Lost reproductive value reveals a high burden of juvenile road mortality in a long-lived species.

Adult mortality is often the most sensitive vital rate affecting at-risk wildlife populations. Therefore, road ecology studies often focus on adult mortality despite the possibility for roads to be hazardous to juvenile individuals during natal dispersal. Failure to quantify concurrent variation in mortality risk and population sensitivity across demographic states can mislead the efforts to understand and mitigate the effects of population threats. To compare relative population impacts from road mortality among demographic classes, we weighted mortality observations by applying reproductive value analysis to quantify expected stage-specific contributions to population growth. We demonstrate this approach for snapping turtles (Chelydra serpentina) observed on roads at two focal sites in Ontario, Canada, where we collected data for both live and dead individuals observed on roads. We estimated reproductive values using stage-classified matrix models to compare relative population-level impacts of adult and juvenile mortality. Reproductive value analysis is a tractable approach to assessing demographically variable effects for applications covering large spatial scales, nondiscrete populations, or where abundance data are lacking. For one site with long-term life-history data, we compared demographic frequency on roads to expected general population frequencies predicted by the matrix model. Our application of reproductive value is sex specific but, as juvenile snapping turtles lack external secondary sex characters, we estimated the sex ratio of road-crossing juveniles after dissecting and sexing carcasses collected on roads at five sites across central Ontario, Canada. Juveniles were more abundant on roads than expected, suggesting a substantial dispersal contribution, and the road-killed juvenile sex ratio approached 1:1. A higher proportion of juveniles were also found dead compared with adults, and cumulative juvenile mortality had similar population-level importance as adult mortality. This suggests that the impact of roads needs to be considered across all life stages, even in wildlife species with slow life histories, such as snapping turtles, that are particularly sensitive to adult mortality.

Maternal provisioning and fluctuating thermal regimes enhance immune response in a reptile with temperature-dependent sex determination.

The Charnov-Bull model of differential fitness is often used to explain the evolution and maintenance of temperature-dependent sex determination (TSD). Most tests of the model focus on morphological proxies of fitness, such as size traits, whereas early life physiological traits that are closely related to lifetime fitness might provide a framework for generalizing the Charnov-Bull model across taxa. One such trait is the strength of the early-life immune response, which is strongly linked to early-life survival and fitness. Here, we manipulated temperature, variance in temperature, and sex to test the Charnov-Bull model using a physiological trait, immune system strength, in the snapping turtle (Chelydra serpentina). We found no evidence of sex-specific differences in bactericidal capacity of hatchling blood, and no evidence that mean temperature influences bactericidal capacity. However, we did find that fluctuating incubation temperature (i.e. a more naturalized incubation regime) is associated with a greater bactericidal capacity compared with constant temperature incubation. We also found that egg mass, a proxy for maternal provisioning, is positively associated with bactericidal capacity. Our findings suggest that the evolution of temperature-dependent sex determination in reptiles is unrelated to our measure of early-life innate immunity. Our study also underlines how immune response is condition dependent in early life, and questions the biological relevance of constant temperature incubation in experimental studies on ectotherm development.

Anthropogenic nest sites provide warmer incubation environments than natural nest sites in a population of oviparous reptiles near their northern range limit.

Oviposition site choice affects a host of offspring phenotypes and directly impacts maternal fitness. Recent evidence suggests that oviparous reptiles often select nest sites where the landscape has been altered by anthropogenic activity, whereas natural nest sites are less often used. We leverage a long-term study of snapping turtle (Chelydra serpentina) to identify natural nest sites and anthropogenic nest sites and to compare habitat variables among nest site types. Natural and anthropogenic nest sites did not differ in average canopy closure, distance to nearest water, substrate composition, or aspect. However, anthropogenic nest sites had less ground-level vegetation and greater soil brightness, and were 3.3 °C warmer than natural nests during incubation. We used the Schoolfield model of poikilotherm development to assess differences in development rate between natural and anthropogenic nests. Because of the difference in temperature, embryos in anthropogenic nests were predicted to have undergone nearly twice as much development as embryos in natural nests during incubation. We outline why the evolution of fast embryonic development rate cannot compensate indefinitely for the low temperature incubation regimes that become increasingly prevalent at northern range margins, thereby underlining why maternal nest site choice of relatively warm anthropogenic sites may help oviparous reptiles persist in thermally constrained environments. Future research should aim to quantify both the thermal benefits of anthropogenic nest sites, as well as associated fitness costs (e.g., increased adult mortality) to elucidate whether anthropogenic disturbance of the landscape can be an ecological trap or serve a net benefit to some reptiles in northern environments.

First report of ranavirus mortality in a common snapping turtle Chelydra serpentina.

An adult male snapping turtle with marked palpebral edema and multifocal skin ulceration was found alive in a marsh in southern Ontario in summer 2017. The turtle was transported to a rehabilitation facility and died 4 d after arrival. The carcass was submitted to the Canadian Wildlife Health Cooperative for post-mortem examination. Gross lesions included ulcerative conjunctivitis, necrotizing stomatitis, and splenomegaly. Microscopically, this corresponded to multisystemic fibrinonecrotizing vasculitis and severe fibrinous splenic necrosis. Liver tissue tested positive for frog virus 3-like ranavirus and negative for herpesvirus via polymerase chain reaction. The gross and microscopic lesions were consistent with previous reports of ranavirus infection in turtles and were severe enough to have been the cause of death in this case. This is the first report of morbidity and mortality in a common snapping turtle with a ranavirus infection, and the first reported case of ranavirus infection in a reptile in Canada. Ranaviruses are considered to be an emerging infectious disease in chelonians as they are increasing in distribution, prevalence, and host range.

SURGICAL ANATOMY OF CELIOTOMY APPROACHES TO THE STOMACH IN THE COMMON SNAPPING TURTLE (CHELYDRA SERPENTINA).

Entanglement in or ingestion of fishing gear is a common cause of morbidity and mortality in chelonians. Commercial and recreational fishing activities exert bycatch pressures sufficient to cause population declines in the common snapping turtle (Chelydra serpentina) and the alligator snapping turtle (Macrochelys spp.). Gastrotomy for the removal of fish hook foreign bodies from the stomach of freshwater turtles typically requires a plastron osteotomy but in sea turtles has been successfully accomplished via soft tissue approaches. This study compared the anatomy and feasibility of soft tissue surgical approaches to the stomach in the common snapping turtle in both the prefemoral and the axillary regions. Anatomical dissections were performed on cadavers of five adult common snapping turtles, and the surgical anatomy of the left axillary and left prefemoral regions was characterized. The left axillary approach required extensive transection of musculature and provided poor exposure of the coelomic cavity and stomach. In contrast, the left prefemoral approach was simple to perform and provided sufficient access to the stomach for gastrotomy. The prefemoral celiotomy has significant advantages over the axillary celiotomy in the common snapping turtle and should be considered the surgical approach of choice for gastrotomy in this species.

Reptile Embryos Lack the Opportunity to Thermoregulate by Moving within the Egg.

Historically, egg-bound reptile embryos were thought to passively thermoconform to the nest environment. However, recent observations of thermal taxis by embryos of multiple reptile species have led to the widely discussed hypothesis that embryos behaviorally thermoregulate. Because temperature affects development, such thermoregulation could allow embryos to control their fate far more than historically assumed. We assessed the opportunity for embryos to behaviorally thermoregulate in nature by examining thermal gradients within natural nests and eggs of the common snapping turtle (Chelydra serpentina; which displays embryonic thermal taxis) and by simulating thermal gradients within nests across a range of nest depths, egg sizes, and soil types. We observed little spatial thermal variation within nests, and thermal gradients were poorly transferred to eggs. Furthermore, thermal gradients sufficiently large and constant for behavioral thermoregulation were not predicted to occur in our simulations. Gradients of biologically relevant magnitude have limited global occurrence and reverse direction twice daily when they do exist, which is substantially faster than embryos can shift position within the egg. Our results imply that reptile embryos will rarely, if ever, have the opportunity to behaviorally thermoregulate by moving within the egg. We suggest that embryonic thermal taxis instead represents a play behavior, which may be adaptive or selectively neutral, and results from the mechanisms for behavioral thermoregulation in free-living stages coming online prior to hatching.

EPINEPHRINE OR GV-26 ELECTRICAL STIMULATION REDUCES INHALANT ANESTHESTIC RECOVERY TIME IN COMMON SNAPPING TURTLES (CHELYDRA SERPENTINA).

Prolonged anesthetic recovery times are a common clinical problem in reptiles following inhalant anesthesia. Diving reptiles have numerous adaptations that allow them to submerge and remain apneic for extended periods. An ability to shunt blood away from pulmonary circulation, possibly due to changes in adrenergic tone, may contribute to their unpredictable inhalant anesthetic recovery times. Therefore, the use of epinephrine could antagonize this response and reduce recovery time. GV-26, an acupuncture point with reported ß-adrenergic and respiratory effects, has reduced anesthetic recovery times in other species. In this prospective randomized crossover study, six common snapping turtles (Chelydra serpentina) were anesthetized with inhalant isoflurane for 90 min. Turtles were assigned one of three treatments, given immediately following discontinuation of isoflurane: a control treatment (0.9% saline, at 0.1 ml/kg i.m.), epinephrine (0.1 mg/kg i.m.), or acupuncture with electrical stimulation at GV-26. Each turtle received all treatments, and treatments were separated by 48 hr. Return of spontaneous ventilation was 55% faster in turtles given epinephrine and 58% faster in the GV-26 group versus saline (P < 0.001). The times to movement and to complete recovery were also significantly faster for both treatments than for saline (P < 0.02). Treated turtles displayed increases in temperature not documented in the control (P < 0.001). Turtles administered epinephrine showed significantly increased heart rates and end-tidal CO(2) (P < 0.001). No adverse effects were noted in the study animals. The mechanisms of action were not elucidated in the present investigation. Nevertheless, the use of parenteral epinephrine or GV-26 stimulation in the immediate postanesthetic period produces clinically relevant reductions in anesthetic recovery time in common snapping turtle. Further research is necessary to evaluate the effects of concurrent GV-26 and epinephrine administration and to assess responses in other reptilian species.

Exogenous application of estradiol to eggs unexpectedly induces male development in two turtle species with temperature-dependent sex determination.

Steroid hormones affect sex determination in a variety of vertebrates. The feminizing effects of exposure to estradiol and the masculinizing effects of aromatase inhibition during development are well established in a broad range of vertebrate taxa, but paradoxical findings are occasionally reported. Four independent experiments were conducted on two turtle species with temperature-dependent sex determination (Chrysemys picta and Chelydra serpentina) to quantify the effects of egg incubation temperature, estradiol, and an aromatase inhibitor on offspring sex ratios. As expected, the warmer incubation temperatures induced female development and the cooler temperatures produced primarily males. However, application of an aromatase inhibitor had no effect on offspring sex ratios, and exogenous applications of estradiol to eggs produced male offspring across all incubation temperatures. These unexpected results were remarkably consistent across all four experiments and both study species. Elevated concentrations of estradiol could interact with androgen receptors or inhibit aromatase expression, which might result in relatively high testosterone concentrations that lead to testis development. These findings add to a short list of studies that report paradoxical effects of steroid hormones, which addresses the need for a more comprehensive understanding of the role of sex steroids in sexual development.

Diverse Response Pattern to Anoxia in Three Freshwater Turtle Species.

With increasing water eutrophication and global warming, anoxia and hypoxia are becoming more and more common in water environments. Most vertebrates have a limited tolerance to anoxia of only a few minutes, but some species, such as turtles, can survive for months being exposed to anoxia. Antioxidant defense systems may have a potential role in resisting anoxia stress in freshwater turtles. The three-keeled pond turtle Chinemys reevesii, the snapping turtle Chelydra serpentina and the soft-shelled turtle Pelodiscus sinensis are three popular aquaculture species and share similar habitats in China. While C. reevesii and C. serpentina are hard-shelled turtles with poor skin permeability, P. sinensis is soft-shelled turtle whose skin permeability is good. We examined the antioxidant defense responses in different tissues of the three turtle species under acute anoxia stress for 10 h and subsequently recovered for 24 h in order to reveal the response patterns of the antioxidant defense system of the three turtle species that differed in morphological structure and life history strategy. We found that the antioxidant response patterns to acute anoxia stress were tissue- and species-specific. The soft-shelled turtle was more sensitive to anoxia than the hard-shelled turtles. Under anoxia stress, the three species kept the activities of most antioxidant enzymes stable. C. reevesii and P. sinensis were highly dependent on vitamin C in antioxidant defense, while high activities of structural antioxidant enzymes were found in the tissues of C. serpentina. The above diverse patterns may be related with adaptive evolution of morphological structure and physiological functions of the three turtle species.

Draft Genome of the Common Snapping Turtle, Chelydra serpentina, a Model for Phenotypic Plasticity in Reptiles.

Turtles are iconic reptiles that inhabit a range of ecosystems from oceans to deserts and climates from the tropics to northern temperate regions. Yet, we have little understanding of the genetic adaptations that allow turtles to survive and reproduce in such diverse environments. Common snapping turtles, Chelydra serpentina, are an ideal model species for studying adaptation to climate because they are widely distributed from tropical to northern temperate zones in North America. They are also easy to maintain and breed in captivity and produce large clutch sizes, which makes them amenable to quantitative genetic and molecular genetic studies of traits like temperature-dependent sex determination. We therefore established a captive breeding colony and sequenced DNA from one female using both short and long reads. After trimming and filtering, we had 209.51Gb of Illumina reads, 25.72Gb of PacBio reads, and 21.72 Gb of Nanopore reads. The assembled genome was 2.258 Gb in size and had 13,224 scaffolds with an N50 of 5.59Mb. The longest scaffold was 27.24Mb. BUSCO analysis revealed 97.4% of core vertebrate genes in the genome. We identified 3.27 million SNPs in the reference turtle, which indicates a relatively high level of individual heterozygosity. We assembled the transcriptome using RNA-Seq data and used gene prediction software to produce 22,812 models of protein coding genes. The quality and contiguity of the snapping turtle genome is similar to or better than most published reptile genomes. The genome and genetic variants identified here provide a foundation for future studies of adaptation to climate.

Morphometric data for five freshwater turtles in south, central, and west Texas.

From 2008 to 2013, we sampled freshwater turtle populations at 66 sites in south, central, and west Texas, USA. Sampling sites included ponds, lakes, resacas (oxbow lakes), canals, and rivers. We sampled turtle populations using baited hoop nets (66 sites) and basking traps (3 sites), and captured turtles by hand opportunistically in terrestrial habitat. We measured carapace length and width, plastron length and width, body depth, and weight of captured turtles. Excluding recaptures, we measured 356 Apalone spinifera emoryi (Texas Spiny Softshell), 24 Chelydra serpentina (Snapping Turtle), 20 Kinosternon flavescens (Yellow Mud Turtle), 47 Trachemys gaigeae (Big Bend Slider), and 1070 Trachemys scripta elegans (Red-eared Slider). Carapace length of Apalone spinifera emoryi ranged from 85 to 426 mm (mean = 182 mm). Carapace length of Chelydra serpentina ranged from 74 to 320 mm (mean = 233 mm). Carapace length of Kinosternon flavescens ranged from 64 to 147 mm (mean = 114 mm). Carapace length of Trachemys gaigeae ranged from 54 to 203 mm (mean = 141 mm). Carapace length of Trachemys scripta elegans ranged from 30 to 328 mm (mean = 171 mm). These data are useful for assessing spatial and temporal variation in size and body condition of freshwater turtles.

EXPERIMENTAL EVIDENCE FOR THE EVOLUTIONARY SIGNIFICANCE OF TEMPERATURE-DEPENDENT SEX DETERMINATION.

The evolutionary significance of sex-determining mechanisms, particularly temperature-dependent sex determination (TSD) in reptiles, has remained unresolved despite extensive theoretical work. To investigate the evolutionary significance of this unusual sex-determining mechanism, I incubated eggs of the common snapping turtle (Chelydra serpentina) at a male-producing temperature (26°C), a female-producing temperature (30°C), and an intermediate temperature that produced both sexes about equally (28°C). Laboratory experiments indicated that two performance variables, but no morphological measurements, were significantly influenced by incubation temperature (P ≤ 0.05): hatchlings from cooler incubation treatments swam faster than turtles from warmer incubation treatments, and hatchlings from 28°C exhibited a greater propensity to run than did individuals from 26°C and 30°C. In the field, hatchlings from the all-male and all-female producing temperatures had significantly higher first-year survivorship than did consexuals from the incubation temperature that produced both sexes (G = 6.622, P = 0.03). Significant directional selection was detected on propensity of hatchlings to run (ß' = -0.758, P = 0.05): turtles that tended to remain immobile had a higher probability of first-year survivorship than did individuals that moved readily. Thus, the effects of the gender × incubation temperature interaction on survivorship of hatchling turtles observed in the field experiment may have been mediated by temperature-dependent antipredator behavior. These results provide a possible functional explanation for the evolutionary significance of TSD in turtles that is consistent with predictions of theoretical models.