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.

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.

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.

Effects of Constant and Fluctuating Temperatures on Gene Expression During Gonadal Development.

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.

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.

Inpatient admissions and outpatient appointments in the first year post cancer diagnosis: A population based study from England.

BACKGROUND: Time spent in hospital (length of stay) is an important component of patient experience and the financial cost of cancer care. This study documents the length of stay across English cancer diagnoses at a national level and reports on variation by patient demographics and tumour characteristics. METHODS: Data on all diagnoses of malignant neoplasms from the English National Cancer Registration and Analysis Service for 252,202 patients first diagnosed in 2015 was linked with NHS Digital's Admitted Patient Care and Outpatient Hospital Episode Statistics datasets to quantify length of stay within one year following diagnosis. Length of stay was modelled using linear regression adjusted for sex, age, tumour type, stage, time spent alive during the study period, vital status at end of study period, region, deprivation and ethnicity. RESULTS: Patients spend a mean of 25 days (median = 17 days; IQR = 8-34 days) in hospital in their first year. Tumour type, stage, age and vital status corrections had the strongest effects in the model adjusting for other independent variables. Younger patients tended towards longer stays. CONCLUSION: Length of stay varies among patients by tumour type, age and stage. Estimating future health service demands should account for changes in incident tumour characteristics.

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.

Is Thermal Responsiveness Affected by Maternal Estrogens in Species with Temperature-Dependent Sex Determination?

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.

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.

The World Is Not Flat: Accounting for the Dynamic Nature of the Environment as We Move Beyond Static Experimental Manipulations.

Although we have long understood that environmental variation affects both physiology and behavior, historically, most studies have limited or simplified environmental variation to focus more directly on traits of interest. Recently, a number of investigators have turned their focus toward attempting to incorporate such variation into studies of physiology and behavior, and not surprisingly, are finding that the results from studies that include more realistic variation, both from the environment as well as in physiological processes within individuals, can differ substantially from those of studies that attempt to hold the parameters constant. Understanding the role that this dynamic variation plays in shaping phenotypes is critical given that, under most predictions from future climate change models, increased variability in factors such as temperature and rainfall are predicted.

Posthatching Parental Care and Offspring Growth Vary with Maternal Corticosterone Level in a Wild Bird Population.

Corticosterone is the primary metabolic steroid in birds and is vital for maintaining homeostasis. However, the relationship between baseline corticosterone and reproduction is unclear, and we lack an understanding of how differences in baseline corticosterone at one stage of the breeding cycle influence reproductive effort at later stages. In a wild population of house wrens, we quantified the concentration of corticosterone in yolks of freshly laid eggs as an integrated measure of maternal physiology and related this to a behavioral measure of stress reactivity made during the nestling period, namely, the latency with which females resumed parental activities following a standardized disturbance at their nest (setting up a camera to record provisioning). Females that recently produced eggs containing higher corticosterone concentrations, which were significantly repeatable within females, took longer to resume activity related to parental care (i.e., feeding and brooding young) following the disturbance. Moreover, a female's latency to resume parental activities negatively predicted her provisioning of nestlings with food and the condition of these young at fledging but did not predict the number fledged. We cross-fostered offspring prior to hatching so these effects on maternal behavior are independent of any prenatal maternal effects on nestlings via the egg. These results are consistent with earlier findings, suggesting that females with higher baseline corticosterone during egg laying or early incubation tend to prioritize self-maintenance over reproduction compared with females with lower baseline corticosterone and suggest that a female's latency to return to her nest and resume parental care following a disturbance might represent a simple, functional measure of maternal stress reactivity.

Red-eared slider hatchlings (Trachemys scripta) show a seasonal shift in behavioral types.

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.

Learning and behavior in hatchling Trachemys scripta exposed to bisphenol-a during embryonic development.

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.

Intestinal B cells in the red-eared slider turtle, Trachemys scripta: Anatomical distribution and implications for ecological interactions with pathogenic microbes.

Disease is a significant threat in the global decline of reptile species. Many aquatic reptiles live in habitats with high levels of opportunistic microbial pathogens, yet little is known about their immune system. Gut-associated lymphoid tissue is vital for protection against ingested pathogens and maintenance of normal gut microbiota. In mammals, gut mucosal immunity is well-characterized and mucosal surfaces are coated in protective antibodies. However, reptiles lack lymph nodes and Peyer's patches, which are the major sites of mammalian B cell responses. The presence or distribution of mucosal B cells in reptiles is unknown. In this study, we first set out to determine if B cells could be detected in intestinal tissues of red-eared slider turtles, Trachemys scripta. Using whole-mount immunochemistry and a primary antibody to turtle antibody light chains, we identified widely distributed B cell aggregates within the small intestine of hatchling turtles. These aggregates appeared similar to isolated lymphoid follicles (ILFs) in mammals and the frequency was much higher in distal intestinal sections than in proximal sections. To determine if these structures were inducible in the presence of microbes, we introduced an enteric Salmonella species through oral gavage. Analysis of intestinal tissues revealed that hatchlings exposed to Salmonella exhibited significantly more of these aggregates when compared with those that did not receive bacteria. These studies provide the first evidence for B cell-containing ILF-like structures in reptiles and provide novel information about gut immunity in nonmammalian vertebrates that could have important implications for ecological interactions with pathogens.

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.

Temperature fluctuations and maternal estrogens as critical factors for understanding temperature-dependent sex determination in nature.

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.