Rachel M. Bowden

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

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

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

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

Understanding the vertebrate immune system: insights from the reptilian perspective

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

Climate and predation dominate juvenile and adult recruitment in a turtle with temperature-dependent sex determination.

Conditions experienced early in life can influence phenotypes in ecologically important ways, as exemplified by organisms with environmental sex determination. For organisms with temperature-dependent sex determination (TSD), variation in nest temperatures induces phenotypic variation that could impact population growth rates. In environments that vary over space and time, how does this variation influence key demographic parameters (cohort sex ratio and hatchling recruitment) in early life stages of populations exhibiting TSD? We leverage a 17-year data set on a population of painted turtles, Chrysemys picta, to investigate how spatial variation in nest vegetation cover and temporal variation in climate influence early life-history demography. We found that spatial variation in nest cover strongly influenced nest temperature and sex ratio, but was not correlated with clutch size, nest predation, total nest failure, or hatching success. Temporal variation in climate influenced percentage of total nest failure and cohort sex ratio, but not depredation rate, mean clutch size, or mean hatching success. Total hatchling recruitment in a year was influenced primarily by temporal variation in climate-independent factors, number of nests constructed, and depredation rate. Recruitment of female hatchlings was determined by stochastic variation in nest depredation and annual climate and also by the total nest production. Overall population demography depends more strongly on annual variation in climate and predation than it does on the intricacies of nest-specific biology. Finally, we demonstrate that recruitment of female hatchlings translates into recruitment of breeding females into the population, thus linking climate (and other) effects on early life stages to adult demographics.

Variation in the seasonal patterns of innate and adaptive immunity in the red-eared slider (Trachemys scripta)

SUMMARY 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.

Geometric Morphometric Sex Estimation for Hatchling Turtles: A Powerful Alternative for Detecting Subtle Sexual Shape Dimorphism

Abstract Identifying sex of hatchling turtles is difficult because juveniles are not obviously externally dimorphic, and current techniques to identify sex are often logistically unfeasible for field studies. We demonstrate a widely applicable and inexpensive alternative to detect subtle but significant sexual dimorphism in hatchlings, using landmark-based geometric morphometric methods. With this approach, carapace landmarks were digitized from photographs of each hatchling, and shape variables were generated after variation in size, location and orientation were eliminated. These variables were then analyzed for sexual dimorphism, and used in discriminant function analysis to estimate sex of each hatchling. Using this approach on two species (Chrysemys picta and Podocnemis expansa), we found this method had high accuracy in assigning sex when compared with true sex (98% and 90%, respectively), and cross-validation revealed a correct classification rate of 85%. These correct classification rates were considerably higher than those found on the same species using linear distance measurements as data. We also explored two alternative statistical approaches for assessing sex (K-means clustering and multiple logistic regression) and found that these alternative approaches were accurate only 61% and 78% of the time, respectively, in C. picta and 69% and 77% of the time in P. expansa. These findings are similar to classification rates found for turtle species using approaches based on linear distance measurements. We also found that the observed sexual dimorphism differed between the two species. In P. expansa, males displayed relatively more expansion of the central region of the carapace relative to females, whereas in C. picta this pattern was reversed. We conclude that discriminant analysis of morphology quantified using geometric morphometrics provides researchers with a powerful tool to discriminate between male and female hatchling turtles.

A proposed role of the sulfotransferase/sulfatase pathway in modulating yolk steroid effects

Steroid hormones have long been studied by behavioral ecologists as a nongenetic means whereby females can influence the development of their offspring. In oviparous vertebrates, steroids are present in the yolk at the time of oviposition and have been shown to affect numerous traits of the offspring. To date, most studies have focused on the functional relationship between yolk steroids and offspring development. In this article we used a mechanistic approach to investigate the effects of yolk steroids in an attempt to decipher how lipophilic steroids may make it from the lipid-rich yolk to the developing embryo. First, we examined the distribution of radioactive and nonradioactive estradiol following the exogenous application of each to developing eggs of the red-eared slider. Second, we quantified sulfotransferase activity in various components of the egg as a potential mechanism for the metabolism of steroids. Results indicate that exogenous estradiol is converted to a water-soluble form during the first 15 days of development, concurrent with an increase of sulfotransferase activity in the yolk and extra-embryonic membranes. Based on these data, we propose a mechanistic model based upon the sulfotransferase/sulfatase pathway as a means through which developing eggs can convert steroids to a water-soluble form that can be transported to the embryo. These sulfonated steroids may then serve as precursors for subsequent steroid production via sulfatase activity. This model utilizes a mechanism known to be important for the modulation of maternal steroid signals in placental mammals, at the same time addressing several previously unanswered questions regarding the mechanisms underlying the effects of yolk steroids.

Experience pays: offspring survival increases with female age

Life-history theory predicts that, in long-lived organisms, effort towards reproduction will increase with age, and research from oviparous vertebrates largely supports this prediction. In reptiles, where parental care occurs primarily via provisioning of the egg, older females tend to produce larger eggs, which in turn produce larger hatchlings that have increased survival. We conducted an experimental release study and report that maternal age positively influences offspring survivorship in the painted turtle (Chrysemys picta) and predicts offspring survival at least as well as hatchling body size does. These data suggest that, although increasing hatchling size is a major component of reproductive success in older individuals, other factors also contribute.

The impact of behavioral and physiological maternal effects on offspring sex ratio in the common snapping turtle, Chelydra serpentina

Theory suggests that maternal effects are especially important in organisms with environmentally-sensitive sex-determining mechanisms. However, there is no substantive body of empirical evidence to confirm this conjecture. We integrated field and laboratory studies to jointly evaluate the significance of behavioral (nest-site choice) and physiological (yolk hormone allocation) maternal effects on offspring sex ratio in the common snapping turtle (Chelydra serpentina), a species with temperature-dependent sex determination (TSD). Of the 16 microhabitat variables measured, only three (south, east, and total overstory vegetation cover) were significantly correlated with nest temperature: cooler nests were located under more vegetation cover. In turn, these microhabitat predictors of nest temperature, and nest temperature itself, may influence nest sex ratio: shadier, cooler nests were more likely to produce a higher proportion of male offspring than less shady, warmer nests. Analysis of eggs from these same nests incubated in a common garden design in the laboratory revealed that clutch sex ratio was unaffected by levels of yolk estradiol, yolk testosterone, or their interaction. Examination of both behavioral and physiological maternal effects revealed no concordant impact on offspring sex ratio. However, eggs from nests that produced male-biased sex ratios in the field yielded higher proportions of males under constant-temperature conditions in the laboratory. Our study confirms the importance of behavioral maternal effects in nature on offspring sex ratios in species with TSD, while also revealing the potential presence of a predisposition for sex-ratio production underlying TSD in this system.

Living at extremes: development at the edges of viable temperature under constant and fluctuating conditions.

In the painted turtle (Chrysemys picta) and the red‐eared slider (Trachemys scripta), the temperature that eggs are exposed to during incubation influences many traits of the developing embryo. We tested the effect of fluctuating‐ versus constant‐temperature incubation regimes at the high and low ends of the viable developmental temperature range to assess the effect of incubation environment on offspring development. Eggs were incubated in four treatments: 23°C constant, 23° ± 3°C, 31°C constant, and 31° ± 3°C. We assessed incubation duration, hatchling survival, growth, and immune function via a delayed‐type hypersensitivity test. We predicted that fluctuations would accelerate developmental time at 23°C and decelerate it at 31°C and that these changes in incubation duration would influence offspring phenotype. We found that fluctuating incubation conditions affected developmental time at both temperatures and that survival, growth, and immune response were increased by temperature fluctuations. These results demonstrate that fluctuating temperatures have a differential impact on offspring phenotype when compared to constant temperatures, and they suggest that hatchling fitness is enhanced under conditions that more closely mimic natural incubation conditions.