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When females experience stress during reproduction, developing embryos can be exposed to elevated levels of glucocorticoids, which can permanently affect offspring development, physiology, and behavior. However, the embryo can regulate exposure to glucocorticoids. In placental species, the placenta regulates embryonic exposure to maternal steroids via metabolism. In a comparable way, recent evidence has shown the extraembryonic membranes of avian species also regulate embryonic exposure to a number of maternal steroids deposited in the yolk via metabolism early in development. However, despite the known effects of embryonic exposure to glucocorticoids, it is not yet understood how glucocorticoids are metabolized early in development. To address this knowledge gap, we injected corticosterone into freshly laid chicken (Gallus gallus) eggs and identified corticosterone metabolites, located metabolomic enzyme transcript expression, tracked metabolomic enzyme transcript expression during the first six days of development, and determined the effect of corticosterone and metabolites on embryonic survival. We found that yolk corticosterone was metabolized before day four of development into two metabolites: 5β-corticosterone and 20β-corticosterone. The enzymes, AKR1D1 and CBR1 respectively, were expressed in the extraembryonic membranes. Expression was dynamic during early development, peaking on day two of development. Finally, we found that corticosterone exposure is lethal to the embryos, yet exposure to the metabolites is not, suggesting that metabolism protects the embryo. Ultimately, we show that the extraembryonic membranes of avian species actively regulate their endocrine environment very early in development., (Copyright © 2023 Elsevier Inc. All rights reserved.)

During times of maternal stress, developing embryos can be exposed to elevated levels of glucocorticoids, which can affect development and permanently alter offspring phenotype. In placental species, the placenta mediates fetal exposure to maternal glucocorticoids via metabolism, yet the placenta itself responds to glucocorticoids to regulate offspring growth and development. In oviparous species, maternal glucocorticoids can be deposited into the egg yolk and are metabolized early in development. This metabolism is mediated by the extraembryonic membranes, but it is unknown if the extraembryonic membranes also respond to maternal glucocorticoids in a way comparable to the placenta. In this study, we quantified the expression of acyl-CoA thioesterase 13 (Acot13) as an initial marker of the membrane's response to corticosterone in chicken (Gallus gallus) eggs. Acot13 regulates fatty acid processing in the embryo, to potentially regulate resource availability during development. We addressed the following questions using Acot13 expression: 1) Do the extraembryonic membranes respond to yolk corticosterone early in development? 2) Is the response to corticosterone dependent on the dose of corticosterone? 3) What is the duration of the response to corticosterone? 4) Does a metabolite of corticosterone (5β-corticosterone) elicit the same response as corticosterone? We found that corticosterone significantly induces the expression of Acot13 on day four of development and that expression of Acot13 increases with the dose of corticosterone. Further, we found expression of Acot13 is significantly elevated by corticosterone on days four and six of development compared to oil treated eggs, but not on days eight and ten. Although this response is transient, it occurs during a critical period of development and could initiate a cascade of events that ultimately alter offspring phenotype. Finally, we found that 5β-corticosterone does not increase the expression of Acot13, indicating that metabolism inactivates corticosterone. Ultimately, this study provides insight into the mechanisms underlying how maternally deposited glucocorticoids can affect embryonic development., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Predation or brood parasitism risks can change the behaviors and reproductive decisions in many parental animals. For oviparous species, mothers can mitigate their reproductive success in at least three ways: (1) by avoiding nest sites with high predation or parasitism risks, (2) through hormonal maternal effects that developmentally prime offspring for survival in risky environments, or (3) by investing less in reproduction when predation or parasitism risks are high. Here, we tested if perceived predation and parasitism risks can induce any of these behavioral or physiological responses by exposing female red-winged blackbirds (Agelaius phoeniceus) to playbacks of two major nest threats, a predator (Cooper's hawk, Accipiter cooperii) and an obligate brood parasite (brown-headed cowbird; Molothrus ater), as well as two controls (harmless Eastern meadowlark, Sturnella magna; and silence). We found that female blackbirds did not avoid nesting at sites treated with predator or brood parasite playbacks, nor were females more likely to abandon nesting attempts at these sites. Egg size and yolk hormone profiles, which are common proxies for maternal investment in oviparous species, were statistically similar across treatment sites. Instead, we found intraclutch variation in yolk steroid hormone profiles: concentrations of three progestogens (pregnanedione, 17α-hydroxypregnenolone, and deoxycorticosterone) and two androgens (testosterone and androstenedione) were higher in third-laid than first-laid eggs. Our study largely confirms previous findings of consistent intraclutch yolk hormone variation in this species, in birds in general, and in other oviparous lineages, but uniquely reports on several yolk steroid hormones largely overlooked in the literature on hormone-mediated maternal effects., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Maternal stress during reproduction can influence how offspring respond to stress later in life. Greater lifetime exposure to glucocorticoid hormones released during stress is linked to greater risks of behavioral disorders, disease susceptibility, and mortality. The immense variation in individual's stress responses is explained, in part, by prenatal glucocorticoid exposure. To explore the long-term effects of embryonic glucocorticoid exposure, we injected Japanese quail (Coturnix japonica) eggs with corticosterone. We characterized the endocrine stress response in offspring and measured experienced aggression at three different ages. We found that prenatal glucocorticoid exposure affected (1) the speed at which the stress response was terminated suggesting dysregulated negative feedback, (2) baseline corticosterone levels in a manner dependent on current environmental conditions with higher levels of experienced aggression associated with higher levels of baseline corticosterone, (3) the magnitude of an acute stress response based on baseline concentrations. We finish by proposing a framework that can be used to test these findings in future work. Overall, our findings suggest that the potential adaptive nature of prenatal glucocorticoid exposure is likely dependent on environmental context and may also be tempered by the negative effects of longer exposure to glucocorticoids each time an animal faces a stressor., (© 2023. The Author(s).)

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., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

There is ample research demonstrating that temperature can have complex effects on biological processes, including the timing of when organisms respond to temperature; some responses occur rapidly while others require an extended exposure time. However, most of what we know about temperature effects comes from studies using constant temperature conditions, which are not reflective of natural, fluctuating temperatures. Species with temperature-dependent sex determination (TSD) present an ideal system to study the temporal aspects of the temperature response because prior research has established a number of temperature-responsive genes involved in TSD, albeit under constant temperatures. To investigate potential differences in timing of sexual development between constant and fluctuating incubation temperatures, we exposed Trachemys scripta embryos to two conditions that produce males (constant 26°C and 26 ± 3°C) and two that produce females (constant 31°C and 31 ± 3°C), and sampled embryonic gonads for gene expression analysisvia qPCR. We analyzed three genes involved in testis differentiation (Kdm6b,Dmrt1, andSox9) and two genes involved in ovary differentiation (Foxl2andCyp19A1). Results show that Kdm6b expression was significantly lower under fluctuating temperatures compared to constant temperatures. Foxl2 and Cyp19A1 expression were also lower under fluctuating temperatures, but not at all stages of development. These results suggest that constant temperatures caused increases in both Foxl2 and Cyp19A1 expression earlier (developmental stage 20) than fluctuating temperatures (stages 22 and 23). Dmrt1 and Sox9 expression did not differ between constant and fluctuating temperatures. These results highlight that not all genes in a temperature-dependent process respond to temperature in the same manner. Whether there are functional consequences of this variation remains to be determined., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology.)

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., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Exposure to maternally derived steroids during embryonic development can elicit phenotypic effects in the resulting offspring. Studies of maternal steroid effects, especially rich in mammals and birds, have offered exciting insights into the evolution of maternal effects in vertebrates. To extend this literature, we quantified levels of steroids in the eggs of four neotropical dendrobatid frogs that lay terrestrial clutches, a reproductive strategy that has evolved multiple times in amphibians. Building on our observational results, we then manipulated levels of pregnenolone and progesterone in eggs of one species and examined how this affected steroid levels during development. Eggs of all four species had detectable steroids levels, with progestogens being more abundant than androgens and glucocorticoids. Estrogens could not be detected. Immersion of frog eggs in a solution containing pregnenolone and progesterone resulted in elevated levels of both steroids early in development, but levels declined and were similar to those in unmanipulated eggs by the end of development. Treated eggs also exhibited a transient increase in levels of steroids that can be produced from pregnenolone and progesterone. Overall, our findings demonstrate that frog eggs contain steroids similar to what has been observed in other egg-laying vertebrates. During development, steroid levels are dynamic, further suggesting developing embryos regulate exposure to maternal steroids. These results set the stage for investigating the causes and consequences of maternal steroid effects in frogs., (© 2021 Wiley Periodicals LLC.)

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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Maternal hormones can shape offspring development and increase survival when predation risk is elevated. In songbirds, yolk androgens influence offspring growth and begging behaviors, which can help mitigate offspring predation risk in the nest. Other steroids may also be important for responding to nest predation risk, but non-androgen steroids have been poorly studied. We used a nest predator playback experiment and liquid chromatography with tandem mass spectrometry (LC-MS-MS) to assess whether nest predation risk influences deposition of 10 yolk steroids. We found no clear evidence that yolk androgen deposition changed when perception of nest predation risk was experimentally increased. However, elevated nest predation risk led to decreased yolk progesterone deposition. Overall, our results suggest yolk progesterone may be more important than yolk androgens in responses to offspring predation risk and highlight new avenues for research., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

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.

In species with temperature-dependent sex determination (TSD), incubation temperatures regulate the expression of genes involved in gonadal differentiation and determine whether the gonads develop into ovaries or testes. For most species, natural incubation conditions result in transient exposure to thermal cues for both ovarian and testis development, but how individuals respond to this transient exposure varies and can drive variation in the resulting sex ratios. Here, we argue that variation in the timing to respond to temperature cues, or thermal responsiveness, is a trait needing further study. Recent work in the red-eared slider turtle (Trachemys scripta) has found that when embryos experience transient exposure to warm conditions (i.e., heatwaves), some embryos show high responsiveness, requiring only short exposures to commit to ovarian development, while others show low responsiveness, developing testes even after more extended exposures to warm conditions. We discuss how maternal estrogens might influence thermal responsiveness for organisms that develop under thermal fluctuations. Examining the interplay of molecular responses to more subtle thermal and endocrine environments may reveal significant insights into the process of sex determination in species with TSD., (© 2021 S. Karger AG, Basel.)

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.

Hosts of avian brood parasites may reduce or forego the costs of caring for foreign young by rejecting parasitic eggs from the nest. Yet, many host species accept parasitic eggs and, even among rejecter species, some individuals go on to incubate and hatch them. The factors explaining the variation in egg rejection between species have received much theoretical and empirical attention, but the causes of intraspecific variation in different individuals' propensity for accepting parasitic eggs are less well understood. Here we tested the maternal investment hypothesis, which predicts that hosts with costlier clutches will be more likely to reject parasitic eggs from their nest. We studied variation in the egg rejection responses of American robins ( Turdus migratorius ), a robust egg-rejecter host of the brood parasitic brown-headed cowbird ( Molothrus ater ), to 3D-printed cowbird-sized eggs which were painted dark blue, a color known to induce variable and repeatable egg rejection responses in individual robins. Costlier clutch investment was estimated by earlier laying date, larger clutch size, heavier unincubated yolk mass, and variable yolk steroid hormone concentrations. There was no statistical support for most of our predictions. However, we detected more concentrated and greater overall amount of deoxycorticosterone deposited in egg yolks of rejecters relative to acceptors, although this accounted for no more than 14% of variance in the data. Future work should test experimentally the potential physiological linkage between maternal egg yolk steroid investment and egg rejection propensity in this and other host species of avian brood parasites., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology.)

Predation often has consistent effects on prey behavior and morphology, but whether the physiological mechanisms underlying these effects show similarly consistent patterns across different populations remains an open question. In vertebrates, predation risk activates the hypothalamic-pituitary-adrenal (HPA) axis, and there is growing evidence that activation of the maternal HPA axis can have intergenerational consequences via, for example, maternally-derived steroids in eggs. Here, we investigated how predation risk affects a suite of maternally-derived steroids in threespine stickleback eggs across nine Alaskan lakes that vary in whether predatory trout are absent, native, or have been stocked within the last 25 years. Using liquid chromatography coupled with mass spectroscopy (LC-MS/MS), we detected 20 steroids within unfertilized eggs. Factor analysis suggests that steroids covary within and across steroid classes (i.e. glucocorticoids, progestogens, sex steroids), emphasizing the modularity and interconnectedness of the endocrine response. Surprisingly, egg steroid profiles were not significantly associated with predator regime, although they were more variable when predators were absent compared to when predators were present, with either native or stocked trout. Despite being the most abundant steroid, cortisol was not consistently associated with predation regime. Thus, while predators can affect steroids in adults, including mothers, the link between maternal stress and embryonic development is more complex than a simple one-to-one relationship between the population-level predation risk experienced by mothers and the steroids mothers transfer to their 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., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

The steroid 17β-estradiol (herein "estradiol") is a potent regulator of sexual differentiation that exerts wide-ranging effects on the developing brain and other tissues. The developing gonads are an important source of estradiol but most, if not all, vertebrate embryos are also exposed to maternally derived estradiol during development. In birds, this maternally derived estradiol is present in the egg at the time of oviposition but very little is known about how this source of estradiol influences development. A critical aspect of understanding yolk estradiol effects is deciphering how steroid metabolism may regulate embryonic exposure to yolk estradiol. In this study, we examine the metabolic fate of estradiol during the first five days of incubation in chicken (Gallus gallus) eggs. Using tritiated estradiol to trace the movement and metabolism of estradiol, we demonstrate that estradiol is metabolized to estrone, which is subsequently conjugated to estrone sulfate as the primary metabolite. Estrone sulfate then accumulates in the albumen by day five of incubation. Overall, these findings have important implications for how yolk estradiol may influence development and alter offspring phenotype. Mechanisms through which estradiol, as well as estrone sulfate, might elicit effects are discussed., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Acute rises in glucocorticoid hormones allow individuals to adaptively respond to environmental challenges but may also have negative consequences, including oxidative stress. While the effects of chronic glucocorticoid exposure on oxidative stress have been well characterized, those of acute stress or glucocorticoid exposure have mostly been overlooked. We examined the relationship between acute stress exposure, glucocorticoids and oxidative stress in Japanese quail ( Coturnix japonica ). We (i) characterized the pattern of oxidative stress during an acute stressor in two phenotypically distinct breeds; (ii) determined whether corticosterone ingestion, in the absence of acute stress, increased oxidative stress, which we call glucocorticoid-induced oxidative stress (GiOS); and (iii) explored how prior experience to stressful events affected GiOS. Both breeds exhibited an increase in oxidative stress in response to an acute stressor. Importantly, in the absence of acute stress, ingesting corticosterone caused an acute rise in plasma corticosterone and oxidative stress. Lastly, birds exposed to no previous acute stress or numerous stressful events had high levels of GiOS in response to acute stress, while birds with moderate prior exposure did not. Together, these findings suggest that an acute stress response results in GiOS, but prior experience to stressors may modulate that oxidative cost.

Correlated and repeatable patterns of behavior, termed behavioral types, can affect individual fitness. The most advantageous behavioral type may differ across predictable environments (e.g., seasonally), and maternally mediated effects may match hatchling behavior to the environment. We measured righting response, an indicator of behavioral type, of juvenile red-eared slider turtles (Trachemys scripta) emerging from early and late season clutches to understand if the production of behavioral types differs across the nesting season. There was a significant effect of season, with early season hatchlings righting more quickly than late season hatchlings, and we explored two potential underlying mechanisms, maternal estrogens and maternal investment (e.g., yolk allocation). We dosed early season eggs with an estrogen mixture to mimic late season eggs and assayed hatchling righting response, but found no significant effect of this maternal effect. We assessed maternal investment by measuring egg, hatchling, and residual yolk masses. We found a seasonal pattern in yolk allocation, where early season eggs have more yolk than late season eggs. Early season hatchlings used more yolk for growth rather than maintenance of existing tissues, resulting in larger hatchlings. Interestingly, across both seasons, hatchlings that received less maternal yolk appeared to be more efficient at converting yolk to tissue, but we found no direct correlation with righting behavior. We demonstrate that the prevalence of behavioral types varies across the nesting season, creating correlated suites of seasonal phenotypes in turtle hatchlings, but it appears that neither maternal estrogens or investment in yolk directly underlie this shift in behavior., (© 2019 Wiley Periodicals, Inc.)

Progesterone has received substantial attention for the essential role it plays in establishing and maintaining pregnancy in placental vertebrates. Despite the prevalence of progesterone during development, relatively little is known about how embryos respond to progesterone. This is true of placental vertebrates as well as egg-laying vertebrates where levels of progesterone in the yolk tend to be higher than most other steroids in the yolk. Bird eggs provide an opportunity to investigate the effects of progesterone on embryonic development because progesterone can be easily manipulated without any confounding effects on maternal physiology. To understand how progesterone might influence embryonic development, it is important to characterize the metabolic fate of progesterone given its potential to be converted to a wide range of steroids. We investigated the metabolic fate of tritiated progesterone over the first four days of development using chicken eggs (Gallus gallus) and identified 5β-pregnanedione as the primary metabolite during this period. After only one day of development, 5β-pregnanedione could be detected within the yolk. Levels of 5β-pregnanedione in both the yolk and albumen tended to rise early in development but conjugated metabolites began to accumulate towards the end of our sampling period. Additionally, in vitro assays using embryo homogenates collected after 72 h of development demonstrated that embryos were capable of carrying out the conversion of progesterone to 5β-pregnanedione. Overall these results have important implications for deciphering the mechanisms through which yolk progesterone might influence embryonic development. Effects could arise via progesterone receptors or receptors capable of binding 5β-pregnanedione but we found no evidence that progesterone is serving as a precursor for androgen or estrogen production., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Steroids play an integral role in orchestrating embryonic development, and they can affect a suite of phenotypic traits, including learning and behavior. Endocrine disrupting compounds (EDCs) can alter steroid-dependent phenotypic traits during embryonic development. Bisphenol-A (BPA) is an EDC that disrupts the action of estrogen, and recent work indicates that BPA can affect learning and behavior similarly to estrogen. We exposed red-eared slider turtle (Trachemys scripta) eggs to BPA during embryonic development and tested hatchlings for effects on learning and behavior in modified T-mazes over the course of two weeks. We found that behavioral patterns changed within a day and over the course of the experiment, but we found no effect of BPA treatment. Further, we found that hatchling turtles were highly consistent in their behaviors. These behaviors varied among individuals, suggesting that there are discrete behavioral types in T. scripta hatchlings. The highly repetitive nature of behaviors in the hatchlings might explain the innate biases that we observed and warrants further study., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved. For permissions please email: journals.permissions@oup.com.)

Vertebrates release glucocorticoids during stressful events. If stress occurs during reproduction, the resulting offspring can show altered phenotypes that are thought to arise from increased exposure to maternal glucocorticoids. Developing offspring can metabolize maternal glucocorticoids, which can alter the pattern of exposure they encounter. For egg laying vertebrates, we are just beginning to understand how embryonic steroid metabolism impacts embryonic exposure to maternal glucocorticoids. Here we injected three doses of radioactive corticosterone into Japanese quail (Coturnix japonica) eggs to determine the degree of embryonic exposure at days six and nine of development. We found that increasing injection dose increased the amount of radioactivity found in the embryo at day six but by day nine the effect of injection dose disappeared as the amount of radioactivity within the embryo dropped to equivalent levels for all three doses. Interestingly, when examined as a percentage of initial dose, there were no differences between treatment groups at any time points. Importantly, using thin-layer chromatography we characterized that some free steroid, putatively identified as corticosterone, does reach the developing embryo. Together, our data suggest that the in ovo metabolism of maternal corticosterone can eventually eliminate it from the egg, but before this happens, embryos developing in eggs with elevated amounts of maternal corticosterone are exposed to higher levels early in development. This has important implications for how we understand the developmental steroid environment and the mechanisms underlying maternal stress effects., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Vertebrates with temperature-dependent sex determination (TSD) have justifiably received a lot of attention when it comes to the potential effects of climate change. Freshwater turtles have long been used to characterize the physiological and genetic mechanisms underlying TSD and provide a great system to investigate how changing climatic conditions will affect vertebrates with TSD. Unfortunately, most of what we know about the mechanisms underlying TSD comes from laboratory conditions that do not accurately mimic natural conditions (i.e., constant incubation temperatures and supraphysiological steroid manipulations). In this paper, we review recent advances in our understanding of how TSD operates in nature that arose from studies using more natural fluctuating incubation temperatures and natural variation in maternal estrogens within the yolk. By incorporating more natural conditions into laboratory studies, we are better able to use these studies to predict how changing climatic conditions will affect species with TSD., (© 2018 Wiley Periodicals, Inc.)

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.

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., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

Brown-headed cowbirds (Molothrus ater) deposit their eggs into the nests of other birds, which then raise the cowbird chick. Female cowbirds thus have limited options for impacting their offspring's development via maternal effects compared to most other passerines. Cowbirds can impact their offspring's phenotype by choosing among potential host nests, and by adjusting egg resources based on host characteristics. To examine whether cowbirds exhibit either or both of these strategies, we investigated rates of cowbird parasitism and egg investment (egg size, yolk-to-albumen ratio, and yolk testosterone and androstenedione) among and within host species in a shrubland bird community. We found that the probability of being parasitized by cowbirds, controlling for host status as a cowbird egg accepter or rejecter and ordinal date, varied significantly among host species, indicating an apparent preference for some hosts. Parasitism rates did not differ with host size, however, and despite variation in cowbird egg size among host species, this variation was not related to host size or cowbird preference. Among host species with eggs that are larger than those of the cowbird, cowbirds were significantly more likely to parasitize nests with relatively smaller eggs, whereas parasitism rates did not vary with relative egg size in host species with smaller eggs. There was no evidence for variation in cowbird egg components among or within host species. Our data indicate that cowbirds discriminate among host nests, but do not appear to adjust the composition of their eggs based on inter- or intraspecific host variation.

Sex-specific maternal effects can be adaptive sources of phenotypic plasticity. Reptiles with temperature-dependent sex determination (TSD) are a powerful system to investigate such maternal effects because offspring phenotype, including sex, can be sensitive to maternal influences such as oestrogens and incubation temperatures.In red-eared slider turtles ( Trachemys scripta ), concentrations of maternally derived oestrogens and incubation temperatures increase across the nesting season; we wanted to determine if sex ratios shift in a seasonally concordant manner, creating the potential for sex-specific maternal effects, and to define the sex ratio reaction norms under fluctuating temperatures across the nesting season.Eggs from early and late season clutches were incubated under a range of thermally fluctuating temperatures, maternally derived oestradiol concentrations were quantified via radioimmunoassay, and hatchling sex was identified. We found that late season eggs had higher maternal oestrogen concentrations and were more likely to produce female hatchlings. The sex ratio reaction norm curves systematically varied with season, such that with even a slight increase in temperature (0.5°C), late season eggs produced up to 49% more females than early season eggs.We found a seasonal shift in sex ratios which creates the potential for sex-specific phenotypic matches across the nesting season driven by maternal effects. We also describe, for the first time, systematic variation in the sex ratio reaction norm curve within a single population in a species with TSD.

Offspring from females that experience stressful conditions during reproduction often exhibit altered phenotypes and many of these effects are thought to arise owing to increased exposure to maternal glucocorticoids. While embryos of placental vertebrates are known to regulate exposure to maternal glucocorticoids via placental steroid metabolism, much less is known about how and whether egg-laying vertebrates can control their steroid environment during embryonic development. We tested the hypothesis that threespine stickleback (Gasterosteus aculeatus) embryos can regulate exposure to maternal steroids via active efflux of maternal steroids from the egg. Embryos rapidly (within 72 h) cleared intact steroids, but blocking ATP-binding cassette (ABC) transporters inhibited cortisol clearance. Remarkably, this efflux of cortisol was sufficient to prevent a transcriptional response of embryos to exogenous cortisol. Taken together, these findings suggest that, much like their placental counterparts, developing fish embryos can actively regulate their exposure to maternal cortisol. These findings highlight the fact that even in egg-laying vertebrates, the realized exposure to maternal steroids is mediated by both maternal and embryonic processes and this has important implications for understanding how maternal stress influences offspring development., (© 2016 The Author(s).)

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., (Copyright © 2015 Elsevier Inc. All rights reserved.)

How and when turtles first acquire gut microflora is largely speculative. In this study, the eggshell and hatching process were evaluated for their role in the initial acquisition of Salmonella, by red-eared slider turtles (Trachemys scripta elegans). First, we examined whether the eggshell is a viable substrate for bacterial persistence during incubation, and if internal egg components (i.e., albumen, yolk, and embryo) have detectable bacterial loads. Second, we experimentally manipulated Salmonella by treating eggs with combinations of Salmonella and gentamicin, an effective Gram-negative antibiotic. We found that the eggshell is a viable substrate for maintaining bacteria, as well as an effective barrier to Salmonella transmission as internal egg components were largely bacteria-free. Water samples collected 18 days post-hatch from individuals that were experimentally inoculated with a topical application of Salmonella as eggs had a higher prevalence of Salmonella than those from eggs inoculated with Salmonella but topically treated with gentamicin prior to hatching, control eggs, and eggs only treated with gentamicin, but by day 35 post-hatch there were no detectable differences among the treatment groups. Though it can also act as a barrier that prevents the bacteria from infecting the embryo prior to hatching these findings suggest that the eggshell is a likely source of Salmonella infection in turtle hatchlings., (© 2016 Wiley Periodicals, Inc.)

Embryonic exposure to steroids often leads to long-term phenotypic effects. It has been hypothesized that mothers may be able to create a steroid environment that adjusts the phenotypes of offspring to current environmental conditions. Complicating this hypothesis is the potential for developing embryos to modulate their early endocrine environment. This study utilized the threespined stickleback (Gasterosteus aculeatus) to characterize the early endocrine environment within eggs by measuring four steroids (progesterone, testosterone, estradiol, and cortisol) of maternal origin. We then examined how the concentrations of these four steroids changed over the first 12 days post fertilization (dpf). Progesterone, testosterone, estradiol, and cortisol of maternal origin could be detected within unfertilized eggs and levels of all four steroids declined in the first 3 days following fertilization. While levels of progesterone, testosterone, and estradiol remained low after the initial decline, levels of cortisol rose again by 8 dpf. These results demonstrate that G. aculeatus embryos begin development in the presence of a number of maternal steroids but levels begin to change quickly following fertilization. This suggests that embryonic processes change the early endocrine environment and hence influence the ability of maternal steroids to affect development. With these findings, G. aculeatus becomes an intriguing system in which to study how selection may act on both maternal and embryonic processes to shape the evolutionary consequence of steroid-mediated maternal effects., (© 2015 Wiley Periodicals, Inc.)

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., (© 2015 The Author(s) Published by the Royal Society. All rights reserved.)

Maternal effects have gained attention as a method by which mothers may alter the physiological condition and phenotype of their offspring based upon current environmental conditions. The physiological and phenotypic outcomes of glucocorticoid-mediated maternal effects have been extensively studied in a variety of vertebrates; however, the underlying mechanism is currently unclear. Here, we injected tritiated corticosterone into the yolks of freshly laid Japanese quail eggs (Coturnix japonica) and traced its movement and metabolism through the in ovo development period. We found that corticosterone was extensively conjugated throughout the egg by the end of development, and while minimal corticosterone was detected within the embryo during development, accumulation of a conjugated metabolite in the embryo started to occur on day 6 of development. Because no movement and metabolism of corticosterone occurred in infertile eggs, our findings suggest that embryos are not passive recipients of maternal steroids, but instead appear to possess extensive metabolic capabilities, which may modulate their exposure to maternal steroids., (© 2014 The Author(s) Published by the Royal Society. All rights reserved.)

Variable environmental conditions can alter the phenotype of offspring, particularly in ectothermic species such as reptiles. Despite this, the majority of studies on development in reptiles have been carried out under constant conditions in the laboratory, raising the question of just how applicable those investigations are to natural conditions? Here, we first review what we have learned from these constant-temperature studies. Second, we examine the importance of temperature fluctuations for development in reptiles and highlight the outcomes of studies conducted under fluctuating conditions. Next, we report our findings from a new study that examines how the frequency of fluctuations in temperature experienced during development affects phenotype. Finally, we suggest some areas in need of additional research so that we can better understand the complex interactions of temperature and physiology, particularly in species with temperature-dependent sex determination. For questions aimed at understanding the complex effects of the environment on phenotype, we must move toward studies that better capture environmental variation. By taking such an approach, it may be possible to predict more accurately how these thermally sensitive organisms will respond to environmental perturbations, including climatic change., (© The Author 2014. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved. For permissions please email: journals.permissions@oup.com.)

All vertebrate embryos develop in the presence of maternally derived steroids, and maternal steroids have been hypothesized to link phenotype of the offspring to maternal physiology. In placental vertebrates, it is known that maternally derived steroids are metabolized during development via the sulfonation pathway. We used eggs from the red-eared slider turtle (Trachemys scripta) to determine whether the same metabolic pathway is used to metabolize maternally derived steroids in an oviparous vertebrate. To examine the relationship between estradiol and estrogen sulfates during development, levels of maternally derived estradiol were compared with levels of estradiol sulfate, estrone sulfate, and estriol sulfate at oviposition and after 20 days of embryonic development. Estrone sulfate was the only detectable estrogen sulfate. At oviposition, levels of both estradiol and estrone sulfate varied seasonally with clutches from later in the nesting season having significantly higher concentrations of both steroids. Levels of estrone sulfate increased during development, demonstrating that the sulfonation of maternally derived steroids occurs in oviparous vertebrates as well as in placental vertebrates. We also found that exogenous estrone sulfate increases the production of female hatchlings, thereby demonstrating the ability of this metabolite to influence embryonic development. To examine the role of sulfonation in the metabolism of maternal progesterone and testosterone, we characterized the metabolic fate of both steroids by applying tritiated forms of each steroid at oviposition and characterizing metabolites after 20 days of incubation. Similar to what was demonstrated for estradiol, both progesterone and testosterone are converted to sulfonated metabolites during embryonic development. These data suggest that steroid sulfates, both those that are maternally derived and those resulting from the metabolism of maternal steroids, are a key component of the mechanism underlying steroid-mediated maternal effects.

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.

Background: We describe the genome of the western painted turtle, Chrysemys picta bellii, one of the most widespread, abundant, and well-studied turtles. We place the genome into a comparative evolutionary context, and focus on genomic features associated with tooth loss, immune function, longevity, sex differentiation and determination, and the species' physiological capacities to withstand extreme anoxia and tissue freezing., Results: Our phylogenetic analyses confirm that turtles are the sister group to living archosaurs, and demonstrate an extraordinarily slow rate of sequence evolution in the painted turtle. The ability of the painted turtle to withstand complete anoxia and partial freezing appears to be associated with common vertebrate gene networks, and we identify candidate genes for future functional analyses. Tooth loss shares a common pattern of pseudogenization and degradation of tooth-specific genes with birds, although the rate of accumulation of mutations is much slower in the painted turtle. Genes associated with sex differentiation generally reflect phylogeny rather than convergence in sex determination functionality. Among gene families that demonstrate exceptional expansions or show signatures of strong natural selection, immune function and musculoskeletal patterning genes are consistently over-represented., Conclusions: Our comparative genomic analyses indicate that common vertebrate regulatory networks, some of which have analogs in human diseases, are often involved in the western painted turtle's extraordinary physiological capacities. As these regulatory pathways are analyzed at the functional level, the painted turtle may offer important insights into the management of a number of human health disorders.

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.

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., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Oviparous amniotes, particularly birds, have become model systems in which to study how mothers may utilize steroids to adaptively adjust offspring development. Although there is now ample evidence that maternally derived steroids in the egg at oviposition can influence offspring phenotype, very little is known about how these steroids elicit such effects. Of the major avian steroid hormones found in yolk, progesterone is by far the most abundant at oviposition, but has received little research attention to date. In this study, we examine the metabolism of [(3)H]-progesterone injected into freshly laid European starling eggs throughout the first 5 days of development by characterization of radioactivity within the egg homogenate. We also introduce a technique that utilizes a focal, freeze/thaw cycle to prevent embryonic development and allows us to assess the role of the embryo in metabolizing progesterone during early incubation. Two major findings result. First is that [(3)H]-progesterone is metabolized in eggs possessing a developing embryo, but not in eggs with disrupted embryonic development. Second is that the change in the distribution of radioactivity within eggs possessing an embryo is the result of metabolism of [(3)H]-progesterone to a more polar form that is subsequently conjugated. Together, these data suggest live embryos are necessary for metabolism of progesterone during early incubation, underscoring the potentially important contribution of embryos to functional modulation or mediation of maternal yolk steroid effects., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Eggs of oviparous amniotes can contain substantial quantities of several steroids at the time of oviposition. These maternally derived steroids appear to affect the phenotype of developing offspring, but not all steroid sensitive traits are affected by maternal steroids, and little is known about how these effects may arise. In this study, we applied tritiated estradiol to the eggs of red-eared sliders (Trachemys scripta) at the time of oviposition and characterized the subsequent metabolism and movement throughout embryonic development. Results indicate that very early in development, estradiol is converted to a variety of water-soluble estrogen sulfates that reside in the yolk and extraembryonic fluids until late in development. Within the final stages of development, we observe a significant decline in the total amount of metabolites present in the yolk and extraembryonic fluids and a significant increase in the amount of metabolites present in the embryo. While estradiol metabolism occurs during the early stages of development, the later stages appear to be the most dynamic with regards to the movement of estradiol metabolites. Our findings have important implications for studies investigating the effect of maternally derived steroids on offspring development., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Understanding the many factors that underlie phenotypic variation is of profound importance to evolutionary biologists. The embryonic endocrine environment is one such factor that has received much attention. In placental amniotes, the dynamic interaction of maternal and embryonic steroid production and metabolism is critical to regulating the endocrine environment. Less is known about how embryos of oviparous amniotes regulate their endocrine environment because most studies have focused on relating initial steroid levels in the yolk at oviposition to offspring phenotype. We tested the hypothesis that embryos of oviparous amniotes regulate their endocrine environment by conjugating maternal steroids and subsequently using the metabolites as precursors for steroid production later in development. Using the red-eared slider turtle (Trachemys scripta), we first characterized the conjugation of exogenous oestradiol to either oestradiol glucuronide or oestradiol sulphate (E(2)-S) in ovo during the first 15 days of development. Results show that oestradiol is primarily conjugated to E(2)-S. We then examined whether E(2)-S influenced sex determination and report that E(2)-S increases the production of female offspring. These data demonstrate that oviparous amniotes can both sulphonate steroids and respond to sulphonated steroids during embryonic development in a manner similar to placental amniotes.

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.

In birds, maternally derived yolk steroids are a proposed mechanism by which females can adjust individual offspring phenotype to prevailing conditions. However, when interests of mother and offspring differ, parent-offspring conflict will arise and embryonic interests, not those of the mother, should drive offspring response to maternal steroids in eggs. Because of this potential conflict, we investigated the ability of developing bird embryos to process maternally derived yolk steroids. We examined how progesterone, testosterone and oestradiol levels changed in both the yolk/albumen (YA) and the embryo of European starling eggs during the first 10 days of development. Next, we injected tritiated testosterone into eggs at oviposition to characterize potential metabolic pathways during development. Ether extractions separated organic and aqueous metabolites in both the embryo and YA homogenate, after which major steroid metabolites were identified. Results indicate that the concentrations of all three steroids declined during development in the YA homogenate. Exogenous testosterone was primarily metabolized to an aqueous form of etiocholanolone that remained in the YA. These results clearly demonstrate that embryos can modulate their local steroid environment, setting up the potential for parent-offspring conflict. Embryonic regulation must be considered when addressing the evolutionary consequences of maternal steroids in eggs.

The primary function of the immune system is to protect the organism from invading pathogens. In vertebrates, this has resulted in a multifaceted system comprised of both innate and adaptive components. The immune system of all jawed vertebrates is complex, but unlike the endothermic vertebrates, relatively little is known about the functioning of the ectothermic vertebrate immune system, especially the reptilian system. Because turtles are long-lived ectotherms, factors such as temperature and age may affect their immune response, but comprehensive studies are lacking. We investigated variation in immune responses of adult male and female red-eared sliders (Trachemys scripta) across the entire active season. We characterized seasonal variation in innate, cell-mediated and humoral components via bactericidal capacity of plasma, delayed-type hypersensitivity and total immunoglobulin levels, respectively. Results indicate that all immune measures varied significantly across the active season, but each measure had a different pattern of variation. Interestingly, temperature alone does not explain the observed seasonal variation. Immune measures did not vary between males and females, but immunoglobulin levels did vary with age. This study demonstrates the highly dynamic nature of the reptilian immune system, and provides information on how biotic and abiotic factors influence the immune system of a long-lived ectotherm.