Ryan T. Paitz

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.

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.

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.

Embryonic modulation of maternal steroids in European starlings (Sturnus vulgaris)

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.

Rapid decline in the concentrations of three yolk steroids during development: is it embryonic regulation?

Maternally derived yolk steroids have been found to elicit both short-term and long-term effects on offspring phenotype. Paradoxically, their effects can be strikingly specific given the often substantial concentrations present at oviposition, and they do not appear to uniformly affect all steroid-sensitive processes. To better understand the dynamics of yolk steroids across embryonic development, we quantified levels of progesterone, testosterone, and estradiol at 5-day intervals throughout development in eggs of the red-eared slider turtle (Trachemys scripta) incubated at both male- and female-producing temperatures. We also assessed the effect of season on yolk steroid levels. For all steroids assayed, the concentrations in yolk declined significantly by day 15 of embryonic development despite large differences in initial concentrations among steroids. We found that estradiol was the only steroid whose initial concentration varied significantly with season, while only the decline in testosterone was affected by incubation temperature. These findings illustrate the complex nature of yolk steroid dynamics and suggest that maternal steroids may be rapidly degraded or subject to embryonic processing, emphasizing the need for studies aimed at understanding the mechanisms through which yolk steroids may elicit their effects.

Age and Season Impact Resource Allocation to Eggs and Nesting Behavior in the Painted Turtle

Theory predicts that in long‐lived organisms females should invest less energy in reproduction and more in growth and self‐maintenance early in life, with this balance shifting as females age and the relative value of each reproductive event increases. We investigated this potential trade‐off by characterizing within‐population variation in resource allocation to eggs by female painted turtles (Chrysemys picta) and relating this variation to their nesting ecology and life history. We examined lipid and protein allocation to yolks, accounting for both relative female age and seasonal effects (first vs. second clutches within a female). Older females appear to increase their investment in reproduction by producing larger eggs, but these eggs are not disproportionately more lipid or protein rich than the smaller eggs from younger females. Within the nesting season, first clutches have more lipid and protein than second clutches. We also found that younger females nest closer to the water than older females. Our results indicate that trade‐offs involving resource allocation and nesting behavior do occur both seasonally and with age, suggesting ontogenetic variation in life‐history strategies in this long‐lived organism.

Constancy in an Inconstant World: Moving Beyond Constant Temperatures in the Study of Reptilian Incubation

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.

Temperature, phenotype, and the evolution of temperature-dependent sex determination: how do natural incubations compare to laboratory incubations?

Phenotypic variation is a fundamental component of the process of evolution and understanding the factors that create this variation is critical to investigations of this process. We test the hypothesis that phenotypic variation created under natural incubation conditions will differ from that created under constant laboratory conditions in a reptile species with temperature-dependent sex determination (TSD), the red-eared slider turtle (Trachemys scripta). Using a split clutch design, we demonstrate that offspring morphology, behavior, and sex differed between hatchlings incubated in the field and those from the laboratory, but immune response did not. The interactions between different phenotypic parameters will ultimately determine how natural selection acts upon offspring, and consequently our data suggest that offspring developing under these differing conditions should have different fitnesses. The relationship between offspring sex and phenotype serves as the theoretical foundation on which most investigations into the evolution of TSD are built. Thus, it may be necessary to use natural incubation conditions to accurately examine how offspring sex relates to other phenotypic parameters if we are to understand the evolution of this sex determining mechanism.

Temperature fluctuations affect offspring sex but not morphological, behavioral, or immunological traits in the Northern Painted Turtle (Chrysemys picta)

Evolutionary theory predicts that when phenotypic variation arises during development that differently influences the fitness of each sex, selection should favor the maternal ability to match offspring phenotype to the sex that incurs a fitness benefit from that phenotype. In reptiles with temperature-dependent sex determination, the temperatures experienced during incubation can influence numerous phenotypic parameters, including sex. To mimic more naturalistic conditions, this experiment examined how variation in temperature fluctuations affects offspring sex as well as a suite of phenotypic parameters having putative fitness consequences in the Northern Painted Turtle (Chrysemys picta (Schneider, 1783)). We also characterized variation in natural nest temperatures, including the daily temperature range, related to the vegetation cover surrounding the nest. We found that temperature fluctuations did not affect hatchling morphology, immune response, or behavior, but did significantly affect offspring sex...