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Reptiles, the only ectothermic amniotes, employ a wide variety of physiological adaptations to adjust to their environments but remain vastly understudied in the field of immunology and ecoimmunology in comparison to other vertebrate taxa. To address this knowledge gap, we assessed the current state of research on reptilian innate immunology by conducting an extensive literature search of peer-reviewed articles published across the four orders of Reptilia (Crocodilia, Testudines, Squamata, and Rhynchocephalia). Using our compiled dataset, we investigated common techniques, characterization of immune components, differences in findings and type of research among the four orders, and immune responses to ecological and life-history variables. We found that there are differences in the types of questions asked and approaches used for each of these reptilian orders. The different conceptual frameworks applied to each group has led to a lack of unified understanding of reptilian immunological strategies, which, in turn, have resulted in large conceptual gaps in the field of ecoimmunology as a whole. To apply ecoimmunological concepts and techniques most effectively to reptiles, we must combine traditional immunological studies with ecoimmunological studies to continue to identify, characterize, and describe the reptilian immune components and responses. This review highlights the advances and gaps that remain to help identify targeted and cohesive approaches for future research in reptilian ecoimmunological studies.

Toxin-resistant predators may suffer costs from eating chemically defended prey and do not feed exclusively on toxic prey. Common Gartersnakes (Thamnophis sirtalis (Linnaeus, 1758)) have been considered the drivers of an evolutionary arms race with highly toxic newts (genus Taricha Gray, 1850), which they consume with few or no deleterious effects. However, how frequently newts are consumed in nature is less clear. To address this question, we investigated the diets of Th. sirtalis at a site in central Oregon where snakes have high levels of resistance and newts have high levels of tetrodotoxin in the skin. Because snake diets are difficult to quantify using traditional means, we used stable isotopes to estimate the proportion of Th. sirtalis diets made up of newts. Our estimate for the proportion of Th. sirtalis diet made up of Rough-skinned Newts (Taricha granulosa (Skilton, 1849)) at this site is 3.2%. Mole Salamanders (genus Ambystoma Tschudi, 1838) were predicted to be the most important prey, followed by slugs, chorus frogs, and mice, with a very minor role for earthworms. Our results demonstrate that even though Th. sirtalis are physiologically capable of consuming toxic prey, they do not often do so. Generalist predators can be exposed to very strong selection from, and exert reciprocal selection on even rarely eaten, chemically defended prey.

Teaching evolution in high school and in entry-level college courses can be challenging due to the inherent misinformation, misunderstanding, and biases with which students approach the topic. In this setting, it is critical to both teach the basic concepts and address common student misconceptions about evolution. We present two paired activities that allow students to (1) explore the processes of natural selection in a direct and experiential way and (2) address common misconceptions in evolutionary theory. The first activity, the “Natural Selection Game,” has students simulate a bird population and experience shifts in phenotype frequency as a result of selective pressures. Following the end of the game, students discuss the outcomes and connect them to real-life examples. The second activity encourages students to actively research common misconceptions with the use of personal technology in order to distinguish between scientifically supported data and poor information online. Both activities can be incorporated in high school and university-level general biology curricula. They will allow students to connect their firsthand experiences to lecture-based instruction and, as a result, develop a stronger understanding of the mechanisms of evolution.

Climate change and anthropogenic modifications to the landscape can have both positive and negative effects on an animal. Linking landscape change to physiological stress and fitness of an animal is a fundamental tenet to be examined in applied ecology. Cortisol is a glucocorticoid hormone that can be used to indicate an animal’s physiological stress response. In the Sierra Nevada Mountains of California, fishers (Pekania pennanti) are a threatened mesocarnivore that have been subjected to rapid landscape changes due to anthropogenic modifications and tree mortality related to a 4-year drought. We measured cortisol concentrations in the hair of 64 fishers (41 females, 23 males) captured and radio-collared in the Sierra National Forest, California. We addressed two main questions: (1) Is the physiological stress response of fishers influenced by anthropogenic factors, habitat type, canopy cover, and tree mortality due to drought in their home range? (2) Does the physiological stress response influence survival, reproduction, or body condition? We examined these factors within a fisher home range at 3 scales (30, 60, 95% isopleths). Using model selection, we found that tree mortality was the principle driver influencing stress levels among individual fishers with female and male fishers having increasing cortisol levels in home ranges with increasing tree mortality. Most importantly, we also found a link between physiological stress and demography where female fishers with low cortisol levels had the highest annual survival rate (0.94), whereas females with medium and high cortisol had lower annual survival rates, 0.78 and 0.81, respectively. We found no significant relationships between cortisol levels and body condition, male survival, or litter size. We concluded that tree mortality related to a 4-year drought has created a “landscape of stress” for this small, isolated fisher population.

Polar bears (Ursus maritimus) use sea ice to access marine mammal prey. In Alaska’s Southern Beaufort Sea, the declining availability of sea ice habitat in summer and fall has reduced opportunities for polar bears to routinely hunt on the ice for seals, their primary prey. This reduced access to prey may result in physiological stress with subsequent potential consequences to reproductive function (physiological changes that accompany reproduction), which can be measured via reproductive hormones. Hormone concentrations in hair can be used as a minimally invasive alternative to serum concentrations, which must come from animal captures. Hair samples also provide a long-term average measurement of hormone concentrations that is not influenced by short-term fluctuations like that of serum. The aim of this study was (1) to determine if a radioimmunoassay could be used to measure adrenal and reproductive hormones in polar bear hair, and (2) to determine what the relationship is between these hormones and other reproductive, condition, and demographic parameters of polar bears. We successfully validated this method for cortisol, progesterone, estradiol, and testosterone through the analysis of hair and serum of 141 free-ranging polar bears. We found that while hair cannot be used to estimate serum hormone concentrations during the breeding season, hormone concentrations in hair can be used to measure reproductive function in polar bears. Further, our findings support trends in previous studies measuring hormone concentrations in serum. We found that adrenal and some reproductive hormones were positively correlated in hair samples of females. Associations between hormone concentrations in hair and serum did not vary relative to reproductive status of adult females. Serum testosterone increased throughout the breeding season for adult males and was significantly associated with body mass index (BMI). Our research supports the use of hair as a measure of reproductive function in polar bears and allows us to monitor the future effects of climate change on polar bear physiology.

While there is huge promise in monitoring physiological parameters in free-living organisms, we also find high amounts of variability over time and space. This variation requires us to capitalize on long-term physiological monitoring to adequately address questions of population health, conservation status, or evolutionary trends as long-term sampling can examine ecoimmunological and endocrine interactions in wild populations while accounting for the variation that often makes ecophysiological field studies difficult to compare. In this study, we tested how immune efficacy and endocrinology interact while accounting for ecological context and environmental conditions in two snake species. Specifically, we measured bacterial killing ability, steroid hormones, and morphological characteristics in multiple populations of the Western Terrestrial Gartersnake (Thamnophis elegans) and Common Gartersnake (T. sirtalis) for multiple seasons over 6 years. Leveraging this long-term dataset, we tested how a broad immune measure and endocrine endpoints interact while accounting for individual traits, sampling date, and environmental conditions. Across both species, we found bacterial killing ability to be directly related to corticosterone (CORT) and temperature and greater overall in the spring compared to the fall. We found CORT and testosterone yielded relationships with individual sex, sampling temperature, and time of year. Wild populations can exhibit high amounts of variation in commonly collected physiological endpoints, highlighting the complexity and difficulty inherent in interpreting single endpoints without taking ecological and environmental conditions into account. Our study emphasizes the importance of reporting the environmental conditions under which the sampling occurred to allow for better contextualization and comparison between studies.

The glucocorticoid hormone corticosterone (CORT) has classically been used in ecophysiological studies as a proxy for stress and energy mobilization, but rarely are CORT and the energy metabolites themselves concurrently measured. To examine CORT's role in mobilizing glucose in a wild reptile, we conducted two studies. The first study measured natural baseline and stress-induced blood-borne CORT and glucose levels in snakes during spring emergence and again when snakes return to the denning sites in fall. Our second study manipulated the hypothalamic pituitary adrenal axis in male snakes in the fall by taking a baseline blood sample, then subjecting individuals to one of five treatments (no injection, saline, CORT, adrenocorticotropin hormone, and metyrapone). Subsequent samples were taken at 30 minutes and 60 minutes. In both studies, we found that glucose levels do increase with the acute stress, but that the relationship was not directly related to CORT elevation. In the second study, we found that none of the HPA axis manipulations directly affected blood glucose levels, further indicating that CORT may play a complex, but not direct role in glucose mobilization in snakes. This study highlights the need for testing mechanisms in wild organisms by combining in situ observations with manipulative studies.

Behavioral fever in reptiles is often considered an adaptive response used to eliminate pathogens, yet empirical data showing the wide-spread use of this response is mixed. This behavioral change can be beneficial by enhancing the host’s immune response and increasing the animal’s chance of survival, but it can also be detrimental in terms of host energetic requirements and enzymatic performance. Thus, we examined whether captive-bred African house snakes (Lamprophis fuliginosus) employed behavioral fever in response to pathogen stimulus. Twenty-one African house snakes were injected separately with three different strains of ultraviolet (UV) light-killed bacteria (Escherichia coli, Staphylococcus aureus, Salmonella enterica). We found an increased variance of hourly cloacal temperatures following exposure to pathogens in male but not female house snakes. We did not, however, find a significant febrile response to pathogen exposure as measured via mean cloacal temperature. This research adds critical information to the field of reptilian physiology as this field remains understudied. Reptilian immune function and its relationship with thermal biology is ever more pertinent as new challenges arise, such as novel pathogens and changing climate.

Human population and thus anthropogenic disturbance of natural systems are increasing at staggering rates. However, the impact of disturbance on natural systems likely varies with the extent of disturbance. While a considerable amount of research has been conducted on high-impact disturbances (e.g., environmental contamination, urban development), much less is known about the effects of less obtrusive activities such as ecotourism and outdoor recreation. Past research has documented behavioral and hormonal changes to non-consumptive wildlife activities; however, these studies rarely provide direct health and survival consequences, or examine impacts over a gradient of disturbances. In the current study, we quantified endocrine and immune responses and reproductive investment in six Galapagos marine iguana ( Amblyrhynchus cristatus ) populations located across a gradient of human disturbance stemming from the ecotourism industry, including urban development, boat traffic, and ecotourist activities. We regressed fitness-related physiological metrics to the intensity of human disturbance across these populations. As predicted, immunological capabilities decreased and oxidative stress increased with increasing intensity of human disturbance and development. Sexes also differed in their endocrine responses to disturbance intensity, with only males showing elevated baseline corticosterone at sites impacted by tourism. Testosterone and estradiol were both positively related to disturbance intensity. There was also a significant overall negative effect of the presence of human disturbance, but not its intensity, on reproductive investment. These results are the first to demonstrate that measures of physiological performance are sensitive to the intensity, and not merely the presence/absence of human disturbance, and that immunological measures in addition to more traditional methods can serve as important indicators of stress and potentially compromised fitness in natural populations.

Toads are chemically defended by cardiotonic steroids known as bufadienolides. Resistance to the acute effects of bufadienolides in snakes that prey on toads is conferred by target-site insensitivity of the toxin’s target enzyme, the Na+/K+-ATPase. Previous studies have focused largely on the molecular mechanisms of resistance but have not investigated the physiological mechanisms or consequences of exposure to the toxins. Adrenal enlargement in snakes often is associated with specialization on a diet of toads. These endocrine glands are partly composed of interrenal tissue, which produces the corticosteroids corticosterone and aldosterone. Corticosterone is the main hormone released in response to stress in reptiles, and aldosterone plays an important role in maintaining ion balance through upregulation of Na+/K+-ATPase. We tested the endocrine response of select species of snakes to acute cardiotonic steroid exposure by measuring circulating aldosterone and corticosterone concentrations. We found that Rhabdophis tigrinus, which specializes on a diet of toads, responds with lower corticosterone and higher aldosterone compared to other species that exhibit target-site resistance to the toxins but do not specialize on toads. We also found differences between sexes in R. tigrinus, with males generally responding with higher corticosterone and aldosterone than females. This study provides evidence of physiological adaptations, beyond target-site resistance, associated with tolerance of bufadienolides in a specialized toad-eating snake.

Assessing the health and condition of animals in their natural environment can be problematic. Many physiological metrics, including immunity, are highly influenced by specific context and recent events to which researchers may be unaware. Thus, using a multifaceted physiological approach and a context-specific analysis encompassing multiple time scales can be highly informative. Ecoimmunological tools in particular can provide important indications to the health of animals in the wild. We collected blood and hair samples from free-ranging polar bears (Ursus maritimus) in the southern Beaufort Sea and examined the influence of sex, age, and reproductive status on metrics of immunity, stress, and body condition during 2013-2015. We examined metrics of innate immunity (bactericidal ability and lysis) and stress (hair cortisol, reactive oxygen species, and oxidative barrier), in relation to indices of body condition considered to be short term (urea to creatinine ratio; UC ratio) and long term (storage energy and body mass index). We found the factors of sex, age, and reproductive status of the bear were critical for interpreting different physiological metrics. Additionally, the metrics of body condition were important predictors for stress indicators. Finally, many of these metrics differed between years, illustrating the need to examine populations on a longer time scale. Taken together, this study demonstrates the complex relationship between multiple facets of physiology and how interpretation requires us to examine individuals within a specific context.

The neuropeptide kisspeptin and its receptor are essential for activation of the hypothalamic-pituitary-gonadal (HPG) axis and regulating reproduction. While the role of kisspeptin in regulating the HPG axis in mammals has been well established, little is known about the functional ability of kisspeptins to activate the HPG axis and associated behavior in non-mammalian species. Here we experimentally examined the effects of kisspeptin on downstream release of testosterone and associated aggression and display behaviors in the side-blotched lizard (Uta stansburiana). We found that exogenous treatment with kisspeptin resulted in an increase in circulating testosterone levels, castration blocked the kisspeptin-induced increase in testosterone, and testosterone levels in kisspeptin-treated animals were positively related to frequency of aggressive behaviors. This evidence provides a clear link between kisspeptin, testosterone, and aggressive behavior in lizards. Thus, it is likely that kisspeptin plays an important role more broadly in non-mammalian systems in the regulation of reproductive physiology and related behaviors.

Endocrine-immune interactions are variable across species and contexts making it difficult to discern consistent patterns. There is a paucity of data in non-model systems making these relationships even more nebulous, particularly in reptiles. In the present study, we have completed a more comprehensive test of the relationship among steroid hormones and ecologically relevant immune measures. We tested the relationship between baseline and stress-induced levels of sex and adrenal steroid hormones and standard ecoimmunological metrics in both female and male Galápagos marine iguanas (Amblyrhynchus cristatus). We found significant associations between adrenal activity and immunity, whereby females that mounted greater corticosterone responses to stress had lower basal and stress-induced immunity (i.e., bactericidal ability). Males showed the opposite relationship, suggesting sex-specific immunomodulatory actions of corticosterone. In both sexes, we observed a stress-induced increase in corticosterone, and in females a stress-induced increase in bactericidal ability. Consistent with other taxa, we also found that baseline corticosterone and testosterone in males was inversely related to baseline bactericidal ability. However, in females, we found a positive relationship between both testosterone and progesterone and bactericidal ability. Multivariate analysis did not discern any further endocrine-immune relationships, suggesting that interactions between adrenal, sex steroid hormones, and the immune system may not be direct and instead may be responding to other common stimuli, (i.e., reproductive status, energy). Taken together, these data illustrate significant endocrine-immune interactions that are highly dependent on sex and the stress state of the animal.

Organisms must balance energy invested into self-maintenance, reproduction, and somatic growth over their lifetime. In this study, the effects of diet restriction and diet complexity on side-blotched lizards (Uta stansburiana) were analyzed. Thirty male lizards, housed in the laboratory, were fed either an ad libitum or a restricted diet for 18 days (phase 1). Individuals from both treatments were then assigned to a diet of the same quantity of food that was either simple (only crickets) or complex (crickets, cockroaches, waxworms, and mealworms) for 35 days (phase 2). We evaluated (1) how diet restriction affected life history strategies and (2) how diet complexity affected recovery from diet restriction as measured at the end of phase 2 by body mass, snout-vent length, calculated body condition score, wound healing, tail regrowth, bacterial killing ability, oxidative stress, and plasma testosterone and corticosterone concentrations. Lizards without diet restriction allocated more energy to self-maintenance (i.e., maintaining higher body condition scores, healing wounds more quickly) than lizards with diet restriction. Lizards with diet restriction had higher plasma testosterone concentrations and larger increases in snout-vent lengths than those fed ad libitum, which may reflect allocations toward reproduction and somatic growth. A complex diet resulted in better body condition and faster tail regrowth than a simple diet, suggesting that a complex diet enhanced recovery from diet restriction, although long-term life history choices remained unaltered. Finally, lizards on a complex diet consumed substantially less food while maintaining higher body condition, suggesting that key nutrients may be lacking from a simple diet.

While the use of nanoparticles has dramatically increased in recent years, the ecological consequences are not well known. In particular, little research has been done to investigate the potentially detrimental effects of nanoparticles on amphibians, especially across all life-history stages of salamanders and newts (caudates). To address this dearth in knowledge, we examined the effects of zinc oxide (ZnO) nanoparticles on egg, larval, and adult Rough-skinned Newts (Taricha granulosa). Chronic toxicity was tested on eggs and larvae, and acute toxicity was tested on eggs, larvae, and adults. For eggs, chronic exposure to ZnO nanoparticles caused higher mortality at 10.0 and 100.0 mg L(-1) compared to 0.0, 0.1, and 1.0 mg L(-1) . When given an acute exposure (24 hr) to 10.0 mg L(-1) nanoparticles at a late developmental stage, larvae hatched 5 days early, at a decreased developmental stage, and smaller size compared to the control. Chronic and acute exposure of larvae increased mortality up to 75% at both 10.0 and 100.0 mg L(-1) and exhibited sublethal effects, most dramatically, severe gill degradation. These results suggest nanoparticles can have lethal and sublethal effects on all life stages of amphibians.

Synthetic chemicals, such as pesticides, are used in a variety of ways in the agricultural industry. Anthropogenic chemicals pose a unique challenge to organisms because of the lack of evolutionary history between the chemical and the organism. However, research has shown that some organisms have a resistance to these synthetic chemicals due to their evolved resistance to a natural compound with a similar structure or mode of action. Indoxacarb (INDOX) is a relatively new pesticide with a similar mode of action to that of tetrodotoxin (TTX). Tetrodotoxin is a naturally occurring toxin that is used as an antipredator defense in the rough-skinned newt (Taricha granulosa). Some populations of the common garter snake (Thamnophis sirtalis) have developed a resistance to tetrodotoxin. Here, we investigated the correlation between TTX and INDOX resistance in snakes. We hypothesized that INDOX would induce a much higher stress response than the naturally occurring TTX. We injected each snake with tetrodotoxin (1 mass-adjusted mouse unit). We did the same with mass-adjusted units of INDOX. We measured corticosterone, testosterone, and bactericidal ability. Our results show an acute stress response to INDOX, but not TTX through an elevate corticosterone and innate immune response, although there was no difference in testosterone concentration. These results suggest that, although INDOX may have a similar mechanism of action, garter snakes do not react in a similar manner as to TTX. This research gives a physiological perspective on the differences between naturally occurring compounds and synthetic compounds.

The field of ecoimmunology has made it clear that individual and ecological contexts are critical for interpreting an animal's immune response. In an effort to better understand the relevance of commonly used immunological assays, we tested how different metrics of immunity and physiological function were interrelated in naturally parasitized individuals of a well-studied reptile, the common gartersnake (Thamnophis sirtalis). Overall, we found that bactericidal ability, an integrative measure of innate immunity, was often correlated with more specific immunological and physiological tests (lysis and oxidative stress) but was not related to tissue-level inflammation that was determined by histopathology. The only hematological metric that correlated with tissue-level inflammation was the prevalence of monocytes in blood smears. Finally, using histological techniques, we describe natural parasitism throughout the organ systems in these individuals, finding that neither the presence nor the burden of parasite load affected the physiological and immune metrics that we measured. By performing comprehensive assessments of physiological and immune processes, we are better able to draw conclusions about how to interpret findings from specific assays in wild organisms.

Polybrominated diphenyl ethers (PBDEs) are used as flame retardants and are persistent contaminants found in virtually every environment and organism sampled to date, including humans. There is growing evidence that PBDEs are the source of thyroid, neurodevelopmental, and reproductive toxicity. Yet little work has focused on how this pervasive contaminant may influence the reproduction and physiology of non-traditional model species. This is especially critical because in many cases non-model species, such as reptiles, are most likely to come into contact with PBDEs in nature. We tested how short-term, repeated exposure to the PBDE congener BDE-47 during pregnancy affected physiological processes in pregnant female gartersnakes (thyroid follicular height, bactericidal ability, stress responsiveness, reproductive output, and tendency to terminate pregnancy) and their resulting offspring (levels of corticosterone, bactericidal ability, and size differences). We found potential effects of BDE-47 on both the mother, such as increased size and higher thyroid follicular height, and her offspring (increased size), suggesting the effects on physiological function of PBDEs do indeed extend beyond the traditional rodent models.

Summary Glucocorticoids are important mediators of energy utilization for key physiological processes, including immune function. Much work has focused on the effects of energy limitation and stress for key physiological processes such as reproduction and immunity. However, it is unclear how stress alters energy use across different energy states, and the physiological ramifications of such effects are even less clear. In this study, we altered energy and stress states of an infrequent feeder, the terrestrial gartersnake (Thamnophis elegans), using fasting and repeated restraint stress (chronic stressors) to test how these challenges interacted to affect immune function, energy metabolites and glucocorticoid reactivity (a traditional indicator of stress state) to restraint stress, a standardized, acute stressor. After this acute stressor, the snakes which had received chronic stress had increased glucocorticoid reactivity, and both treatments altered energy metabolite use and storage. Evidence of interaction of food restriction and chronic stress treatments on innate immune function and energy metabolites (triglycerides and glycerol) suggests that stress alters energy use in a manner dependent on the energy state of the animal. Snakes have a remarkable ability to maintain functionality of key physiological processes under stressful conditions but are still susceptible to multiple simultaneous stressors, a situation increasingly prevalent in our ever-changing environment.

A variety of mechanisms are responsible for enabling an organism to escape a predatory attack, including behavioral changes, alterations in hormone levels, and production and/or secretion of toxins. However, these mechanisms are rarely studied in conjunction with each other. The Rough-skinned Newt ( Taricha granulosa ) is an ideal organism to examine the relationships between these mechanisms because its behavioral displays and toxin secretion during a predator attack are well documented and readily characterized. While we found no direct relationship between antipredator behavior and endogenous levels of corticosterone (CORT), antipredator behavior was inhibited when exogenous CORT and adrenocorticotropic hormone (ACTH) were administered, resulting in high circulating concentrations of CORT, indicating that CORT may play a role in mediating the behavior. There was no correlation between the animal’s toxicity and either CORT or behavior. The results of this study provide evidence that CORT plays an important, yet complex, role in the antipredator response of these amphibians.

The activation of the hypothalamic pituitary adrenal (HPA) axis is one of the most important physiological processes in coping with any deviation in an organism's homeostasis. This activation and the secretion of glucocorticoids, such as corticosterone, allow organisms to cope with perturbations and return to optimal physiological functioning as quickly as possible. In this study, we examined the HPA axis activation in common gartersnakes (Thamnophis sirtalis) as a response to a natural toxin, tetrodotoxin (TTX). This neurotoxin is found in high levels in the Rough-skinned Newt (Taricha granulosa), which is a prey item for these snakes. To consume this toxic prey, these snakes have evolved variable resistance. We hypothesized that the more resistant individuals would show a lower HPA axis response than less resistant individuals, as measured by corticosterone (CORT) and bactericidal ability, which is a functional downstream measurement of CORT's activity. We determined "resistance level" for tetrodotoxin from each individual snake by determining the dose which reduced race speed by 50%. Individuals were injected them with an increasing amount of tetrodotoxin (10, 25, and 50 MAMUs) to determine this value. Thirty minutes after every injection, we gathered blood samples from each snake. Our results show that, while there were no significant differences among individual CORT levels in a dose-dependent manner, female snakes did have a larger stress response when compared to both males and juveniles. Different life-histories could explain why females were able to mount a higher HPA axis response. However, TTX had no downstream effects on bactericidal ability, although juveniles had consistently lower values than adults. Our research shows a possible dichotomy between how each sex manages tetrodotoxin and gives way for a more comprehensive analysis of tetrodotoxin in an ecological context.

Knowledge of endocrine stress responses can be advantageous for understanding how animals respond to their environment. One tool in wildlife endocrinology is to measure the adrenocortical activity as a parameter of disturbance of animals. Fecal glucocorticoid metabolites (GCMs) provide a noninvasive assessment of adrenocortical activity. Using an adrenocorticotropic hormone (ACTH) challenge administered to 28 captive coyotes (Canis latrans), we measured the levels of plasma cortisol, and fecal cortisol and corticosterone metabolites (i.e., GCMs). Our goal was to determine the dose-response in the plasma and fecal samples following the injection and determine if there were effects of sex, age, and time of day. Specifically, animals were anesthetized for ~ 90 min with treatment animals intravenously injected with exogenous ACTH and control animals receiving saline. We collected blood samples prior to injection and at 4 different time points post-injection. We also collected fecal samples 2 days pre- and 2 days post-injection to measure fecal GCMs and determine if an endocrine stress response could be detected in fecal samples. We found a definite response in cortisol levels in the plasma for coyotes to the ACTH challenge. There was a response in fecal corticosterone 1 day post-injection, but the control males showed a similar response indicating a handling effect. Fecal cortisol levels did not indicate a response to the ACTH challenge, and were significantly lower than corticosterone concentrations. We also found significant sex, but not age or diurnal, differences in fecal GCMs. Radioimmunoassays for fecal corticosterone levels appeared to be a reliable indicator of physiological stress in coyotes.

Pesticide use results in the widespread distribution of chemical contaminants, which necessites regulatory agencies to assess the risks to environmental and human health. However, risk assessment is compromised when relatively few studies are used to determine impacts, particularly if most of the data used in an assessment are produced by a pesticide’s manufacturer, which constitutes a conflict of interest. Here, we present the shortcomings of the US Environmental Protection Agency’s pesticide risk assessment process, using the recent reassessment of atrazine’s impacts on amphibians as an example. We then offer solutions to improve the risk assessment process, which would reduce the potential for and perception of bias in a process that is crucial for environmental and human health.Keywords: amphibian, atrazine, conflicts of interest, Environmental Protection Agency, risk assessment

Immune system function is affected by a variety of exogenous and endogenous stressors. Most studies have focused on the effect of stressors on immune function, but not necessarily on trade-offs within the immune system and interactions with energy-mobilizing hormones. In this study, we examined how bactericidal ability and corticosterone interacted by applying acute restraint stress in a non-model organism, the side-blotched lizard (Uta stansburiana), 10 days after receiving a cutaneous wound. We found a decrease in bactericidal ability in wounded animals after restraint stress. However, the percentage healed during the first 7 days was positively correlated with bactericidal ability 10 days after wounding. In addition, the magnitude of change in corticosterone concentration during the acute stress was positively correlated with the percentage of wound healing during the first 3 days. These two relationships may demonstrate a "faster is better" strategy. If energy is invested heavily in the initial wound healing stages, the individual may be able to mount a more effective immune and stress response earlier.

Understanding intraspecific variation in habitat use is important for the management of any species. In many studies of reptiles, habitat use by juveniles is poorly understood when compared to their adult conspecifics because of capture biases and logistical constraints. We compared habitat use between sexes and age classes of Common Watersnakes (Nerodia sipedon) at a reservoir in central Illinois. Juvenile N. sipedon occurred more frequently in habitat with high conopy cover, whereas adults N. sipedon, especially reproductive females, were found exclusively in habitat with no canopy cover. Adult males used both locations equally. We emphasize the need to investigate ontogenetic variation in habitat use to better understand how reptiles utilize diverse anthropogenically altered landscapes.

This study examined the effects of sub-lethal exposure of the ubiquitous pesticide malathion on the behavior of the model orthopteran species, the house cricket (Acheta domesticus). Increasing concentrations of malathion caused male crickets to increase periods of non-directional movement, such as twitching and grooming, directional movement, and to seek out shelter less. These are all behavioral alterations that may increase the cricket's chances of being preyed upon, and thus have the potential for serious ecological consequences through trophic transfer. This study also revealed that female crickets appeared to be less affected by malathion than their male conspecifics, indicating a potential sex-bias in both susceptibility and possible predator attack.

Ecotoxicological studies that focus on a single endpoint might not accurately and completely represent the true ecological effects of a contaminant. Exposure to atrazine, a widely used herbicide, disrupts endocrine function and sexual development in amphibians, but studies involving live-bearing reptiles are lacking. This study tracks several effects of atrazine ingestion from female Northern Watersnakes (Nerodia sipedon) to their offspring exposed in utero. Twenty-five gravid N. sipedon were fed fish dosed with one of the four levels of atrazine (0, 2, 20, or 200 ppb) twice weekly for the entirety of their gestation period. Endpoints for the mothers included blood estradiol levels measured weekly and survival more than 3 months. Endpoints for the offspring included morphometrics, clutch sex ratio, stillbirth, and asymmetry of dorsal scales and jaw length. Through these multiple endpoints, we show that atrazine ingestion can disrupt estradiol production in mothers, increase the likelihood of mortality from infection, alter clutch sex ratio, cause a higher proportion of stillborn offspring, and affect scale symmetry. We emphasize the need for additional research involving other reptile species using multiple endpoints to determine the full range of impacts of contaminant exposure.

The energetic cost of immunity depends on many factors, including the type of challenge, the timing of the response, and the state of the animal. We measured changes in the standard metabolic rates of side-blotched lizards (Uta stansburiana Baird and Girard, 1852) in response to different immune challenges and nutritional states. In the first experiment, lizards were randomly assigned to one of four treatments: lipopolysaccharide (LPS) injection (to stimulate the response to a pathogen), cutaneous biopsy (as a proxy to a superficial wound), both injection and biopsy, or neither (control). Four and five days later, we measured the standard metabolic rates of the lizards. In response to healing a cutaneous wound, lizards reduced metabolic rate and lost body mass. Healing rate was also inversely related to weight loss, but LPS had no effect on body mass or metabolic rate. In the second experiment, a new set of lizards were randomly assigned to a high-food or low-food diet and administered a cutaneous biopsy. As in the first experiment, we observed a reduction in metabolic rate after wounding; moreover, this decrease was positively correlated with the rate of healing. We observed higher rates of metabolism in lizards that ate more food, but food consumption was unrelated to the decrease in metabolic rate following the biopsy. These experiments demonstrate the dynamic nature of the immune response in response to immune challenge and the state of the organism.

Freshwater organisms are increasingly exposed to elevated salinity in their habitats, presenting physiological challenges to homeostasis. Amphibians are particularly vulnerable to osmotic stress and yet are often subject to high salinity in a variety of inland and coastal environments around the world. Here, we examine the physiological responses to elevated salinity of rough-skinned newts (Taricha granulosa) inhabiting a coastal stream on the Pacific coast of North America and compare the physiological responses to salinity stress of newts living in close proximity to the ocean with those of newts living farther upstream. Although elevated salinity significantly affected the osmotic (body weight, plasma osmolality), stress (corticosterone), and immune (bactericidal ability) responses of newts, animals found closer to the ocean were generally less reactive to salt stress than those found farther upstream. Our results provide possible evidence for some physiological tolerance in this species to elevated salinity in coastal environments. As freshwater environments become increasingly saline and more stressful, understanding the physiological tolerances of vulnerable groups such as amphibians will become increasingly important to our understanding of their abilities to respond, to adapt, and, ultimately, to survive.

Non-invasive measures of the stress response are used to understand the impacts of natural and anthropogenic disturbances on wild animals. They can, however, be challenging to interpret without additional contextual information and specifics of the animals in question. Here, we used fecal samples collected from the Henry Mountains bison herd in Utah to measure the glucocorticoid hormone corticosterone (CORT), which is indicative of stress. We compared site-specific measures of fecal CORT concentration with measures of covariates related to geography (elevation, slope, aspect, distance to roads, distance to water, food quality, habitat type, season), bison physiology (body condition, parasite load, sex), and human activity (traffic volume at multiple time scales, hunting seasons). Our aim was to determine whether an unexpected habitat selection pattern could be a response to human disturbance, and thus whether ecological covariates could explain variations in fecal CORT concentration in free-ranging bison. No meaningful relationships were found for any of the covariates included in the study. At least some of those covariates should be related to the stress state of the herd, but in large and highly mobile species such as bison there is a scale mismatch between the physiological stress response of an animal and the spatiotemporal distribution of fresh feces left on the landscape. We offer our assessment of fecal CORT in bison as a case study demonstrating the utility and complications associated with using fecal indicators of stress in wildlife populations.

While the use of nanoparticles has dramatically increased in recent years, the ecological consequences are not well known. In particular, little research has been done to investigate the potentially detrimental effects of nanoparticles on amphibians, especially across all life-history stages of salamanders and newts (caudates). To address this dearth in knowledge, we examined the effects of zinc oxide (ZnO) nanoparticles on egg, larval, and adult Rough-skinned Newts (Taricha granulosa). Chronic toxicity was tested on eggs and larvae, and acute toxicity was tested on eggs, larvae, and adults. For eggs, chronic exposure to ZnO nanoparticles caused higher mortality at 10.0 and 100.0 mg L(-1) compared to 0.0, 0.1, and 1.0 mg L(-1) . When given an acute exposure (24 hr) to 10.0 mg L(-1) nanoparticles at a late developmental stage, larvae hatched 5 days early, at a decreased developmental stage, and smaller size compared to the control. Chronic and acute exposure of larvae increased mortality up to 75% at both 10.0 and 100.0 mg L(-1) and exhibited sublethal effects, most dramatically, severe gill degradation. These results suggest nanoparticles can have lethal and sublethal effects on all life stages of amphibians.

Atrazine (2-chloro-4-ethythlamino-6-isopropylamine-1,3,5-tiazine) is a widely used preemergent herbicide for controlling broadleaf plants. Because atrazine (a known endocrine-disrupting chemical) is applied in the late spring and early summer, its incidental effects on species exposed to runoff from terrestrial sources in this time period are of special interest. To examine the possible secondary impact of atrazine, we obtained eggs from 10 nests of two map turtle species, Graptemys ouachitensis and G. pseudogeographica, that nest on riverine sandbars. We incubated two eggs from each nest in sand containing one of four initial concentrations of atrazine (control and 0.1, 10, and 100 µg/L) based on levels measured in the river at the site where eggs were collected. We recorded hatching success, incubation time, external morphological abnormalities, gonadal sex, three measures of body size, righting time, and swimming time for all turtles. We reared a subset of the original neonates individually ...

In an era of rapidly changing environments and greater human mobility and penetration into wild areas, organisms are being discovered in increasingly unexpected places. One such finding is a road-killed juvenile gartersnake (Thamnophis) outside of Haines, Alaska, in August 2005. The poor condition of the specimen prevented a positive identification based on morphology alone. Furthermore, no snakes are known to be native to this region. We therefore undertook a molecular approach to determine the species and geographic origin of the individual. We sequenced two partial loci of mitochondrial DNA (cytochrome b and NADH subunit 2) from the Alaska specimen and seven specimens from localities in the lower northwestern United States. Phylogenetic reconstruction using our sequences and additional GenBank samples unambiguously revealed that the Alaska specimen is Thamnophis ordinoides and that it shares a haplotype with the northernmost sampled Washington population of T. ordinoides. In light of these ana...

Summary The field of ecoimmunology is currently undergoing rapid expansion, whereby biologists from a wide range of ecological disciplines are increasingly interested in assessing immunocompetence in their study organisms. One of the key challenges to researchers is determining what eco-immune measures to use in a given experiment. Moreover, there are limitations depending on study species, requirements for specific antibodies, and relevance of the methodology to the study organism. Here we introduce an improved ex vivo method for microbiocidal activity across vertebrate species. The utility of this assay is that it determines the ability of an organism to remove a pathogen that could be encountered in the wild, lending ecological relevancy to the technique. The applications of this microbiocidal assay are broad, as it is readily adaptable to different types of microbes as well as a wide variety of study species. We describe a method of microbiocidal analysis that will enable researchers across disciplines to effectively employ this method to accurately quantify microbial killing ability, using readily available microplate absorbance readers.

Ecotoxicological studies that focus on a single endpoint might not accurately and completely represent the true ecological effects of a contaminant. Exposure to atrazine, a widely used herbicide, disrupts endocrine function and sexual development in amphibians, but studies involving live-bearing reptiles are lacking. This study tracks several effects of atrazine ingestion from female Northern Watersnakes (Nerodia sipedon) to their offspring exposed in utero. Twenty-five gravid N. sipedon were fed fish dosed with one of the four levels of atrazine (0, 2, 20, or 200 ppb) twice weekly for the entirety of their gestation period. Endpoints for the mothers included blood estradiol levels measured weekly and survival more than 3 months. Endpoints for the offspring included morphometrics, clutch sex ratio, stillbirth, and asymmetry of dorsal scales and jaw length. Through these multiple endpoints, we show that atrazine ingestion can disrupt estradiol production in mothers, increase the likelihood of mortality from infection, alter clutch sex ratio, cause a higher proportion of stillborn offspring, and affect scale symmetry. We emphasize the need for additional research involving other reptile species using multiple endpoints to determine the full range of impacts of contaminant exposure.

Extreme environmental events (EEEs) are likely to exert deleterious effects on populations. From 1996 to 2012 we studied the nesting dynamics of a riverine population of painted turtles ( Chrysemys picta ) that experienced seven years with significantly definable spring floods. We used capture–mark–recapture methods to estimate the relationships between more than 5 m and more than 6 m flood events and population parameters. Contrary to expectations, flooding was not associated with annual differences in survival, recruitment or annual population growth rates of the adult female segment of the population. These findings suggest that female C. picta exhibit resiliency to key EEE, which are expected to increase in frequency under climate change.