Lorin A. Neuman-Lee

Improved ex vivo method for microbiocidal activity across vertebrate species

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.

Food restriction and chronic stress alter energy use and affect immunity in an infrequent feeder

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.

Confirmation and Distribution of Tetrodotoxin for the First Time in Terrestrial Invertebrates: Two Terrestrial Flatworm Species ( Bipalium adventitium and Bipalium kewense )

The potent neurotoxin tetrodotoxin (TTX) is known from a diverse array of taxa, but is unknown in terrestrial invertebrates. Tetrodotoxin is a low molecular weight compound that acts by blocking voltage-gated sodium channels, inducing paralysis. However, the origins and ecological functions of TTX in most taxa remain mysterious. Here, we show that TTX is present in two species of terrestrial flatworm (Bipalium adventitium and Bipalium kewense) using a competitive inhibition enzymatic immunoassay to quantify the toxin and high phase liquid chromatography to confirm the presence. We also investigated the distribution of TTX throughout the bodies of the flatworms and provide evidence suggesting that TTX is used during predation to subdue large prey items. We also show that the egg capsules of B. adventitium have TTX, indicating a further role in defense. These data suggest a potential route for TTX bioaccumulation in terrestrial systems.

The role of corticosterone and toxicity in the antipredator behavior of the Rough-skinned Newt (Taricha granulosa).

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 animals 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.

Atrazine Exposure Impacts Behavior and Survivorship of Neonatal Turtles

Abstract:  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 for 11 mo, during which time nest escape behavior, time to first foraging event, time to capture prey, growth, and survival were evaluated. None of the variables recorded at hatching was significantly affected by atrazine treatment, although abnormalities declined as atrazine levels increased. However, turtles deriving from the lowest atrazine-treated eggs had inhibited nest escape behavior and reduced posthatching survival. These findings reveal persistent fitness-reducing impacts on neonatal turtles resulting from atrazine exposure during embryonic development.

Physiological Responses to Salinity Vary with Proximity to the Ocean in a Coastal Amphibian

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.

Physiological effects of polybrominated diphenyl ether (PBDE-47) on pregnant gartersnakes and resulting offspring.

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.

Swimming against the tide: resilience of a riverine turtle to recurrent extreme environmental events

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.

Endocrine-reproductive-immune interactions in female and male Galápagos marine iguanas.

Abstract 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. HighlightsRelationship among hormones and immunity is contradictory, especially in non‐model organisms.Comprehensive test of in situ associations among hormones and immunity is necessary.Acute stress induces an increase in immunity, and adrenal and sex steroids.Sex and adrenal steroids were related to immunity, but relationships were sex‐specific.

Mutations to the cardiotonic steroid binding site of Na(+)/K(+)-ATPase are associated with high level of resistance to gamabufotalin in a natricine snake.

Although toads are defended by bufadienolide toxins, some snakes have evolved resistance to bufadienolides and feed heavily on toads. We compared resistance in Nerodia rhombifer, which possesses mutations that confer target-site resistance, to Pituophis catenifer, which lacks those mutations. Even at the highest dosage tested, Nerodia showed no effects, whereas the lowest dose was lethal to Pituophis. Our results demonstrate a striking level of resistance to bufadienolides in a species possessing the mutations for resistance.