use both freeze tolerance and freeze avoidance strategies in whatever combination is needed to optimize their winter survival. Indeed, Grenot et al. [40] retrieved viable individuals of L. vivipara from the same field site in the same winter in both supercooled or frozen states. Supercooling might often be the preferable (default) strategy of reptile cold hardiness as it is the less demanding strategy both physically and biochemically but freeze tolerance provides the animals with the means to survive when environmental conditions cause inoculative freezing. Hence, freeze tolerance has developed as an important facet of cold hardiness for a number of species. The metabolic and gene expression responses that support reptile freeze tolerance are the subject of most of the rest of this review.

The ecology, physiology, and biochemistry of winter survival among terrestrially hibernating reptiles has been most extensively studied using hatchling painted turtles, C. picta, as the model animal. Furthermore, the debate over the winter survival strategy used by reptiles—freeze tolerance or freeze avoidance—has also been most hotly contested by researchers studying this species.

Painted turtles lay their eggs in early summer in soil not too far from the edges of rivers or lakes, often on south facing banks. At higher latitudes only one clutch is laid per year and the eggs incubate throughout the summer and then hatch in September. However, instead of digging out of the nest, the hatchlings remain hidden underground over their first winter, living off the remains of a large internalized yolk sac, and emerge in the late spring. Wintering in the nest appears to be a strategy for minimizing time under high risk conditions (cold weather, low food availability, and susceptible to predation) and delays emergence until conditions are conducive to rapid juvenile growth [35]. The strategy is common to a number of turtle species over both warm and cold regions of their range but, in northern regions, is complicated by nest temperatures that can fall below 0 °C and the consequent need for a strategy of cold hardiness. Painted turtles in their second and subsequent years of life overwinter under water where strategies of subzero survival are not an issue but where anoxia tolerance is critical for these lung-breathers. Indeed, freshwater turtles of the Chrysemys and Trachemys genera are the premier facultative anaerobes among vertebrates and they are widely used model species for studying the molecular mechanisms that support natural anoxia tolerance and in medical studies that seek to improve the ischemia/anoxia resistance of mammalian organs [52], [53], [73] and [77].

The mechanisms of cold hardiness used by painted turtle hatchlings have been extensively studied by three groups: ourselves, Costanzo and Lee (University of Miami, Ohio) and Packard and Packard (Colorado State University). Work by the Packard laboratory champions the idea that freeze avoidance is the primary means of overwintering and that freeze tolerance makes little or no contribution to winter survival in the natural environment [65]. They point to nest temperatures that can fall to about −10 °C for up to 2 weeks at a time at their study sites, whole animal supercooling points that decrease in the field over the autumn months (and in the laboratory with cold acclimation), poor long term freezing survival in laboratory studies at temperatures lower than −2 °C, and survival in artificial nest situations of only those animals that did not show a freezing exotherm [63], [64] and [65]. They also linked improved resistance to inoculative freezing over the autumn with the accumulation of a dense lipid layer in the α-keratin layer of the epidermis of the limbs which are in contact with soil in the nest [91].

However, studies by ourselves and the Costanzo/Lee laboratory have shown good freeze tolerance of C. picta hatchlings and conclude that adaptive strategies for freeze tolerance are an important part of winter cold hardiness for this species, at least over parts of the species range [12], [21], [22] and [85]. Indeed, Ontario hatchlings (from Algonquin park near the northern limit of the species range) that were tested immediately after excavation from nests in the spring showed the least supercooling capacity (−1 °C) and greatest freeze tolerance (11 days at −2.5 °C) that has ever been reported for C. picta [12]. Costanzo et al. [21] reported improvement in freeze tolerance over time with few turtles withstanding freezing when sampled from nests just after hatching but most animals sampled later (December to April) readily surviving freezing trials (3 days at −3 °C). Our analysis of Ontario turtles also reported the presence of specific proteinaceous nucleators in plasma of cold acclimated hatchlings, a common observance for freeze tolerant species [84], whereas studies by Packard and Packard [65] reported a clearance of nucleators from body fluids over the autumn months, a common part of a freeze avoidance strategy. Studies by Costanzo and Lee acknowledge that both strategies can contribute to hatchling cold hardiness [21] and [22]; indeed, they found that supercooling capacity, resistance to inoculative freezing, and freeze tolerance all improved during cold acclimation or during the autumn months under field conditions. The thermal minimum for freezing survival appears to be −3 to −4 °C but turtles can survive at temperatures as low as −12 °C if they remain supercooled although supercooling is not always possible due to contact with ice crystals or ice-nucleating agents (INAs) in nesting soil [21]. Given that the risk of inoculative freezing is strongly influenced by moisture content, texture, and other characteristics of nesting soil as well as yearly variation in ambient temperature and snow cover [21], C. picta hatchlings may need to be prepared to use either freeze tolerance or supercooling strategies to deal with the particular conditions of any given winter. Freeze tolerance is probably of greatest importance during wet winters when the high likelihood of inoculation by environmental ice or INAs precludes supercooling. Hence, painted turtle hatchlings show significant freeze tolerance and they are a valid model with which to investigate the physiological, biochemical, and genomic mechanisms that support vertebrate freezing survival. Apart from wood frogs, they are the second most extensively studied freeze tolerant vertebrate species.

We have analyzed the process of whole animal freezing in hatchling C. p. marginata using proton magnetic resonance imaging. Freezing was initiated at a peripheral site (probably due to inoculation via ice forming on the skin) and the freezing front propagated in a directional manner though the turtle’s body with ice forming first in extraorgan spaces such as the brain ventricles and the abdominal space [70]. This is the same pattern as was seen in freeze tolerant wood frogs [71]. Interestingly, in both species, thawing was not directional but melting occurred uniformly throughout the body core and organs melted more rapidly than did the extraorgan ice that surrounded them.

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