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.     

Thermally induced torpor in fullterm lizard embryos synchronizes hatching with ambient conditions

Eggs inside an underground nest have limited access to information about above-ground conditions that might affect the survival of emerging hatchlings. Our measurements of heart rates of embryos inside the intact eggs of montane lizards (Bassiana duperreyi, Scincidae) show that low temperatures induce torpor in fullterm embryos, but do not do so during earlier embryogenesis or later, post-hatching. Because above-ground conditions affect soil temperatures, this stage-dependent torpor effectively restricts hatching to periods of high ambient temperatures above ground. Torpor thus can function not only to synchronize activity with suitable environmental conditions during post-hatching life (as reported for many species), but also can occur in embryos, to synchronize hatching with above-ground conditions that facilitate successful emergence from the nest.     

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.     

Wetness of the nest environment influences cardiac development in pre- and post-natal snapping turtles (Chelydra serpentina)

We dissected hearts from near-term embryos and hatchlings of common snapping turtles (Chelydridae: Chelydra serpentina) whose eggs had incubated on wet or dry substrates, and then dried and individually weighed the heart and yolk-free carcass from each animal. Hearts and carcasses of prenatal and neonatal animals grew at different rates, and the patterns of growth by both heart and carcass differed between wet and dry environments. Hearts grew faster, both in actual mass and in mass adjusted for variation in body size, in embryos and hatchlings whose eggs were incubated on dry substrates than in animals whose eggs were held on wet media. This finding is consistent with a hypothesis that embryos incubating in dry settings experience hypovolemia secondary to dehydration and that enlargement of the heart compensates, in part, for the associated increase in viscosity of the blood. Embryonic turtles seemingly exhibit the same plasticity and response that would be expected from other vertebrate ectotherms subjected to the physiological challenges associated with desiccation and an associated reduction in blood volume.     

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     

Effects of temperature and moisture during incubation on carcass composition of hatchling snapping turtles (Chelydra serpentina)

Summary Flexible-shelled eggs of common snapping turtles (Chelydra serpentina) were incubated on each of two substrates (vermiculite, sand) at each of three temperatures (26.0°C, 28.5°C, 31.0°C) and three moisture regimes (wet, intermediate, dry). Embryos developing in cool, wet environments mobilized the largest amounts of protein from their yolk and attained the largest size before hatching, whereas turtles developing in warm, dry environments mobilized the smallest quantities of protein and were the smallest in body size at hatching. Embryos on wet substrates mobilized more lipid from their yolk than did embryos on dry media, but ambient temperature had no demonstrable influence on patterns of lipid mobilization. The total reserve of neutral lipid available in residual yolk plus carcass to sustain neonates in the interval prior to the beginning of feeding was largest in hatchlings from dry environments and smallest in animals from wet environments, but was unaffected by temperature during incubation. Hydration of tissues in hatchlings was higher when incubation was in cool, moist conditions than when incubation was in warm, dry settings, thereby indicating that some of the effects of moisture and temperature on mobilization of nutrients by embryos may be mediated by differences in intracellular water. Patterns of response to temperature and moisture recorded for turtles emerging from eggs on sand were similar to those recorded for hatchlings on vermiculite, so no important conclusion would have been affected by incubating eggs on one medium instead of the other.     

Leech removal is not the primary driver of basking behavior in a freshwater turtle

Leaving the water to bask (usually in the sun) is a common behavior for many freshwater turtles, with some species also engaging in “nocturnal basking.” Ectoparasite removal is an obvious hypothesis to explain nocturnal basking and has also been proposed as a key driver of diurnal basking. However, the efficacy of basking, day or night, to remove leeches has not been experimentally tested. Therefore, we examined the number of leeches that were removed from Krefft''s river turtles (Emydura macquarii krefftii) after experimentally making turtles bask at a range of times of day, durations, and temperatures. Turtles had high initial leech loads, with a mean of 32.1 leeches per turtle. Diurnal basking under a heat lamp for 3 hr at ~28°C significantly reduced numbers of leeches relative to controls. In diurnal trials, 90.9% of turtles lost leeches (mean loss of 7.1 leeches per turtle), whereas basking for 30 min under the same conditions was not effective (no turtles lost leeches, and all turtles were still visibly wet). Similarly, “nocturnal basking” at ~23°C for 3 hr was not effective at removing leeches. Only 18% of turtles lost leeches (one turtle lost one leech and another lost four leeches). Diurnal basking outdoors under direct sunlight for 20 min (mean temp = 34.5°C) resulted in a small reduction in leeches, with 50% of turtles losing leeches and an average loss of 0.7 leeches per turtle. These results indicate basking can remove leeches if temperatures are high or basking durations are long. However, it was only effective at unusually long basking durations in this system. Our data showed even the 20‐min period was longer than 70.1% of natural diurnal basking events, many of which took place at cooler temperatures. Therefore, leech removal does not appear to be the purpose of the majority of basking events.     

Increased expression of Hsp70 and Hsp90 mRNA as biomarkers of thermal stress in loggerhead turtle embryos (Caretta Caretta)

The survival and viability of sea turtle embryos is dependent upon favourable nest temperatures throughout the incubation period. Consequently, future generations of sea turtles may be at risk from increasing nest temperatures due to climate change, but little is known about how embryos respond to heat stress. Heat shock genes are likely to be important in this process because they code for proteins that prevent cellular damage in response to environmental stressors. This study provides the first evidence of an expression response in the heat shock genes of embryos of loggerhead sea turtles (Caretta caretta) exposed to realistic and near-lethal temperatures (34 °C and 36 °C) for 1 or 3 hours. We investigated changes in Heat shock protein 60 (Hsp60), Hsp70, and Hsp90 mRNA in heart (n=24) and brain tissue (n=29) in response to heat stress. Under the most extreme treatment (36 °C, 3 h), Hsp70 increased mRNA expression by a factor of 38.8 in heart tissue and 15.7 in brain tissue, while Hsp90 mRNA expression increased by a factor of 98.3 in heart tissue and 14.7 in brain tissue. Hence, both Hsp70 and Hsp90 are useful biomarkers for assessing heat stress in the late-stage embryos of sea turtles. The method we developed can be used as a platform for future studies on variation in the thermotolerance response from the clutch to population scale, and can help us anticipate the resilience of reptile embryos to extreme heating events.     

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.     

Past, current and future thermal profiles of green turtle nesting grounds: Implications from climate change

Sex determination and hatching success in sea turtles is temperature dependent and as a result global warming poses a threat to sea turtles. Warmer sand temperatures may skew sea turtle population′s sex ratios towards predominantly females and decrease hatching success. Therefore, understanding the rates at which sand temperatures are likely to increase as climate change progresses is warranted. We recorded sand temperature and used historical sea surface and air temperature to model past and to predict future sand temperature under various scenarios of global warming at key sea turtle nesting grounds (n = 7) used by the northern Great Barrier Reef (nGBR) green turtle, Chelonia mydas, population. Reconstructed temperatures from 1990 to the present suggest that sand temperatures at the nesting sites studied have not changed significantly during the last 18 years. Current thermal profile at the nesting grounds suggests a bias towards female hatchling production into this population. Inter-beach thermal variance was observed at some nesting grounds with open areas in the sand dune at northern facing beaches having the warmest incubating environments. Our model projections suggest that a near complete feminization of hatchling output into this population will occur by 2070 under an extreme scenario of climate change (A1T emission scenario). Importantly, we found that some nesting grounds will still produce male hatchlings, under the most extreme scenario of climate change, this finding differs from predictions for other locations. Information from this study provides a better understanding of possible future changes in hatching success and sex ratios at each site and identifies important male producing regions. This allowed us to suggest strategies that can be used at a local scale to offset some of the impacts of warmer incubating temperatures to sea turtles.     

Choosy cannibals: Targeted consumption of conspecific hatchlings by larval cane toads is triggered by species‐specific defensive toxins

In many species, cannibalism is uncommon and involves nonselective consumption of conspecifics as well as heterospecifics. However, within their invasive Australian range, cane toad larvae (Rhinella marina) specifically target and voraciously consume the eggs and hatchlings of conspecifics, often extirpating entire clutches. In contrast, toad larvae rarely consume the eggs and hatchlings of native frogs. Here, we use laboratory studies to demonstrate that this selective consumption is triggered by species‐specific chemical cues: maternally‐invested bufadienolide toxins that otherwise defend cane toad eggs and hatchlings against predators. We find that these cues stimulate feeding behaviors in toad tadpoles, such that the addition of bufadienolide toxins to the water column increases predation on eggs, not only of conspecifics, but also of native anuran species that are otherwise usually ignored. In contrast, we find that cannibalism rates on conspecific hatchlings are high and unaffected by the addition of bufadienolide cues. The maternally‐invested toxins present in conspecific eggs may therefore be more easily detected post‐hatching, at which point tadpole feeding behaviors are induced whether or not additional toxin cues are present. As bufadienolide cues have previously been found to attract toad tadpoles to vulnerable hatchlings, our present findings demonstrate that the same toxin cues that attract cannibalistic tadpoles also induce them to feed, thereby facilitating cannibalism through multiple behavioral effects. Because native fauna do not produce bufadienolide toxins, the species specificity of these chemical cues in the Australian landscape may have facilitated the evolution of targeted (species‐specific) cannibalism in invasive cane toad populations. Thus, these bufadienolide toxins confer cost (increased vulnerability to cannibalism in early life‐stages) as well as benefit (reduced vulnerability to predation by other taxa).     

Experimental Feeding of Hydrilla verticillata Colonized by Stigonematales Cyanobacteria Induces Vacuolar Myelinopathy in Painted Turtles (Chrysemys picta)

Vacuolar myelinopathy (VM) is a neurologic disease primarily found in birds that occurs when wildlife ingest submerged aquatic vegetation colonized by an uncharacterized toxin-producing cyanobacterium (hereafter “UCB” for “uncharacterized cyanobacterium”). Turtles are among the closest extant relatives of birds and many species directly and/or indirectly consume aquatic vegetation. However, it is unknown whether turtles can develop VM. We conducted a feeding trial to determine whether painted turtles (Chrysemys picta) would develop VM after feeding on Hydrilla (Hydrilla verticillata), colonized by the UCB (Hydrilla is the most common “host” of UCB). We hypothesized turtles fed Hydrilla colonized by the UCB would exhibit neurologic impairment and vacuolation of nervous tissues, whereas turtles fed Hydrilla free of the UCB would not. The ability of Hydrilla colonized by the UCB to cause VM (hereafter, “toxicity”) was verified by feeding it to domestic chickens (Gallus gallus domesticus) or necropsy of field collected American coots (Fulica americana) captured at the site of Hydrilla collections. We randomly assigned ten wild-caught turtles into toxic or non-toxic Hydrilla feeding groups and delivered the diets for up to 97 days. Between days 82 and 89, all turtles fed toxic Hydrilla displayed physical and/or neurologic impairment. Histologic examination of the brain and spinal cord revealed vacuolations in all treatment turtles. None of the control turtles exhibited neurologic impairment or had detectable brain or spinal cord vacuolations. This is the first evidence that freshwater turtles can become neurologically impaired and develop vacuolations after consuming toxic Hydrilla colonized with the UCB. The southeastern United States, where outbreaks of VM occur regularly and where vegetation colonized by the UCB is common, is also a global hotspot of freshwater turtle diversity. Our results suggest that further investigations into the effect of the putative UCB toxin on wild turtles in situ are warranted.     

Field Measurements Indicate Unexpected,Serious Underestimation of Mussel Heart Rates and Thermal Tolerance by Laboratory Studies

Attempts to predict the response of species to long-term environmental change are generally based on extrapolations from laboratory experiments that inevitably simplify the complex interacting effects that occur in the field. We recorded heart rates of two genetic lineages of the brown mussel Perna perna over a full tidal cycle in-situ at two different sites in order to evaluate the cardiac responses of the two genetic lineages present on the South African coast to temperature and the immersion/emersion cycle. “Robomussel” temperature loggers were used to monitor thermal conditions at the two sites over one year. Comparison with live animals showed that robomussels provided a good estimate of mussel body temperatures. A significant difference in estimated body temperatures was observed between the sites and the results showed that, under natural conditions, temperatures regularly approach or exceed the thermal limits of P. perna identified in the laboratory. The two P. perna lineages showed similar tidal and diel patterns of heart rate, with higher cardiac activity during daytime immersion and minimal values during daytime emersion. Comparison of the heart rates measured in the field with data previously measured in the laboratory indicates that laboratory results seriously underestimate heart rate activity, by as much as 75%, especially during immersion. Unexpectedly, field estimates of body temperatures indicated an ability to tolerate temperatures considered lethal on the basis of laboratory measurements. This suggests that the interaction of abiotic conditions in the field does not necessarily raise vulnerability to high temperatures.     

A test of an antipredatory function of conspicuous plastron coloration in hatchling turtles

Bright colorations in animals are sometimes an antipredatory signal meant to startle, warn, or deter a predator from consuming a prey organism. Freshwater turtle hatchlings of many species have bright ventral coloration with high internal contrast that may have an antipredator function. We used visual modeling and field experiments to test whether the plastron coloration of Chrysemys picta hatchlings deters predators. We found that bird predators can easily distinguish hatchling turtles from their backgrounds and can easily see color contrast within the plastron. Raccoons cannot easily discriminate within-plastron color contrast but can see hatchlings against common backgrounds. Despite this, we found that brightly-colored, high contrast, replica turtles were not attacked less than low contrast replica turtles, suggesting that the bright coloration is not likely to serve an antipredatory function in this context. We discuss the apparent lack of innate avoidance of orange coloration in freshwater turtles by predators and suggest that preference and avoidance of colors are context-dependent. Since the bright colors are likely not a signal, we hypothesize that the colors may be caused by pigments deposited in tissue from maternal reserves during development. In most species, these pigments fade ontogenetically but they may have important physiological functions in species that maintain the bright coloration throughout adulthood.     

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 the hydric environment affect the incubation of small rigid-shelled turtle eggs?

Hydric environments are hypothesized to have minor effects on the embryonic development of rigid-shelled turtle eggs due to the low water permeability of the eggshell. However, the water reserve in the eggs may still influence their resistance to environmentally induced dehydration. We incubated rigid-shelled turtle eggs (Pelodiscus sinensis) on different moist substrates (from ? 12 to ? 750 kPa) to test the hypothesis that small rigid-shelled eggs would be sensitive to hydric environments. The hydric treatment significantly affected the incubation period, with eggs incubated in the moistest and driest substrates taking longer to hatch than those on the medium-moisture substrates. Hatching success was slightly lower for eggs incubated in dry conditions than those in wet conditions, but the difference was not statistically significant. The heart rates of early embryos were lower on moist substrates than those on dry substrates, but this difference disappeared in late embryos. Hatchlings from the moistest substrate were larger (in carapace length and width) and heavier than those from drier substrates. However, the dry body mass of the hatchlings did not differ among the hydric treatments. The functional performance (righting response) of the hatchlings was affected by the hydric environment. The time to right was shorter for the hatchlings from the substrate of ? 12 kPa than those from ? 220 kPa. These results are consistent with the hypothesis that the hydric environment may significantly affect developing embryos and the resulting hatchlings in turtle species, such as P. sinensis, with small rigid-shelled eggs.     

ECOLOGY AND PHYSIOLOGY OF HIBERNATION AND OVERWINTERING AMONG FRESHWATER FISHES, TURTLES, AND SNAKES

1. Freshwater fishes are the most northerly of freshwater ectotherms, followed by frogs. North American freshwater snakes, turtles, and salamanders do not range farther north than southernmost Canada. 2. Freezing and desiccation are the main challenges during terrestrial hibernation of ectotherms. Oxygen depletion, water balance, and ionic balance are the major problems for air breathing ectotherms that hibernate underwater. 3. The importance of accumulation of energy stores for overwintering among fishes depends upon the length and severity of the winters, whether or not there is springtime reproduction, body size, latitude, and the availability and use of food during overwintering. 4. Fishes can decrease energy demands during the winter by reductions in activity, metabolic depression, and entrance in semi-torpidity. 5. Adaptations for coping with hypoxia and anoxia among overwintering freshwater fishes may include metabolic depression, a decrease in blood O₂ affinity, microhabitat selection, air breathing, short-distance migration, biochemical modifications aimed at adjusting glycolytic rates, and alcoholic fermentation. 6. Freshwater turtles have a worldwide northern limit of approximately 50° N, which means that some species spend about half of their lives hibernating. 7. Aquatic turtles normally hibernate underwater, although occasionally they hibernate on land. In water they usually hibernate in a hypoxic or anoxic (mud) environment and in relatively shallow water. Wintertime movements of unknown frequency occur in some species. 8. The hatchlings of many turtle species can overwinter in the nest. Among northern species this behaviour is most common among painted turtles, whose hatchlings can withstand freezing. 9. Mortality among adult turtles is probably highest during the hibernation cycle. 10. Temperature appears to the most important cue for entry and exit from hibernation among freshwater turtles. 11. Little is known of the energetics of overwintering turtles. Energy stores for overwintering may be more important at lower latitudes than at higher ones, due to the higher metabolic rates of overwintering, but non-feeding, southern turtles. 12. The ability of turtles to tolerate submergence is a function of temperature, degree of water oxygenation, latitude of origin, efficacy of extrapulmonary respiratory pathways, and metabolic rate. 13. For turtles that hibernate in an anoxic hibernaculum, and for those without sufficient extrapulmonary uptake of O₂ to allow metabolism to be completely aerobic, the most important physiological perturbation is an acidosis developed from a continuing production of lactate. If sufficient O₂ can be obtained, the most likely factors limiting hibernation time are water balance and ion balance. 14. Mechanisms of turtles for coping with acidosis include metabolic depression, integumental CO₂ loss, bicarbonate buffering, and changes in ion concentrations that minimize the decrease in SID (strong ion difference). The most important among the latter are a decrease in plasma [Cl^-] and large increases in plasma calcium and magnesium. 15. Turtles are unique among reptiles in their ability to maintain both cardiovascular and nervous system function during prolonged anoxia. 16. Turtles gain weight from water uptake during submerged hibernation, but apparently maintain some kidney function; however, osmoregulation is one of the least known areas of the physiology of hibernation. 17. Recovery of turtles upon emergence commences with a rapid hyperventilatory compensation of pH, followed by a slower adjustment of ion levels. Basking speeds recovery greatly. 18. While hibernation of turtles in the northern parts of their ranges is most likely very stressful physiologically, northern range limits are more likely to be determined by reproductive restraints than by the rigors of extended hibernation. 19. The superior ability of turtles to tolerate anoxia may be more the result of an annual hibernation than of their diving habits during active periods of the year. 20. Freshwater snakes usually hibernate on land. However, they appear to be capable of aquatic hibernation and may not do so because of the risk of death from anoxia. 21. Some species of terrestrial snakes are known to hibernate underwater, and are able to do so in the laboratory for months. In the field, this behaviour is considered opportunistic, as there is no evidence to suggest that any snakes can tolerate extended anoxia.     

Embryonic communication in the nest: metabolic responses of reptilian embryos to developmental rates of siblings

Incubation temperature affects developmental rates and defines many phenotypes and fitness characteristics of reptilian embryos. In turtles, eggs are deposited in layers within the nest, such that thermal gradients create independent developmental conditions for each egg. Despite differences in developmental rate, several studies have revealed unexpected synchronicity in hatching, however, the mechanisms through which synchrony are achieved may be different between species. Here, we examine the phenomenon of synchronous hatching in turtles by assessing proximate mechanisms in an Australian freshwater turtle (Emydura macquarii). We tested whether embryos hatch prematurely or developmentally compensate in response to more advanced embryos in a clutch. We established developmental asynchrony within a clutch of turtle eggs and assessed both metabolic and heart rates throughout incubation in constant and fluctuating temperatures. Turtles appeared to hatch at similar developmental stages, with less-developed embryos in experimental groups responding to the presence of more developed eggs in a clutch by increasing both metabolic and heart rates. Early hatching did not appear to reduce neuromuscular ability at hatching. These results support developmental adjustment mechanisms of the 'catch-up hypothesis' for synchronous hatching in E. macquarii and implies some level of embryo-embryo communication. The group environment of a nest strongly supports the development of adaptive communication mechanisms between siblings and the evolution of environmentally cued hatching.     

Heat shock protein expression enhances heat tolerance of reptile embryos

The role of heat shock proteins (HSPs) in heat tolerance has been demonstrated in cultured cells and animal tissues, but rarely in whole organisms because of methodological difficulties associated with gene manipulation. By comparing HSP70 expression patterns among representative species of reptiles and birds, and by determining the effect of HSP70 overexpression on embryonic development and hatchling traits, we have identified the role of HSP70 in the heat tolerance of amniote embryos. Consistent with their thermal environment, and high incubation temperatures and heat tolerance, the embryos of birds have higher onset and maximum temperatures for induced HSP70 than do reptiles, and turtles have higher onset and maximum temperatures than do lizards. Interestingly, the trade-off between benefits and costs of HSP70 overexpression occurred between life-history stages: when turtle embryos developed at extreme high temperatures, HSP70 overexpression generated benefits by enhancing embryo heat tolerance and hatching success, but subsequently imposed costs by decreasing heat tolerance of surviving hatchlings. Taken together, the correlative and causal links between HSP70 and heat tolerance provide, to our knowledge, the first unequivocal evidence that HSP70 promotes thermal tolerance of embryos in oviparous amniotes.