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.     

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.     

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.     

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.     

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.     

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     

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.     

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 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.     

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.     

Warm water and cool nests are best. How global warming might influence hatchling green turtle swimming performance

For sea turtles nesting on beaches surrounded by coral reefs, the most important element of hatchling recruitment is escaping predation by fish as they swim across the fringing reef, and as a consequence hatchlings that minimize their exposure to fish predation by minimizing the time spent crossing the fringing reef have a greater chance of surviving the reef crossing. One way to decrease the time required to cross the fringing reef is to maximize swimming speed. We found that both water temperature and nest temperature influence swimming performance of hatchling green turtles, but in opposite directions. Warm water increases swimming ability, with hatchling turtles swimming in warm water having a faster stroke rate, while an increase in nest temperature decreases swimming ability with hatchlings from warm nests producing less thrust per stroke.     

Behavioral responses of hatchling painted turtles (Chrysemys picta) and snapping turtles (Chelydra serpentina) at subzero temperatures

We monitored behavioral responses of cold-acclimated hatchling painted turtles (Chrysemys picta) indigenous to Nebraska and hatchling snapping turtles (Chelydra serpentina) indigenous to Nebraska and Arkansas during cooling (0.1°C/min) to temperatures as low as −19°C. All turtles made exploratory movements during cooling and locomotion occurred at temperatures as low as −2 to −4°C, but C. picta maintained relatively higher levels of locomotor activity than C. serpentina, and no differences in motility occurred between northern and southern groups of C. serpentina. Slow movements of the head and limbs were observed in supercooled hatchling C. picta at temperatures as low as −10°C, whereas at about −5°C, C. serpentina exhibited an increase in spontaneous motor activity followed by muscle contracture, immobility, and spontaneous freezing. C. picta spontaneously froze at about −16°C without exhibiting cold contracture, suggesting that they are better adapted to survive exposure to extreme cold.     

Fuel use and corticosterone dynamics in hatchling green sea turtles (Chelonia mydas) during natal dispersal

During their natal dispersal hatchling sea turtles depart their nest, beach and inshore areas quickly to move into offshore developmental habitat using their finite energy stores. Patterns of fuel use and endocrine responses that could facilitate hatchling sea turtle dispersal activity are poorly understood. This study, examined aspects of intermediary metabolism by measuring plasma fuel use and an endocrine response of hatchling green turtles (Chelonia mydas) during terrestrial and aquatic activity coinciding with natal dispersal. Specifically, we measured plasma concentrations of glucose, non-esterised free fatty acids and protein to gauge the contributions of carbohydrate, lipid and protein metabolism for fuelling natal dispersal. In addition, we measured plasma levels of the steroid hormone corticosterone (CORT) a hormone implicated in regulating a number of metabolic events associated with migration and energy use in vertebrates. During terrestrial activity, hatchlings ascended through the sand from their nests and exhibited significant increases in plasma CORT and lactate indicating intense periods of anaerobic activity. During swimming, all plasma metabolites, with the exception of plasma protein, peaked between 1 and 4 h post-beginning swimming activity. Plasma CORT peaked at between 3 and 5 h of swimming activity. These plasma concentrations are consistent with intensive activity inducing catabolism of carbohydrate, lipid and protein stores to support prolonged activity. These results are similar to other vertebrates and suggest a relatively uniform cascade of physiological processes during such arduous migratory events.     

Acquisition of Salmonella flora by turtle hatchlings on commercial turtle farms

A commercial turtle pond in South Louisiana was studied to identify the mechanism by which turtle hatchlings acquire Salmonella flora. The visceral organs and mature eggs removed from 31 adult gravid female turtles over the course of two egg-laying seasons and from 37 adult females during one winter dormant period were examined bacteriologically for Salmonella. Pond water, egg nest soil, and hatchlings produced by eggs removed from the oviducts and nest soil were also tested. Eighty-eight turtles hatched from eggs removed from the oviducts of 15 turtles at necropsy did not excrete or harbor systemically Salmonella, nor were these pathogens isolated from ovarian tissue or immature eggs. The findings suggest transovarian transmission of these pathogens does not occur frequently. Turtles hatched from eggs retrieved from soil nests 1 to 2 h after deposition harbor and excrete these organisms. This result coupled with the isolation of these pathogens from the cloaca, colon contents, and bursal fluid from 18 females captured in the act of egg laying supports the cloaca to egg and nest soil to egg mode for salmonellae infection in the resultant hatchling. Salmonella arizonae and Salmonella serogroups B, C2, and E1 were isolated from the cloaca, colon contents, pond water, and nest soil, and were excreted by hatchlings produced from eggs removed from the soil nests. These same serogroups were isolated from the colon contents of 19 of 37 females tested during the dormant period, suggesting the salmonellae persist in the pond environment in the adult throughout the year.     

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.     

Environmentally induced variation in body size of turtles hatching in natural nests

Eggs from three snapping turtles (Chelydra serpentina) were divided between two natural nests in a factorial experiment assessing the role of the nest environment as a cause for variation in body size and energy reserves of hatchlings at our study site in northcentral Nebraska. Nest # 1 was located in an unshaded area on the south side of a high sandhill, whereas nest #2 was located in an unshaded area on level ground. Eggs in nest #1 increased in mass over the course of incubation, with eggs at the bottom of the nest gaining more mass than eggs nearer to the surface. In constrast, eggs in nest #2 lost mass during incubation, with eggs at the bottom declining less in mass than eggs at the top of the cavity. Hatchlings from nest #1 were much larger (but contained smaller masses of unused yolk) than hatchlings from nest #2. Additionally, eggs from the lower layers in both nests tended to produce larger hatchings (but with smaller masses of unused yolk) than eggs from the upper layers. Thus, ecologically important variation in body size and nutrient reserves of hatchling snapping turtles results from variation in the environment among and within nests.     

Compass Orientation During Dispersal of Freshwater Hatchling Snapping Turtles (Chelydra serpentina) and Blanding's Turtles (Emydoidea blandingii)

Freshwater turtle hatchlings primarily use visual cues for orientation while dispersing from nests; however, hatchlings rapidly develop a relationship between a sun or geomagnetic compass and a dispersal target that allows them to maintain an established direction of movement when target habitats are not visible. We examined dispersal patterns of hatchling snapping turtles (Chelydra serpentina) and Blanding's turtles (Emydoidea blandingii) dispersing in large arenas in a mowed field and in dense corn. The dispersal of three categories of hatchlings were examined: (1) naïve individuals (no previous dispersal experience), (2) arena‐experienced (limited dispersal experience in arenas in natural habitat), and (3) natural‐experienced hatchling Blanding's turtles (captured after extensive experience dispersing W in natural habitats toward wetlands). Experienced hatchlings were assigned to treatments consisting of having a magnet or a non‐magnetic aluminum sham or nothing glued to their anterior carapace before release in the corn arena. Dispersal patterns of naïve hatchlings of both species were strongly directional in the field arena with visible target horizons and primarily random in the corn arena where typical target horizons were blocked. When released in corn, dispersal patterns were similar for arena‐experienced hatchlings with magnets or shams attached and differed from their prior dispersal headings in the field arena as naïve hatchlings. Natural‐experienced hatchling Blanding's turtles with and without magnets were able to accurately maintain their prior headings to the WNW while dispersing in the field or corn arenas (i.e., the presence of a magnet did not disrupt their ability to maintain their prior heading). Based on the assumption that no other type of compass exists in hatchlings, we conclude that they were not using a geomagnetic compass, but by default were using sun compass orientation to maintain dispersal headings in dense corn where no typical target habitats were visible.