Hatchling painted turtles (Chrysemys picta) survive exposure to subzero temperatures during hibernation by avoiding freezing

Hatchling painted turtles (Chrysemys picta) were placed individually into artificial nests constructed in jars of damp soil and then were cooled slowly to temperatures between-7.7 and-12.7 °C. Distinct exotherms were recorded in all jars when water in the soil began to freeze at temperatures between-0.9 and-2.4 °C. A second (animal) exotherm was subsequently detected in some of the jars when water in hatchlings also began to freeze. An animal exotherm occurred in the temperature records for all 23 hatchlings that died in tests terminating at temperatures between-7.7 and-10.8 °C, but no such exotherm was apparent in the temperature records for the 23 turtles that survived these treatments. Moreover, the 4 hatchlings that produced exotherms in tests terminating between-11.5 and-12.7 °C failed to survive, but 5 of 7 hatchlings that produced no exotherm in these tests also died. Thus, turtles that die at subzero temperatures above-11 °C apparently succumb to freezing when ice propagates across their integument from the frozen soil, but animals that die at temperatures below-11 °C generally perish from some other cause. These findings indicate that hatchling painted turtles overwintering inside their shallow, subterranean nests survive exposure to subzero temperatures by avoiding freezing instead of by tolerating freezing.     

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.     

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.     

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     

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.     

Influence of the nest environment on bone mineral content in hatchling painted turtles (Chrysemys picta)

We performed an experiment at a field site in north-central Nebraska to assess the role of the nest environment in inducing variation in bone mineral content in hatchling painted turtles Chrysemys picta (Schneider 1783). The contents of several newly constructed nests were manipulated by reciprocal transplant, after which the eggs were allowed to incubate for 8 wk under natural conditions. The nests were then excavated, and the eggs were brought into the laboratory to complete incubation and hatch under standard conditions of temperature and moisture. The hatchlings were killed, and their carcasses and residual yolks were analyzed separately for calcium and phosphorus. More of the random variation in carcass calcium and phosphorus was related to the nest in which eggs incubated (37% and 42%, respectively) than was associated with the clutch of origin (21% and 37%). Moreover, hatchlings from some nests contained substantially more calcium and phosphorus than did hatchlings from other nests, both in terms of the absolute amounts of the elements in their carcasses (pointing to variation in body size) and in terms of the concentrations of those elements (pointing to variation in bone density). The amounts of calcium and phosphorus in carcasses of hatchlings were positively correlated with changes in mass of their eggs during the 8 wk that the eggs incubated in nests in the field, thereby indicating that the influence of the nest environment on developing embryos probably was mediated by water exchanges experienced by the eggs. These findings indicate that developmental plasticity underlies a major fraction of the variation in mineral content of hatchling painted turtles emerging from nests in the field. Phenotypic variation attributable to plasticity consequently needs to be addressed in models for life-history evolution of painted turtles and other chelonians producing eggs with soft, flexible shells.     

Influence of water availability during incubation on hatchling size, body composition, desiccation tolerance, and terrestrial locomotor performance in the snapping turtle Chelydra serpentina

The effects of water availability during incubation on the water contents of neonatal snapping turtles at hatching were examined, along with the influence of hatchling water content on desiccation tolerance and terrestrial locomotor performance. The water contents of hatchlings from eggs incubated on wet substrates were both absolutely and proportionally greater than were those of hatchlings from eggs incubated on dry substrates. Hatchlings with greater water contents at hatching were able to survive longer and to lose more water before physiological performance was adversely affected by desiccation. Increased water contents in hatchlings with greater water availability during incubation may enhance survival by increasing the amount of water the animal can afford to lose before dehydration begins to adversely affect whole animal performance.     

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.     

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.     

A novel hypothesis for the adaptive maintenance of environmental sex determination in a turtle

Temperature-dependent sex determination (TSD) is widespread in reptiles, yet its adaptive significance and mechanisms for its maintenance remain obscure and controversial. Comparative analyses identify an ancient origin of TSD in turtles, crocodiles and tuatara, suggesting that this trait should be advantageous in order to persist. Based on this assumption, researchers primarily, and with minimal success, have employed a model to examine sex-specific variation in hatchling phenotypes and fitness generated by different incubation conditions. The unwavering focus on different incubation conditions may be misplaced at least in the many turtle species in which hatchlings overwinter in the natal nest. If overwintering temperatures differentially affect fitness of male and female hatchlings, TSD might be maintained adaptively by enabling embryos to develop as the sex best suited to those overwintering conditions. We test this novel hypothesis using the painted turtle (Chrysemys picta), a species with TSD in which eggs hatch in late summer and hatchlings remain within nests until the following spring. We used a split-clutch design to expose field-incubated hatchlings to warm and cool overwintering (autumn–winter–spring) regimes in the laboratory and measured metabolic rates, energy use, body size and mortality of male and female hatchlings. While overall mortality rates were low, males exposed to warmer overwintering regimes had significantly higher metabolic rates and used more residual yolk than females, whereas the reverse occurred in the cool temperature regime. Hatchlings from mixed-sex nests exhibited similar sex-specific trends and, crucially, they were less energy efficient and grew less than same-sex hatchlings that originated from single-sex clutches. Such sex- and incubation-specific physiological adaptation to winter temperatures may enhance fitness and even extend the northern range of many species that overwinter terrestrially.     

High thermal variance in naturally incubated turtle nests produces faster offspring

The effects of climate change on populations are complex and difficult to predict, and can result in mismatches between interdependent organisms or between organisms and their environment. Reptiles with temperature-dependent sex determination may be able to compensate for potential skews in offspring sex ratio caused by climate change by selecting cooler (i.e., shadier) nest sites. Although changing nest location may prevent sex ratio skews, it may also affect thermally sensitive performance traits in offspring. I tested righting, sprinting, and swimming performance in hatchling painted turtles (Chrysemys picta), produced by female turtles from five populations across the species’ geographic range, nesting in a common-garden environment. I found that speed of hatchling performance was faster in hatchlings whose mothers originated from warmer climates, and that nests with higher mean daily variation in incubation temperature produced faster hatchlings. These results suggest that the increased temperatures predicted by climate change models could result in hatchling turtles that are faster at sprinting and swimming; however, it is not yet known how these performance measures translate into fitness.     

Does maternal oviposition site influence offspring dispersal to suitable habitat?

Orientation and dispersal to suitable habitat affects fitness in many animals, but the factors that govern these behaviors are poorly understood. In many turtle species, hatchlings must orient and disperse to suitable aquatic habitat immediately after emergence from subterranean nests. Thus, the location of nest sites relative to aquatic habitats ideally should be associated with the direction of hatchling dispersal. At our study site, painted turtles (Chrysemys picta) nest to the west (on an island) and east (on the mainland) of a wetland, which determines the direction that hatchlings must travel to reach suitable aquatic habitat. To determine if hatchling orientation is intrinsically influenced by the location where their mothers nest, we employed a two-part cross-fostering experiment in the field, whereby half the eggs laid in mainland nests were swapped with half the eggs laid in island nests. Moreover, because C. picta hatchlings overwinter inside their nests, we performed a second cross-fostering experiment to fully decouple the effects of (1) the maternally chosen nest location, (2) the embryonic developmental location, and (3) the overwinter location. We released hatchlings into a circular arena in the field and found that turtles generally dispersed in a westerly direction, regardless of the maternally chosen nest location and independent of the locations of embryonic development and overwintering. Although this westerly direction was towards suitable aquatic habitat, we could not distinguish whether naïve hatchling turtles (i) use environmental cues/stimuli to orient their movement, or (ii) have an intrinsic bias to orient west in the absence of stimuli. Nevertheless, these findings suggest that the orientation behavior of naïve hatchling turtles during terrestrial dispersal is not dependent upon the location of maternally-chosen nest sites.     

Responses of hatchling sea turtles to rotational displacements

After emerging from underground nests, sea turtle hatchlings migrate through the surf zone and out to the open ocean. During this migration, both waves and water currents can disrupt hatchling orientation by unpredictably rotating the turtles away from their migratory headings. In addition, waves cause turtles to roll and pitch, temporarily impeding forward swimming by forcing the hatchlings into steeply inclined positions. To maintain seaward orientation and remain upright in the water column, hatchlings must continuously compensate for such displacements. As a first step toward determining how this is achieved, we studied the responses of loggerhead (Caretta caretta L.) sea turtle hatchlings to rotational displacements involving yaw, roll, and pitch. Hatchlings responded to rotations in the horizontal plane (yaw) by extending the rear flipper on the side opposite the direction of rotation. Thus, the flipper presumably acts as a rudder to help turn the turtle back toward its original heading. Turtles responded to rotations in the roll plane with stereotypic movements of the front flippers that act to right the hatchlings with respect to gravity. Finally, hatchlings responded to rotations in the pitch plane with movements of the hind flippers that appear likely to curtail or counteract the pitching motion. Thus, the results of these experiments imply that young sea turtles emerge from their nests possessing a suite of stereotypic behavioral responses that function to counteract rotational displacements, enable the animals to maintain equilibrium, and facilitate efficient movement toward the open sea.     

孵化温度对王锦蛇胚胎代谢和幼体适合度的影响

孵化温度所驱动的爬行动物的表型变异是生理生态学研究的热点。本研究以王锦蛇(Elaphe carinata)为实验动物,检验了24℃和28℃孵化温度对王锦蛇胚胎代谢速率、孵化过程中的卵重量、孵出幼体代谢和行为的影响。研究结果显示:卵重和胚胎的呼吸代谢均与孵化时间呈正相关;28℃下胚胎代谢速率大于24℃;幼蛇孵出15 d内体重随着生长时间的延长而减小,24℃孵出幼体的代谢速率大于28℃孵出幼体,两温度下孵出幼体的呼吸代谢速率和生长时间无显著关系;28℃孵出幼体的疾游速和吐信频次均大于24℃;两孵化温度孵出幼体的选择体温无显著差异,但在消耗完体内的剩余卵黄后28℃孵出幼体有60%的个体摄食,而24℃孵出幼体无摄食个体。总体而言,王锦蛇28℃孵出幼体适合度优于24℃孵出幼体。     

Accumulation of lactate by frozen painted turtles (Chrysemys picta) and its relationship to freeze tolerance

Hatchling painted turtles (Chrysemys picta) survived freezing at -2 degrees C for 4 d, few recovered from freezing lasting 6 d, and none survived being frozen for 8 d. Whole-body glucose and lactate were low in animals that had not been subjected to cold and ice but increased precipitously in animals that were frozen for 2 d. Both metabolites continued to increase, but at a somewhat lower rate, in animals frozen for 4, 6, or 8 d. The increase in whole-body lactate reflects a reliance by frozen hatchlings on anaerobiosis, whereas the increase in glucose presumably results from mobilization of glycogen reserves to support anaerobic metabolism. Mortality of frozen hatchlings is correlated with the increase in whole-body lactate. Factors that may contribute to the observed correlation include a compromised capacity for individual organs to cope with the lactic acidosis that accompanies anaerobic metabolism and organ-specific depletion of energy reserves. Individual organs must rely on buffering and glucose reserves available in situ because blood of frozen hatchlings does not circulate. Thus, buffer from the shell cannot be transported to other organs, lactate cannot be sequestered in the shell, and glucose mobilized from liver glycogen is not available to supplement glucose reserves of other tissues. This integrated suite of physiological disruptions may limit tolerance of freezing to conditions with little or no ecological relevance.     

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.     

Hatching success, delay of emergence and hatchling biometry of the spur-thighed tortoise, Testudo graeca, in south-western Spain

Hatching success, egg incubation, emergence and hatchling characteristics were assessed for 44 naturally incubating nests of Testudo graeca in south-western Spain. Nest predation rate was 4.5% and overall hatching success was 82.4%. Incubation periods ranged from 78 to 114 days, and hatchlings delayed emergence from the nest from one to 23 days. Emergences occurred from mid August to late September, and were not correlated with nesting dates, but earlier laid nests had longer incubation times, which was probably owing to lower temperatures experienced by clutches laid at the beginning of the nesting season. Variance of hatchling body size and mass was high and was mainly influenced by the gravid female. Mean straight carapace length was 34.14mm, and mean body mass 10.8g. Hatchlings from clutches laid last in the nesting season had significantly better physical condition. Hatchling mass was positively correlated with egg mass, and both variables were positively correlated with emergence date. Both better physical condition and relatively late emergence may confer advantages to hatchlings in the face of unfavourable environmental conditions in autumn.