Anthropogenic and climatic factors interact to influence reproductive timing and effort.

Reproduction, although absolutely essential to a species' persistence, is in itself challenging. As anthropogenic change increasingly affects every landscape on Earth, it is critical to understand how specific pressures impact the reproductive efforts of individuals, which directly contribute to the success or failure of populations. However, organisms rarely encounter a single burden at a time, and the interactions of environmental challenges can have compounding effects. Understanding environmental and physiological pressures is difficult because they are often context-dependent and not generalizable, but long-term monitoring across variable landscapes and weather patterns can improve our understanding of these complex interactions. We tested the effects of urbanization, climate, and individual condition on the reproductive investment of wild side-blotched lizards (Uta stansburiana) by measuring physiological/reproductive metrics from six populations in urban and rural areas over six consecutive years of variable precipitation. We observed that reproductive stage affected body condition, corticosterone concentration, and oxidative stress. We also observed that reproductive patterns differed between urban and rural populations depending on rainfall, with rural animals increasing reproductive investment during rainier years compared to urban conspecifics, and that reproductive decisions appeared to occur early in the reproductive process. These results demonstrate the plastic nature of a generalist species optimizing lifetime fitness under varying conditions.

Lack of vitamin B12 impairs innate and adaptive immunity of Cuban tree frog (Osteopilus septentrionalis) tadpoles.

Vitamin B12 is a micronutrient required by a variety of organisms for healthy cellular functioning. Despite the systemic effects observed in cases of B12 deficiency, relatively little is known about how vitamin B12 affects immune health, especially in amphibians, which are declining at unprecedented rates. In this study, we tested how supplementing an algae diet with B12 affects the innate and adaptive immunity of Cuban tree frog (Osteopilus septentrionalis) tadpoles. We found that innate immunity, as measured by a bacterial killing assay, was significantly more robust in B12-supplemented tadpoles than control tadpoles, but no significant differences were found in natural antibody production or hematocrit between groups. Adaptive immunity, as measured by Aeromonas hydrophila-specific IgY antibodies, was significantly greater in tadpoles challenged with A. hydrophila and supplemented with B12 than in control tadpoles, those only challenged with A. hydrophila, and those only given B12. Our results suggest that vitamin B12 is an important factor in maintaining a functional immune system in tadpoles, which may also be true for all vertebrates.

Complex tourism and season interactions contribute to disparate physiologies in an endangered rock iguana.

To promote survival and fitness, organisms use a suite of physiological systems to respond to both predictable and unpredictable changes in the environment. These physiological responses are also influenced by changes in life history state. The continued activation of physiological systems stemming from persistent environmental perturbations enable animals to cope with these challenges but may over time lead to significant effects on the health of wildlife. In the present study, we tested how varying environmental perturbations driven by tourism and associated supplemental feeding affects the energetics, corticosterone and immunity of six discrete populations of the northern Bahamian rock iguana (Cyclura cychlura inornata and Cyclura cychlura figginsi). We studied populations within and outside the reproductive season and quantified tourist numbers during sample collection. Specifically, we measured clutch size, body condition, plasma energy metabolites, reactive oxygen species, baseline corticosterone concentrations and immune function of male and female iguanas from each population to address whether (i) disparate physiologies are emerging across a gradient of tourism and feeding, (ii) both subspecies respond similarly and (iii) responses vary with season/reproductive condition. We found significant effects of tourism level, season and their interaction on the physiology of both C. c. inornata and C. c. figginsi, supporting the idea that tourism is leading to the divergence of phenotypes. Specifically, we found elevated plasma energy metabolites, oxidative stress and a measure of innate immunity (bactericidal ability), but reduced corticosterone concentrations with increasing tourism in both subspecies of rock iguanas. These physiological metrics differ according to the level of tourism in both subspecies and persist across seasons despite variation with natural seasonal and reproductive changes. These findings suggest that anthropogenic disturbance results in disparate physiologies in northern Bahamian rock iguanas.

Effect of light exposure on growth rate of veterinary clinical dermatophyte isolates.

BACKGROUND: Veterinary textbooks and literature suggest that exposure to light is inhibitory to growth of clinical dermatophyte isolates. HYPOTHESIS/OBJECTIVES: We hypothesized that this idea was derived from experiments that examined the effect of high doses of ultraviolet and visible light exposure on dermatophyte growth, and that exposure to typical room lighting would not adversely affect dermatophyte growth rate. METHODS AND MATERIALS: Isolates of common veterinary dermatophytes (three each of Microsporum canis, Nannizia gypsea and Trichophyton benhamiae) were exposed to typical fluorescent room lighting, incubated in a closed drawer, or exposed at close range to fluorescent wide-spectrum light. Dermatophytes were grown on Sabouraud Dextrose Agar (SAB) and Dermatophyte Test Medium (DTM). Colony diameter was measured and growth rate (expressed as colony diameter increase mm/day) calculated at the linear portion of the culture growth curve. Statistical analyses compared growth rates across the various incubation conditions and among dermatophyte isolates. RESULTS: There was little difference in growth rate between cultures incubated under typical fluorescent room lighting and those placed in the dark. Exposure to the close-range light increased growth rate as a consequence of the elevated incubation temperature created by the lamp. Significant differences in growth rate were noted among strains of the same dermatophyte species. Dermatophytes grew more rapidly on SAB than DTM agar. CONCLUSIONS AND CLINICAL RELEVANCE: Exposure to typical lighting conditions in a clinical environment does not inhibit growth of dermatophyte colonies. Veterinary clinicians may conduct routine dermatophyte cultures without incubating them in the dark.

Effects of elevated corticosterone on humoral innate and antibody-mediated immunity in southern leopard frog (Lithobates sphenocephalus) tadpoles.

As a free-living larval stage of a vertebrate, tadpoles are good subjects for the study of the development of physiological systems and the study of evolutionarily conserved, context-dependent responses to variable environments. While the basic components of innate and adaptive immune defenses in tadpoles are known, the impact of glucocorticoids on immune defenses in tadpoles is not well-studied. We completed four experiments to assess effects of elevation of corticosterone on humoral innate defenses and antibody-mediated immunity in southern leopard frog tadpoles (Lithobates sphenocephalus). To test humoral innate defense within the tadpoles exposed to short-term and long-term elevation of glucocorticoids, we exposed tadpoles to exogenous corticosterone for different lengths of time in each experiment (0-84 days). We used bacterial killing assays to assess humoral innate immune defense. To test antibody-mediated immune responses, we again exposed tadpoles to exogenous corticosterone, while also exposing them to Aeromonas hydrophila. We used A. hydrophila ELISA comparing IgM and IgY responses among groups. Plasma from corticosterone-dosed tadpoles killed more A. hydrophila than control tadpoles each following a short-term (14 day) and long-term (56 day) exposure to exogenous corticosterone. Conversely, corticosterone-dosed tadpoles had significantly lower IgM and IgY against A. hydrophila after 12 weeks. Our fourth experiment revealed that the lower IgY response is a product of weaker, delayed isotype switching compared with controls. These results show that elevated corticosterone has differential effects on innate and acquired immunity in larval southern leopard frogs, consistent with patterns in more derived vertebrates and in adult frogs.

Effects of ammonium sulfate on stress physiology and innate immunity of Western mosquitofish (Gambusia affinis).

Fertilizers increase yield of crops but may have unintended negative effects on fish as a byproduct of runoff into bodies of freshwater. The objective of this study was to determine if environmentally relevant concentrations of an ammonium fertilizer impacts stress and innate immunity in Western mosquitofish (Gambusia affinis). The mosquitofish were exposed to different concentrations of ammonium sulfate fertilizer: 0 ppm, 40 ppm, and 80 ppm. To test the effects of ammonium sulfate on stress physiology, cortisol released into water by individual fish was collected after 1 week of exposure and again after 2 weeks of exposure and quantified with an enzyme immunoassay. Cortisol levels in the 0-ppm group were not significantly different over the course of the study, but we found a significant increase in cortisol levels in the fish exposed to 40 ppm and 80 ppm. We found reduced survival in fish from the 40 ppm and 80 ppm of ammonium sulfate groups compared with the 0-ppm group. We also used blood samples to complete a lysozyme assay as a measure of innate immune defense. Higher concentrations of ammonium sulfate correlated with significantly lower lysozyme activity in the fish. Overall, our results suggest that relatively low amounts of ammonium sulfate runoff into bodies of water are likely to have negative sublethal and lethal effects on small fishes.

Effects of imidacloprid on Rana catesbeiana immune and nervous system.

Imidacloprid (IMD), a neonicotinoid, is generally considered to be of low toxicity in vertebrates. However, the inhibition of acetylcholine (ACh) receptors can have a profound effect on both the immune and nervous system due to the anti-inflammatory effects of ACh. Vertebrates, such as amphibians, might be affected by IMD because they breed in wetlands where the concentration of IMD is high. In our study, we experimentally exposed Rana catesbeiana tadpoles to environmentally relevant IMD and then quantified the ACh and antibody to non-replicating antigens. We hypothesized that IMD exposure would result in higher AChE and antibody levels. We completed a factorial experiment in which tadpoles were divided into four groups, two of which were exposed to 100 ng/L of IMD. After five weeks, two groups were injected with the novel antigen keyhole limpet hemocyanin (KLH) and two injected with a control. Three weeks later, tadpoles were euthanized and blood samples collected. At 100 ng/L, IMD exposure did not cause a significant difference in AChE levels or KLH-specific IgY antibodies. However, tadpoles injected with KLH had slightly higher levels of AChE. In addition, we saw a trend in total IgM with higher levels in tadpoles exposed to IMD. While we found no effect of IMD at 100 ng/L on antibody response to a novel, non-replicating antigen nor on ACh production, further research is needed to determine if higher concentrations of IMD or parasite infection can influence development of R. catesbeiana.

Immunoglobulin detection in wild birds: effectiveness of three secondary anti-avian IgY antibodies in direct ELISAs in 41 avian species.

Immunological reagents for wild, non-model species are limited or often non-existent for many species.In this study, we compare the reactivity of a new anti-passerine IgY secondary antibody with existing secondary antibodies developed for use with birds. Samples from 41 species from the following six avian orders were analysed: Anseriformes (1 family, 1 species), Columbiformes (1 family, 2 species), Galliformes (1 family, 1 species), Passeriformes (16 families, 34 species), Piciformes (1 family, 2 species) and Suliformes (1 family, 1 species). Direct ELISAs were performed to detect total IgY using goat anti-passerine IgY, goat anti-chicken IgY or goat anti-bird IgY secondary antibodies.The anti-passerine antibody exhibited significantly higher IgY reactivity compared to the anti-chicken and/or anti-bird antibodies in 80% of the passerine families tested. Birds in the order Piciformes (woodpeckers) and order Suliformes (cormorants) were poorly detected by all three secondary antibodies. A comparison of serum and plasma IgY levels was made within the same individuals for two passerine species (house finch and white-crowned sparrow), and serum exhibited significantly more IgY than the plasma for all three secondary antibodies. This result indicates that serum may be preferred to plasma when measuring total antibody levels in blood.This study indicates that the anti-passerine IgY secondary antibody can effectively be used in immunological assays to detect passerine IgY for species in most passerine families and is preferred over anti-chicken and anti-bird secondary antibodies for the majority of passerine species. This anti-passerine antibody will allow for more accurate detection and quantification of IgY in more wild bird species than was possible with previously available secondary antibodies.

Effects of bird-feeding activities on the health of wild birds.

Among the most popular reasons that people feed wild birds is that they want to help birds. The extent to which supplemental food helps birds, however, is not well established. From spring 2011 to spring 2014, we examined how feeding of wild birds influences the health of individual birds at forested sites in central Illinois, USA. Specifically, we compared three forested sites where we provided supplemental food with three forested sites for which no supplemental food was available and monitored changes in the individual health of birds. In addition, we determined whether any changes in bird health had occurred after feeders had been removed from sites 10 months before. Generally, the individual health of birds improved with supplemental feeding, including increased antioxidant levels, reduced stress (heterophil-to-lymphocyte ratio) and more rapid feather growth. In some species, we also found improved body condition index scores and innate immune defense. The difference among sites was not present 10 months after feeders were removed, suggesting that the impact on health was indeed related to supplemental feeding. Potential negative effects of supplemental feeding were also found, including an increase in infectious disease prevalence among individual birds at forested sites where supplemental food was offered. Birds with clear signs of pathology showed deficits in most of the physiological metrics in which birds at feeder sites typically showed improved health condition. At the peak of prevalence of infectious disease, 8.3% of all birds at feeders exhibited symptoms of conjunctivitis, pox, dermal disease or cloacal disease. We found both positive and negative impacts of wild bird feeding, and that, in general, birds that had access to supplemental food were in better physiological condition. Moreover, the negative effects we found may be mitigated by hobbyists engaging in safer bird-feeding practices.

Physiology of reproductive senescence in Florida scrub-jays: results from a long-term study and GnRH challenge.

In most vertebrates, production of reproductive hormones wanes with age, co-occurring with a decline in reproductive output. Measurement of these hormones can serve as a key marker of the onset of reproductive senescence. Longitudinal studies of physiological parameters in populations of free-living animals are relatively uncommon; however, we have monitored baseline concentrations of hormones for nine years in a population of Florida scrub-jays (Aphelocoma coerulescens). We hypothesized that concentrations of circulating reproductive hormones change with age, and predicted declines in reproductive hormones in the oldest jays. We found that baseline levels of luteinizing hormone (LH) and testosterone (T) were relatively low in both young and old male breeders and reach their highest levels in birds aged 4-7years. Conversely, we found no age-related patterns in baseline levels of LH or estradiol in female jays. In males we determined which component of the hypothalamo-pituitary-gonadal (HPG) axis is responsible for observed age-based differences, by challenging males of different ages with gonadotropin-releasing hormone (GnRH); thereby allowing assessment of pituitary and gonadal responsiveness by measuring plasma concentrations of luteinizing hormone and testosterone, respectively. The magnitude of increase in levels of both LH and T in response to GnRH challenge decreased with age in male breeders. Combined with the baseline levels, the results from the GnRH challenge suggest that younger birds have the capability to produce higher levels of reproductive hormone, whereas the old birds may be constrained by senescence in their ability to produce these hormones.

Dietary protein restriction impairs growth, immunity, and disease resistance in southern leopard frog tadpoles.

The immune system is a necessary, but potentially costly, defense against infectious diseases. When nutrition is limited, immune activity may consume a significant amount of an organism's energy budget. Levels of dietary protein affect immune system function; high levels can enhance disease resistance. We exposed southern leopard frog [Lithobates sphenocephalus (=Rana sphenocephala)] tadpoles to high and low protein diets crossed with the presence or absence of the pathogenic amphibian chytrid fungus (Batrachochytrium dendrobatidis; Bd) and quantified: (1) tadpole resistance to Bd; (2) tadpole skin-swelling in response to phytohaemagglutinin (PHA) injection (a measure of the T cell-mediated response of the immune system); (3) bacterial killing ability (BKA) of tadpole blood (a measure of the complement-mediated cytotoxicity of the innate immune system); and (4) tadpole growth and development. Tadpoles raised on a low-protein diet were smaller and less developed than tadpoles on a high-protein diet. When controlled for developmental stage, tadpoles raised on a low-protein diet had reduced PHA and BKA responses relative to tadpoles on a high-protein diet, but these immune responses were independent of Bd exposure. High dietary protein significantly increased resistance to Bd. Our results support the general hypothesis that host condition can strongly affect disease resistance; in particular, fluctuations in dietary protein availability may change how diseases affect populations in the field.

Age-related differences in baseline and stress-induced corticosterone in Florida scrub-jays.

In physiological studies of free-living species, it is essential to consider the context of the life history stage at which an individual was observed in order to link measures of physiology with ecological parameters. One such measure that is important to consider is the age of an individual. We tested whether baseline or stress-induced corticosterone levels vary with age in free-living Florida scrub-jays (Aphelocoma coerulescens) during the pre-breeding period. Corticosterone (CORT), the primary avian stress hormone, is released in response to stressful stimuli, and stimulates gluconeogenesis; however, it also serves as a chemical messenger that can influence other physiological processes, reproduction, and behavior. We monitored both baseline CORT levels longitudinally throughout a five-year period and stress-induced CORT responses over a shorter two-year period. We predicted that older jays would have lower baseline CORT levels and a dampened stress response compared to younger birds, as has been shown in other avian species. We found no significant differences in baseline CORT levels with age. We found a decrease in total corticosterone responses to a stressor with age, however, the oldest birds in the population showed greater total corticosterone responses to a stressor. These results may be a product of age-related changes in physiological processes related to the stress response or a result of selection acting on the population, resulting in only the most responsive individuals surviving to old age.

Corticosterone, brood size, and hatch order in free-living Florida scrub-jay (Aphelocoma coerulescens) nestlings.

It is well known that variation in developmental conditions can have profound effects upon lifetime fitness. In altricial avian species, nestlings undergo a substantial portion of development in the nest after hatching, often in the presence of nest-mates. This can result in the formation of brood hierarchies based on age, size, and competitive ability. Measurement of baseline corticosterone (CORT) levels in developing birds may provide a means to assess whether individuals within a brood experience stress due to sibling competition or nutritional state. However, few studies have attempted to correlate corticosterone concentrations with brood hierarchies in free-living, developing birds. We investigated the degree to which corticosterone levels in nestling Florida scrub-jays (Aphelocoma coerulescens) were predicted by brood size, hatching order, and body mass. Nestling corticosterone levels were inversely correlated with body mass but did not differ among brood sizes. Within broods of two, second-hatched nestlings had lower CORT than first-hatched nestlings, although there was no effect of hatch order in broods of three or four nestlings. The results of this study suggest that conditions within the nest, particularly those associated with within-brood hierarchies, are manifested through differential body condition and nestling corticosterone secretion. The consequences of this variation in nestling corticosterone are unknown, but development of the adult phenotype, as well as life-long survival, may be impacted.

Selection on innate immunity and body condition in Florida scrub-jays throughout an epidemic.

Opportunities to investigate selection in free-living species during a naturally occurring epidemic are rare; however, we assessed innate immunocompetence in Florida scrub-jays before the population suffered the greatest over-winter mortality in 20 years of study. Propitiously, three months prior to the epidemic, we had sampled a number of male breeders to evaluate a suite of physiological measures that are commonly used to estimate the overall health-state of an individual. There was a significant, positive selection gradient for both Escherichia coli bacterial killing capability and body condition, suggesting that directional selection had occurred upon each of these traits during the disease epidemic.

The influence of nest attendance and provisioning on nestling stress physiology in the Florida scrub-jay.

Stressful conditions during development may have lasting consequences for an animal's lifetime fitness. We investigated the degree to which parental provisioning and nest attendance influenced baseline levels of the stress hormone corticosterone in nestling Florida scrub-jays. Provisioning rates of male and female breeders and nest attendance of female breeders were recorded during focal watches conducted between days 3 and 5 post-hatch. A small blood sample was taken from each nestling on day 11 post-hatch and used to quantify levels of baseline corticosterone. The proportion of time spent by female breeders at a considerable distance from the nest was positively related to nestling corticosterone levels. Nestling corticosterone was also negatively related to parental provisioning rate, although this effect seemed to be secondary to the effect of the female's time away from the nest. These results indicate that parental behavior contributes to nestling stress physiology, which may in turn direct the formation of the adult phenotype and influence an individual's chances of survival.

Environment, glucocorticoids, and the timing of reproduction.

Glucocorticoids mediate glucose availability under stressful and non-stressful conditions and, therefore, are essential for life. However, data across taxa demonstrate that chronic or elevated secretion of corticosterone or cortisol (CORT) can have negative effects at many levels and can trigger physiological or behavioral responses that may delay or, even halt reproduction. We present a brief overview of the effects that glucocorticoids, primarily the avian form, corticosterone, can have on the reproductive axis. Considerable data have demonstrated that environmental perturbations can result in elevated CORT levels that alter a bird's investment in current reproduction. Studies in our laboratory have shown a link between CORT and timing of reproduction in Florida scrub-jays: in "bad" years, clutch initiation dates are positively correlated with baseline CORT levels of female breeders. Also, population-level differences in CORT levels may explain timing of reproduction as lower CORT levels in suburban-dwelling jays are coupled with early breeding. Most research on stress and CORT concentrates on transient effects of CORT secretion. However, developmental CORT exposure, either from the yolk or embryo, may have long-term effects upon adult phenotype. For example, CORT levels in nestling scrub-jays predicts later 'personality,' as levels were highly correlated (r(2)=0.84) with fearfulness at 7 months of age. One can imagine that such 'personality' traits might also translate into differential success in gaining a territory or a mate. While speculative, it may be that early CORT exposure effectively programs adult behaviors that have wide ranging effects, including upon reproduction.

Corticosterone administration does not affect timing of breeding in Florida scrub-jays (Aphelocoma coerulescens).

Providing supplemental food to Florida scrub-jays (Aphelocoma coerulescens) causes a reliable advance in clutch initiation of 1 to 2 weeks. In some years, supplemental food appeared to not only advance laying date but also decrease baseline concentrations of corticosterone (CORT) relative to controls. The coincidence of low CORT levels and early breeding led us to hypothesize that CORT serves to communicate information about environmental conditions to the hypothalamic-pituitary-gonadal (HPG) axis, which ultimately influences the timing of breeding. To test this hypothesis, we administered small oral doses of CORT three times each day to female breeders that were provisioned with supplemental food. We compared clutch initiation dates of the CORT-dosed females to females with supplementation but no exogenous CORT and to females with neither CORT nor supplemental food. CORT administration had a strong temporary effect on circulating CORT concentrations but clutch initiation did not differ between the two groups of supplemented birds, both of which laid eggs approximately 10 days earlier than nonsupplemented birds. Furthermore, during the year of our study we found no reduction in baseline CORT concentrations in our undosed supplemental groups, as had been observed in past studies.