Experiencing short heat waves early in development changes thermal responsiveness of turtle embryos to later heat waves.

Although physiological responses to the thermal environment are most frequently investigated using constant temperatures, the incorporation of thermal variability can allow for a more accurate prediction of how thermally sensitive species respond to a rapidly changing climate. In species with temperature-dependent sex determination (TSD), developmental responses to incubation temperature are mediated by several genes involved in gonadal differentiation. Kdm6b and Dmrt1 respond to cool incubation temperatures and are associated with testis development, while FoxL2 and Cyp19A1 respond to warm incubation temperatures and are associated with ovary development. Using fluctuating incubation temperatures, we designed two studies, one investigating how conflicting thermal cues affect the timing of commitment to gonadal development, and another investigating the rapid molecular responses to conflicting thermal cues in the red-eared slider turtle (Trachemys scripta). Using gene expression as a proxy of timing of commitment to gonadal fate, results from the first study show that exposure to high amounts of conflicting thermal cues during development delays commitment to gonadal fate. Results from the second study show that Kdm6b splice variants exhibit differential responses to early heat wave exposure, but rapidly (within 2 days) recover to pre-exposure levels after the heat wave. Despite changes in the expression of Kdm6b splice variants, there was no effect on Dmrt1 expression. Collectively, these findings demonstrate how short exposures to heat early in development can change how embryos respond to heat later in development.

Understanding how variable thermal environments affect the molecular mechanisms underlying temperature-sensitive phenotypes: lessons from sex determination.

The thermal environment that organisms experience can affect many aspects of their phenotype. As global temperatures become more unpredictable, it is imperative that we understand the molecular mechanisms by which organisms respond to variable, and often transient, thermal environments. Beyond deciphering the mechanisms through which organisms respond to temperature, we must also appreciate the underlying variation in temperature-dependent processes, as this variation is essential for understanding the potential to adapt to changing climates. In this Commentary, we use temperature-dependent sex determination as an example to explore the mechanistic processes underlying the development of temperature-sensitive phenotypes. We synthesize the current literature on how variable thermal conditions affect these processes and address factors that may limit or allow organisms to respond to variable environments. From these examples, we posit a framework for how the field might move forward in a more systematic way to address three key questions: (1) which genes directly respond to temperature-sensitive changes in protein function and which genes are downstream, indirect responders?; (2) how long does it take different proteins and genes to respond to temperature?; and (3) are the experimental temperature manipulations relevant to the climate the organism experiences or to predicted climate change scenarios? This approach combines mechanistic questions (questions 1 and 2) with ecologically relevant conditions (question 3), allowing us to explore how organisms respond to transient thermal environments and, thus, cope with climate change.

Temperature fluctuations and estrone sulfate affect gene expression via different mechanisms to promote female development in a species with temperature-dependent sex determination.

Variation in developmental conditions can affect a variety of embryonic processes and shape a number of phenotypic characteristics that can affect offspring throughout their lives. This is particularly true of oviparous species where development typically occurs outside of the female, and studies have shown that traits such as survival and behavior can be altered by both temperature and exposure to steroid hormones during development. In species with temperature-dependent sex determination (TSD), the fate of gonadal development can be affected by temperature and by maternal estrogens present in the egg at oviposition, and there is evidence that these factors can affect gene expression patterns. Here, we explored how thermal fluctuations and exposure to an estrogen metabolite, estrone sulfate, affect the expression of several genes known to be involved in sexual differentiation: Kdm6b, Dmrt1, Sox9, FoxL2 and Cyp19A1. We found that most of the genes responded to both temperature and estrone sulfate exposure, but that the responses to these factors were not identical, in that estrone sulfate effects occur downstream of temperature effects. Our findings demonstrate that conjugated hormones such as estrone sulfate are capable of influencing temperature-dependent pathways to potentially alter how embryos respond to temperature, and highlight the importance of studying the interaction of maternal hormone and temperature effects.

Sex-specific effects of hatching order on nestling baseline corticosterone in a wild songbird.

Variation in nestling growth and survival is often influenced by hatching order, with first-hatched offspring having an advantage over later-hatched younger siblings. In house wrens (Troglodytes aedon), this effect of hatching order is especially evident in asynchronously hatched broods and can lead to sex-specific differences in the size and condition of nestlings. Females appear to allocate the sex of their offspring across the laying order to capitalize on these differences. We hypothesized that levels of circulating corticosterone, the primary metabolic hormone in birds, mediates these sex-specific effects in nestlings. We predicted that: i) baseline levels of corticosterone in nestlings should vary along the hatching order, ii) effects of hatching order on baseline corticosterone should be sex specific, and iii) any sex-specificity of hatching order on baseline corticosterone could be contingent on the degree of hatching synchrony. We tested these predictions in a study in which we measured baseline corticosterone in first- and last-hatched nestlings in synchronously and asynchronously hatching broods. To assess whether any differences in nestling baseline corticosterone levels could be attributed to pre-natal maternal effects, the post-natal environment, or both, we conducted two additional studies in which we measured i) yolk corticosterone in first- and last-laid eggs and ii) baseline corticosterone in nestlings that were cross-fostered to create simulated 'asynchronously' hatched broods. There was a significant interaction between sex and relative hatching order in their effects on nestling baseline corticosterone, but no effect of hatching synchrony. Corticosterone levels remained relatively constant across the hatching order in males but decreased in females. There was a significant effect of laying order on yolk corticosterone, with first-laid eggs containing significantly higher levels of yolk corticosterone than last-laid eggs. Cross-fostering of nestlings at different points of development had no significant effect on nestling corticosterone levels. These results indicate that sex-dependent differences in corticosterone levels across the hatching order may arise, at least in part, from embryonic exposure to maternally derived corticosterone, whereas the post-natal rearing environment plays, at best, a minimal role in determining nestling baseline corticosterone levels.

Brief exposure to warm temperatures reduces intron retention in Kdm6b in a species with temperature-dependent sex determination.

Animals with temperature-dependent sex determination (TSD) respond to thermal cues during early embryonic development to trigger gonadal differentiation. TSD has primarily been studied using constant temperature incubations, where embryos are exposed to constant male- or female-producing temperatures, and these studies have identified genes that display sex-specific expression in response to incubation temperature. Kdm6b, a histone demethylase gene, has received specific attention as it is among the initial genes to respond to incubation temperature and is necessary for testis development. Interestingly, Kdm6b retains an intron when eggs are incubated at a constant male-producing temperature, but the role of thermal variability in this developmental process is relatively understudied. Species with TSD regularly experience thermal cues that fluctuate between male- and female-producing temperatures throughout development but it is unclear how Kdm6b responds to such variable temperatures. In this study, we investigate temperature-sensitive splicing in Kdm6b by exposing embryos to male- and female-producing thermal conditions. We show a rapid decrease in levels of the intron retaining transcript of Kdm6b upon exposure to female-producing conditions. These results demonstrate that, under ecologically relevant conditions, temperature-sensitive splicing can differentially regulate genes critical to TSD.

Using naturalistic incubation temperatures to demonstrate how variation in the timing and continuity of heat wave exposure influences phenotype.

Most organisms are exposed to bouts of warm temperatures during development, yet we know little about how variation in the timing and continuity of heat exposure influences biological processes. If heat waves increase in frequency and duration as predicted, it is necessary to understand how these bouts could affect thermally sensitive species, including reptiles with temperature-dependent sex determination (TSD). In a multi-year study using fluctuating temperatures, we exposed Trachemys scripta embryos to cooler, male-producing temperatures interspersed with warmer, female-producing temperatures (heat waves) that varied in either timing during development or continuity and then analysed resulting sex ratios. We also quantified the expression of genes involved in testis differentiation (Dmrt1) and ovary differentiation (Cyp19A1) to determine how heat wave continuity affects the expression of genes involved in sexual differentiation. Heat waves applied during the middle of development produced significantly more females compared to heat waves that occurred just 7 days before or after this window, and even short gaps in the continuity of a heat wave decreased the production of females. Continuous heat exposure resulted in increased Cyp19A1 expression while discontinuous heat exposure failed to increase expression in either gene over a similar time course. We report that even small differences in the timing and continuity of heat waves can result in drastically different phenotypic outcomes. This strong effect of temperature occurred despite the fact that embryos were exposed to the same number of warm days during a short period of time, which highlights the need to study temperature effects under more ecologically relevant conditions where temperatures may be elevated for only a few days at a time. In the face of a changing climate, the finding that subtle shifts in temperature exposure result in substantial effects on embryonic development becomes even more critical.

Characterizing the timing of yolk testosterone metabolism and the effects of etiocholanolone on development in avian eggs.

Maternal transfer of steroids to eggs can elicit permanent effects on offspring phenotype. Although testosterone was thought to be a key mediator of maternal effects in birds, we now know that vertebrate embryos actively regulate their exposure to maternal testosterone through steroid metabolism, suggesting testosterone metabolites, not testosterone, may elicit the observed phenotypic effects. To address the role steroid metabolism plays in mediating yolk testosterone effects, we used European starling (Sturnus vulgaris) eggs to characterize the timing of testosterone metabolism and determine whether etiocholanolone, a prominent metabolite of testosterone in avian embryos, is capable of affecting early embryonic development. Tritiated testosterone was injected into freshly laid eggs to characterize steroid movement and metabolism during early development. Varying levels of etiocholanolone were also injected into eggs, with incubation for either 3 or 5 days, to test whether etiocholanolone influences the early growth of embryonic tissues. The conversion of testosterone to etiocholanolone was initiated within 12 h of injection, but the increase in etiocholanolone was transient, indicating that etiocholanolone is also subject to metabolism, and that exposure to maternal etiocholanolone is limited to a short period during early development. Exogenous etiocholanolone manipulation had no significant effect on the growth rate of the embryos or extra-embryonic membranes early in development. Thus, the conversion of testosterone to etiocholanolone may be an inactivation pathway that buffers the embryo from maternal steroids, with any effects of yolk testosterone resulting from testosterone that escapes metabolism; alternatively, etiocholanolone may influence processes other than growth or take additional time to manifest.

Year-round plasma steroid hormone profiles and the reproductive ecology of gopher tortoises (Gopherus polyphemus) at the southernmost edge of their range.

Populations of wide ranging ectotherms often exhibit variation in traits that are influenced by local environmental conditions. Although the gopher tortoise, Gopherus polyphemus, is well studied in pine flatwoods habitats across their range, little attention has been given to coastal populations existing in the southern extreme portion of the range. We examined the reproductive physiology of a coastal dune population in southwest Florida to determine if reproductive cycles vary across populations. Here we present the first year-round sex hormone profiles for a wild population of gopher tortoises. Male testosterone concentrations varied across the year (F11,54 = 2.52, P = 0.015) with elevated values from September to December and minimal levels from April to July, with the exception of a secondary peak during the month of June. Female testosterone and estradiol concentrations varied across the sampling period (T: F11,66 = 8.54, P < 0.001, E: F11,66 = 4.57, P < 0.001) with highest values from August to February, and lowest levels from May to July. Female progesterone concentrations varied over the year (F11,64 = 3.29, P = 0.002) and increased in late fall with a peak in March. These data suggest this population has an extended breeding season from fall through spring with mating likely occurring from September through March, and nesting in winter through spring. This pattern is similar to reproductive patterns described for tropical and sub-tropical chelonians but differs from that of gopher tortoise populations in northern portions of the range where hibernation may last for five months and a single clutch of eggs are deposited in late spring.

Evidence of embryonic regulation of maternally derived yolk corticosterone.

In recent years, the potential for maternal stress effects to adaptively alter offspring phenotype has received considerable attention. This research has identified offspring traits that are labile in response to maternal stress; however, an understanding of the mechanisms underlying these effects is lagging and is crucial to appreciating the significance of this maternal effect. In the present study, we sought to better understand maternal stress effects by examining the potential for embryonic regulation of corticosterone exposure, determining the phenotypic consequences of elevated corticosterone during development, and characterizing the levels of maternally transferred corticosterone in unmanipulated eggs using Trachemys scripta By dosing eggs with tritiated corticosterone and tracking the steroid throughout development, we found that most corticosterone is metabolized, and less than 1% of the corticosterone dose reaches the embryo as free corticosterone. We also found that exogenous dosing of corticosterone, in concentrations sufficient to overwhelm embryonic metabolism, reduces embryonic survival and negatively impacts hatchling traits important to fitness. Our results demonstrate that concentrations of maternal corticosterone in the yolks of unmanipulated eggs are low and are significantly lower than the doses of corticosterone required to elicit phenotypic effects in hatchlings. Taken together, these results provide evidence that both the embryo and the female may minimize corticosterone accumulation in the embryo to avoid reductions in embryonic survival and negative impacts on offspring phenotype and fitness.

The effect of environmental temperature on reptilian peripheral blood B cell functions.

Recent studies have identified phagocytic B cells in a variety of species, yet little is understood about their function and how it is influenced by natural environmental variation, such as temperature. Phagocytic B-cells are present in red-eared slider turtles, Trachemys scripta, and the wide range of temperatures experienced by these ectotherms may have an effect on immunity, including B cell antibody secretion and phagocytosis. We examined the impact of environmental temperature on B cell function in vitro using phagocytic and ELISpot assays conducted at biologically relevant temperatures. We found a significant effect of temperature on antibody secretion, with maximal antibody secretion occurring at intermediate temperatures (estimated maximum of 28.8°C). There was no effect of temperature on phagocytosis. We also noted a difference in the efficiency of phagocytosis in this assay between B cells and non-B cells. Interestingly, in our in vitro assay, phagocytic B cells engulfed more foreign fluorescent beads per cell than phagocytes lacking surface immunoglobulin. This work sheds light on our understanding of phagocytic B cells and the importance of environmental temperature on the behavior of reptilian immune cells, which may have relevance for organismal fitness.

Characterizing the distribution of steroid sulfatase during embryonic development: when and where might metabolites of maternal steroids be reactivated?

All vertebrate embryos are exposed to maternally derived steroids during development. In placental vertebrates, metabolism of maternal steroids by the placenta modulates embryonic exposure, but how exposure is regulated in oviparous vertebrates is less clear. Recent work in oviparous vertebrates has demonstrated that steroids are not static molecules, as they can be converted to more polar steroid sulfates by sulfotransferase enzymes. Importantly, these steroid sulfates can be converted back to the parent compound by the enzyme steroid sulfatase (STS). We investigated when and where STS was present during embryonic development in the red-eared slider turtle, Trachemys scripta We report that STS is present during all stages of development and in all tissues we examined. We conclude that STS activity may be particularly important for regulating maternal steroid exposure in oviparous vertebrates.

Elevated corticosterone during egg production elicits increased maternal investment and promotes nestling growth in a wild songbird.

Glucocorticoids circulating in breeding birds during egg production accumulate within eggs, and may provide a potent form of maternal effect on offspring phenotype. However, whether these steroids affect offspring development remains unclear. Here, we employed a non-invasive technique that experimentally elevated the maternal transfer of corticosterone to eggs in a wild population of house wrens. Feeding corticosterone-injected mealworms to free-living females prior to and during egg production increased the number of eggs that females produced and increased corticosterone concentrations in egg yolks. This treatment also resulted in an increase in the amount of yolk allocated to eggs. Offspring hatching from these eggs begged for food at a higher rate than control offspring and eventually attained increased prefledging body condition, a trait predictive of their probability of recruitment as breeding adults in the study population. Our results indicate that an increase in maternal glucocorticoids within the physiological range can enhance maternal investment and offspring development.

Immune activation generates corticosterone-mediated terminal reproductive investment in a wild bird.

Despite classical expectations of a trade-off between immune activity and reproduction, an emergent view suggests that individuals experiencing activation of their immune system actually increase reproductive effort and allocation to offspring as a form of terminal investment in response to reduced survival probability. However, the components and mechanisms of increased parental investment following immunostimulation are currently unknown. We hypothesize that increased glucocorticoid production following immunostimulation modulates the increase in reproductive effort that constitutes terminal investment. We activated the immune system of breeding female house wrens (Troglodytes aedon) with an immunogen and cross-fostered the eggs that they subsequently produced to separate prenatal and postnatal components of maternal investment. Cross-fostering revealed an increase in both pre- and postnatal allocation from immunostimulated females, which was confirmed by quantification of egg constituents and maternal provisioning behavior. The increase in maternal provisioning was mediated, at least in part, by increased corticosterone in these females. Offspring immune responsiveness was also enhanced through transgenerational immune priming via the egg. Thus, our results indicate that maternal immunostimulation induces transgenerational effects on offspring through both pre- and postnatal parental effects and support an important role for corticosterone in mediating parental investment.

The in ovo conversion of oestrone to oestrone sulfate is rapid and subject to inhibition by Bisphenol A.

Vertebrate embryos develop in the presence of maternally derived steroids. While these steroids can influence development, embryonic enzymes are thought to buffer some steroid sensitive processes, such as gonadal differentiation, from the effects of maternal steroids. Many of these same enzymes may also buffer the embryo from chemicals present in the environment, but this may alter their capacity to metabolize maternal steroids. Here, we characterized the ability of red-eared slider (Trachemys scripta) embryos to metabolize oestrone immediately following oviposition and tested whether a prevalent environmental chemical, Bisphenol A (BPA), would affect the in ovo conversion of oestrone to oestrone sulfate. We found that tritiated oestrone applied at the time of oviposition is mostly converted to oestrone sulfate within 6 h. However, when BPA is present, that conversion is inhibited, resulting in elevated oestrone levels. Our finding of rapid in ovo metabolism of steroids suggests that maternally derived enzymes are present in the egg and can alter embryonic exposure to exogenous chemicals. The disruption of this metabolism by BPA demonstrates how environmental chemicals might change embryonic exposure to endogenous substances within the egg. Taken together, these findings highlight the dynamic nature of the early endocrine environment in developing vertebrates.

In ovo inhibition of steroid metabolism by bisphenol-A as a potential mechanism of endocrine disruption.

During embryonic development, endogenous signals, for example steroid hormones, and exogenous signals, for example endocrine disrupting chemicals (EDCs), have the capacity to produce phenotypic effects that persist into adulthood. As the actions of steroids are mediated through the binding of steroid receptors, most studies of EDCs have assumed that they too elicit their effects by binding steroid receptors. We tested an alternative hypothesis, namely that EDCs elicit their effects during embryonic development by disrupting the metabolism of maternally derived steroids, thereby allowing maternally derived steroids to bind steroid receptors and elicit effects. Specifically, we examined the ability of the EDC, bisphenol-A (BPA) to inhibit the normal metabolism of oestradiol during the first nine days of embryonic development in the red-eared slider turtle (Trachemys scripta). We found that, when BPA was present, oestrogen metabolism was inhibited when compared to control eggs. In particular, the formation of oestrone sulfate was blocked in BPA-treated eggs. We postulate that the oestrogenic effects of EDCs may be driven, at least in part, by inappropriate oestrogen signalling. The retention of oestrogens at points of development when they would normally be metabolized to inactive forms might also help explain low-dose effects frequently reported for EDCs.

Humoral immune responses are maintained with age in a long-lived ectotherm, the red-eared slider turtle.

Aging is typically associated with a decrease in immune function. However, aging does not affect each branch of the immune system equally. Because of these varying effects of age on immune responses, aging could affect taxa differently based on how the particular taxon employs its resources towards different components of immune defense. An example of this is found in the humoral immune system. Specific responses tend to decrease with age while non-specific, natural antibody responses increase with age. Compared with mammals, reptiles of all ages have a slower and less robust humoral immune system. Therefore, they may invest more in non-specific responses and thus avoid the negative consequences of age on the immune system. We examined how the humoral immune system of reptiles is affected by aging and investigated the roles of non-specific, natural antibody responses and specific responses by examining several characteristics of antibodies against lipopolysaccharide (LPS) in the red-eared slider turtle. We found very little evidence of immunosenescence in the humoral immune system of the red-eared slider turtle, Trachemys scripta, which supports the idea that non-specific, natural antibody responses are an important line of defense in reptiles. Overall, this demonstrates that a taxon's immune strategy can influence how the immune system is affected by age.

Yolk-albumen testosterone in a lizard with temperature-dependent sex determination: relation with development.

The leopard gecko (Eublepharis macularius) exhibits temperature-dependent sex determination as well as temperature-influenced polymorphisms. Research suggests that in oviparous reptiles with temperature-dependent sex determination, steroid hormones in the yolk might influence sex determination and sexual differentiation. From captive leopard geckos that were all from the same incubation temperature regime, we gathered freshly laid eggs, incubated them at one of two female-biased incubation temperatures (26 or 34°C), and measured testosterone content in the yolk-albumen at early or late development. No differences in the concentration of testosterone were detected in eggs from different incubation temperatures. We report testosterone concentrations in the yolk-albumen were higher in eggs of late development than early development at 26°C incubation temperatures, a finding opposite that reported in other TSD reptiles studied to date.

Embryonic exposure to corticosterone modifies the juvenile stress response, oxidative stress and telomere length.

Early embryonic exposure to maternal glucocorticoids can broadly impact physiology and behaviour across phylogenetically diverse taxa. The transfer of maternal glucocorticoids to offspring may be an inevitable cost associated with poor environmental conditions, or serve as a maternal effect that alters offspring phenotype in preparation for a stressful environment. Regardless, maternal glucocorticoids are likely to have both costs and benefits that are paid and collected over different developmental time periods. We manipulated yolk corticosterone (cort) in domestic chickens (Gallus domesticus) to examine the potential impacts of embryonic exposure to maternal stress on the juvenile stress response and cellular ageing. Here, we report that juveniles exposed to experimentally increased cort in ovo had a protracted decline in cort during the recovery phase of the stress response. All birds, regardless of treatment group, shifted to oxidative stress during an acute stress response. In addition, embryonic exposure to cort resulted in higher levels of reactive oxygen metabolites and an over-representation of short telomeres compared with the control birds. In many species, individuals with higher levels of oxidative stress and shorter telomeres have the poorest survival prospects. Given this, long-term costs of glucocorticoid-induced phenotypes may include accelerated ageing and increased mortality.

No evidence that estrogens affect the development of the immune system in the red-eared slider turtle, Trachemys scripta.

Exposure to maternally derived substances during development can affect offspring phenotype. In ovo exposure to maternally derived steroids has been shown to influence traits such as growth and behavior in the offspring. The development of the immune system also can be altered by exposure to both androgens and glucocorticoids in a variety of species, but much less is known about the potential for estrogens to influence the development of this system. We examined the effect of estradiol on the development of both innate and adaptive immune components in the red-eared slider turtle (Trachemys scripta). A bacterial killing assay was used to assess innate immunity, a delayed-type hypersensitivity test for cellular immunity, and total immunoglobulin levels to measure the humoral immune response. We found no effect of in ovo estradiol treatment on any of our immune measures despite using doses that are known to influence other phenotypic parameters during development and varying the timing of dosing across development. Our results suggest that maternally derived estradiol does not affect the development of the immune system in T. scripta.