Temperature experienced during incubation affects antioxidant capacity but not oxidative damage in hatchling red-eared slider turtles (Trachemys scripta elegans).

Our understanding of how oxidative stress resistance phenotypes are affected by the developmental environment is limited. One component of the developmental environment, which is likely central to early life oxidative stress among ectothermic and oviparous species, is that of temperature. We investigated how incubation temperature manipulations affect oxidative damage and total antioxidant capacity (TAC) in red-eared slider turtle (Trachemys scripta elegans) hatchlings. First, to determine whether temperature fluctuations elicit oxidative stress, eggs from clutches were randomly assigned to either a constant (29.5 °C) or daily fluctuating temperature incubation (28.7 ± 3 °C) treatment. Second, to assess the effect of temperature fluctuation frequency on oxidative stress, eggs were incubated in one of three fluctuating incubation regimes: 28.7 ± 3 °C fluctuations every 12 h (hyper), 24 h (normal) or 48 h (hypo). Third, we tested the influence of average incubation temperature by incubating eggs in a daily fluctuating incubation temperature regime with a mean temperature of 26.5 °C (low), 27.1 °C (medium) or 27.7 °C (high). Although the accumulation of oxidative damage in hatchlings was unaffected by any thermal manipulation, TAC was affected by both temperature fluctuation frequency and average incubation temperature. Individuals incubated with a low frequency of temperature fluctuations had reduced TAC, while incubation at a lower average temperature was associated with enhanced TAC. These results indicate that although sufficient to prevent oxidative damage, TAC is influenced by developmental thermal environments, potentially because of temperature-mediated changes in metabolic rate. The observed differences in TAC may have important future consequences for hatchling fitness and overwinter survival.

Understanding the vertebrate immune system: insights from the reptilian perspective.

Reptiles are ectothermic amniotes, providing the key link between ectothermic anamniotic fishes and amphibians, and endothermic amniotic birds and mammals. A greater understanding of reptilian immunity will provide important insights into the evolutionary history of vertebrate immunity as well as the growing field of eco-immunology. Like mammals, reptile immunity is complex and involves innate, cell-mediated and humoral compartments but, overall, there is considerably less known about immune function in reptiles. We review the current literature on each branch of the reptilian immune system, placing this information in context to other vertebrates. Further, we identify key areas that are prime for research as well as areas that are lagging because of lack of reagents in non-model systems.

The Devil is in the Details: Identifying Aspects of Temperature Variation that Underlie Sex Determination in Species with TSD.

Most organisms experience thermal variability in their environment; however, our understanding of how organisms cope with this variation is under-developed. For example, in organisms with temperature-dependent sex determination (TSD), an inability to predict sex ratios under fluctuating incubation temperatures in the field hinders predictions of how species with TSD will fare in a changing climate. To better understand how sex determination is affected by thermal variation, we incubated Trachemys scripta eggs using a "heat wave" design, where embryos experienced a male-producing temperature of 25 ± 3°C for the majority of development and varying durations at a female-producing temperature of 29.5 ± 3°C during the window of development when sex is determined. We compared the sex ratios from these incubation conditions with a previous data set that utilized a similar heat wave design, but instead incubated eggs at a male-producing temperature of 27 ± 3°C but utilized the same female-producing temperature of 29.5 ± 3°C. We compared the sex ratio reaction norms produced from these two incubation conditions and found that, despite differences in average temperatures, both conditions produced 50:50 sex ratios after ∼8 days of exposure to female-producing conditions. This emphasizes that sex can be determined in just a few days at female-producing conditions and that sex determination is relatively unaffected by temperatures outside of this short window. Further, these data demonstrate the reduced accuracy of the constant temperature equivalent model (the leading method of predicting sex ratios) under thermally variable temperatures. Conceptualizing sex determination as the number of days spent incubating at female-producing conditions rather than an aggregate statistic is supported by the mechanistic underpinnings of TSD, helps to improve sex ratio estimation methods, and has important consequences for predicting how species with TSD will fare in a changing climate.

Short heatwaves during fluctuating incubation regimes produce females under temperature-dependent sex determination with implications for sex ratios in nature.

Patterns of temperature fluctuations in nature affect numerous biological processes, yet, empirical studies often utilize constant temperature treatments. This can limit our understanding of how thermally sensitive species respond to ecologically relevant temperatures. Research on turtles with temperature-dependent sex determination (TSD) provides good examples of this, since nest temperatures from many populations rarely exceed those necessary to produce females under constant laboratory conditions. We hypothesized that exposure to brief periods of warm temperatures (i.e., heat waves) are integral to sex determination in species with TSD, which requires tests that move beyond constant temperatures. We exposed Trachemys scripta embryos from multiple populations and across the nesting season to heat waves of variable durations and quantified sex ratios. We found that embryos from all populations were highly sensitive to brief exposures to female producing temperatures; only 7.9 days of exposure produced a 50:50 sex ratio, but the response varied across the nesting season. From these findings, a model was developed to estimate sex ratios from field temperature traces, and this model outperformed traditional methods. Overall, these results enhance our understanding of TSD and emphasize the importance of using biologically relevant temperatures when studying thermally sensitive processes.

Turtle hatchlings show behavioral types that are robust to developmental manipulations.

There can be substantial variation among individuals within a species in how they behave, even under similar conditions; this pattern is found in many species and across taxa. However, the mechanisms that give rise to this behavioral variation are often unclear. This study investigated the influence of environmental manipulations during development on behavioral variation in hatchlings of the red-eared slider turtle (Trachemys scripta). First, we examined the effects of three manipulations during incubation (estrone sulfate exposure, corticosterone exposure, and thermal fluctuations) on hatchling righting response and exploration. Second, we determined whether hatchlings showed consistent differences (i.e. behavioral types) in their righting response and exploration across days and months, and whether these behaviors were correlated with one another. Finally, we examined whether righting response was predictive of ecologically relevant behaviors such as habitat choice and dispersal. Hatchling behavior was robust to our early manipulations; none of the pre-hatch treatments affected later behavior. There were significant clutch effects, which due to the split-clutch design suggests genetic underpinnings and/or maternal effects. We found evidence for behavioral types in turtles; both righting response and exploration were strongly repeatable and these behaviors were positively correlated. Righting response was not predictive of dispersal ability in the field, necessitating a revision in the general interpretations of righting response as a proxy for dispersal ability in turtles. Thus, turtle hatchlings show consistent behavioral differences that are robust to early developmental manipulations, and while not necessarily predictive of dispersal, these behavioral types can have important consequences throughout ontogeny.

Environmental sex determination in a reptile varies seasonally and with yolk hormones.

Most hypotheses that have been put forward in order to explain the persistence of environmental sex determination (ESD) in reptiles assume a relatively fixed association of sex with temperature-induced phenotype and no maternal influence on offspring sex. Here we demonstrate the association of maternally derived yolk hormone levels with the offspring sex ratio and describe two new aspects of temperature-dependent sex determination (TSD), i.e. seasonal variation in both thermal response and yolk steroid levels. Eggs from painted turtles (Chrysemys picta) were incubated at 28 degrees C. The hatchling sex ratio at 28 degrees C (i.e. the phenotypic reaction norm for sex at 28 degrees C) shifted seasonally from ca. 72% male to ca. 76% female. Yolk oestradiol (E2) increased seasonally while testosterone (T) decreased. The proportion of males in a clutch decreased as E2 levels increased and the E2:T ratio increased. These new findings are discussed in relation to heritability and adaptive explanations for the persistence of ESD in reptiles. Maternally derived yolk hormones may provide a mechanism for the seasonal shift in the sex ratio which in turn may help explain the persistence of ESD in reptiles. They may also explain those clutches of other reptiles with TSD that fail to yield only males at maximally masculinizing conditions.

The ontogeny of postmaturation resource allocation in turtles.

Resource-allocation decisions vary with life-history strategy, and growing evidence suggests that long-lived endothermic vertebrates direct resources toward growth and self-maintenance when young, increasing allocation toward reproductive effort over time. Few studies have tracked the ontogeny of resource allocation (energy, steroid hormones, etc.) in long-lived ectothermic vertebrates, limiting our understanding of the generality of life-history strategies among vertebrates. We investigated how reproductively mature female painted turtles (Chrysemys picta) from two distinct age classes allocated resources over a 4-yr period and whether resource-allocation patterns varied with nesting experience. We examined age-related variation in body size, egg mass, reproductive frequency, and yolk steroids and report that younger females were smaller and allocated fewer resources to reproduction than did older females. Testosterone levels were higher in eggs from younger females, whereas eggs from second (seasonal) clutches contained higher concentrations of progesterone and estradiol. These allocation patterns resulted in older, larger females laying larger eggs and producing second clutches more frequently than their younger counterparts. We conclude that resource-allocation patterns do vary with age in a long-lived ectotherm.

Concentrations of steroid hormones in layers and biopsies of chelonian egg yolks.

The actions of circulating hormones, although relatively well understood for adults, are largely unknown for their developing embryos. Transfer of maternal hormones to the egg is known to occur in oviparous species, and recently the presence of hormonally heterogeneous yolk layers has been described in two avian species. To investigate the possibility of a similar phenomenon occurring in chelonian species, egg yolk layers were analyzed in the painted turtle (Chrysemys picta marginata) and the red-eared slider turtle (Trachemys scripta elegans), two species that exhibit temperature-dependent sex determination. There was a similar pattern of hormonally heterogeneous yolk layers in both species: concentrations of progesterone and testosterone were significantly higher in the external yolk layer while concentrations of 17beta-estradiol were significantly higher in the intermediate and internal layers. This pattern of hormone deposition concurs with previously published studies of plasma hormone profiles from females of temperate-zone turtle species. Yolks of freshly laid eggs were also sampled using a biopsy technique to examine the concordance of early yolk hormone concentrations and offspring sex. No relationship was found between yolk hormone concentrations and individual offspring sex. Previous work showing that maternally derived yolk estradiol concentrations are correlated with female-biased sex ratios was, however, replicated. These findings suggest that offspring sex is influenced, in part, by the maternal hormone environment.