Improved ex vivo method for microbiocidal activity across vertebrate species

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

The Natural Selection Game: Incorporating Active Learning in Evolution Curricula for General Biology

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

Instability of Glucocorticoid Metabolites in Coyote Scats: Implications for Field Sampling

Studying physiologic stress responses can assist in understanding the welfare of animals. One method of measuring the physiologic stress response is evaluating concentrations of glucocorticoid metabolites in feces. Previously, using an adrenocorticotropic hormone challenge, we found fecal glucocorticoid metabolite levels were a reliable indicator of physiologic stress response in coyotes (Canis latrans). We determine whether glucocorticoid metabolite concentrations remain stable when collecting feces over a 2-week period, a timeframe commonly used in scat surveys for wild canids. We collected feces from 6 captive coyotes maintained at the U.S. Department of Agriculture, Wildlife Services, National Wildlife Research Center, Predator Research Facility near Millville, Utah, USA, and exposed them to the environment for 13 days during summer (August 26 to September 8, 2011) and winter (January 11–24, 2012). Every 2 days, we collected a sub-sample from each individual scat and then quantified the concentration of fecal glucocorticoid metabolites. We found changes in fecal glucocorticoid metabolite concentrations over the 13-day period, with values increasing 45–79% from day 1 to day 3 of sampling. There was also high variation in fecal glucocorticoid metabolites among individuals over time. We provide evidence that fecal samples collected in the field even 3 days after defecation will not provide reliable measures of fecal glucocorticoid metabolites and thus recommend using only fresh fecal samples. We also recommend that, due to high individual variability in fecal glucocorticoid metabolites, a large number of individuals be sampled when a population-wide assessment is desired

Investigating the Relationship Between Corticosterone and Glucose in a Reptile

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\u27s 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 autumn. The second study manipulated the hypothalamic–pituitary–adrenal (HPA) axis in male snakes in the autumn 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 and 60 min. In both studies, we found that glucose levels do increase with 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

Differential Habitat Use by Common Watersnakes (Nerodia sipedon)

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 canopy cover, whereas adult 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

Differential habitat use by Common Watersnakes (Nerodia sipedon)

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

To stress or not to stress: Physiological responses to tetrodotoxin in resistant gartersnakes vary by sex

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

Demographic and temporal variations in immunity and condition of polar bears (\u3ci\u3eUrsus maritimus\u3c/i\u3e) from the southern Beaufort Sea

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

Landscape of stress: Tree mortality influences physiological stress and survival in a native mesocarnivore

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

Anthropogenic and Natural Stressors and Their Effect on Immunity, Reproduction, and the Stress Response

Organisms must be able to cope with many natural and anthropogenic stressors in order to successfully survive and reproduce. These stressors can come in many forms and are increasing as anthropogenic activities become more and more prevalent across the globe. In order to cope with these stressors, organisms must allocate limited energy away from processes such as reproduction to mount a stress response. This stress response involves the activation of the hypothalamic-pituitary-adrenal axis and results in a cascade of hormones and down-stream effects, such as changes in reproduction and immune function. In order to understand how reptiles and amphibians cope with a variety of stressors, I conducted seven experiments. I first validated an immune assay which can be employed across vertebrate taxa and can measure functional immune responses. I then analyzed effects of natural stressors (wounding, predator attacks, natural toxins, and food restriction) and/or anthropogenic stressors (restraint and the anthropogenic toxins polybrominated diphenyl ether and indoxacarb) on reptiles and/or amphibians. In measuring many different stressors and several different taxa (the side blotched-lizard, Uta stansburiana, the rough-skinned newt, Taricha granulosa, the wandering gartersnake, Thamnophis elegans, and the common gartersnake, T. sirtalis), I hoped to determine if patterns in energy allocation and trade-offs existed on a broad scale. I found that while there are some similarities among the responses, each organism exposed to different stressors had to be examined separately. This supports the emerging consensus that the stress response is extremely context-dependent and responses seen in one context cannot be inferred to other organisms with disparate life-histories, sexes, geographic range, or previous experience. Because of this, researchers must focus on the population in question to assess physiological questions before making management decisions