Effect of dietary macronutrients and immune challenge on gut microbiota, physiology and feeding behaviour in zebra finches.

Macronutrients play a vital role in host immunity and can influence host-pathogen dynamics, potentially through dietary effects on gut microbiota. To increase our understanding of how dietary macronutrients affect physiology and gut microbiota and investigate whether feeding behaviour is influenced by an immune threat, we conducted two experiments. First, we determined whether zebra finches (Taeniopygia guttata) exhibit shifts in physiology and gut microbiota when fed diets differing in macronutrient ratios. We found the type and amount of diet consumed affected gut microbiota alpha diversity, where microbial richness and Shannon diversity increased with caloric intake in birds fed a high-fat diet and decreased with caloric intake in birds fed a high protein diet. Diet macronutrient content did not affect physiological metrics, but lower caloric intake was associated with higher complement activity. In our second experiment, we simulated an infection in birds using the bacterial endotoxin lipopolysaccharide (LPS) and quantified feeding behaviour in immune challenged and control individuals, as well as birds housed near either a control pair (no immune threat), or birds housed near a pair given an immune challenge with LPS (social cue of heightened infection risk). We also examined whether social cues of infection alter physiological responses relevant to responding to an immune threat, an effect that could be mediated through shifts in feeding behaviour. LPS induced a reduction in caloric intake driven by a decrease in protein, but not fat consumption. No evidence was found for socially induced shifts in feeding behaviour, physiology or gut microbiota. Our findings carry implications for host health, as sickness-induced anorexia and diet-induced shifts in the microbiome could shape host-pathogen interactions.

Male pathology regardless of behaviour drives transmission in an avian host-pathogen system.

Host sex is an important source of heterogeneity in the severity of epidemics. Pinpointing the mechanisms causing this heterogeneity can be difficult because differences in behaviour among sexes (e.g. greater territorial aggression in males) can bias exposure risk, obfuscating the role of immune function, which can lead to differences in pathology, in driving differential susceptibility between sexes. Thus, sex-biased transmission driven by differences in immune function independent of behaviour is poorly understood, especially in non-mammalian systems. Here we examine the previously unexplored potential for male-biased pathology to affect transmission using an avian host-pathogen system. We employ a sex-dependent multistate transmission model parameterized with isolated, individual-based experimental exposures of domestic canaries and experimental transmission data of house finches. The experiment revealed that male birds have shorter incubation periods, longer recovery periods, higher pathogen burdens and greater disease pathology than females. Our model revealed that male-biased pathology led to epidemic size rapidly increasing with the proportion of male birds, with a nearly 10-fold increase in total epidemic size from an all-female to an all-male simulation. Our results demonstrate that female-biased resistance, independent of male behaviour, can drive sex-dependent transmission in wildlife, indicating that sex-based differences in immune function, not just differences in exposure risk, can shape epidemic dynamics.

Perception of infection: disease-related social cues influence immunity in songbirds.

While avoidance of sick conspecifics is common among animals, little is known about how detecting diseased conspecifics influences an organism's physiological state, despite its implications for disease transmission dynamics. The avian pathogen Mycoplasma gallisepticum (MG) causes obvious visual signs of infection in domestic canaries (Serinus canaria domestica), including lethargy and conjunctivitis, making this system a useful tool for investigating how the perception of cues from sick individuals shapes immunity in healthy individuals. We tested whether disease-related social information can stimulate immune responses in canaries housed in visual contact with either healthy or MG-infected conspecifics. We found higher complement activity and higher heterophil counts in healthy birds viewing MG-infected individuals around 6-12 days post-inoculation, which corresponded with the greatest degree of disease pathology in infected stimulus birds. However, we did not detect the effects of disease-related social cues on the expression of two proinflammatory cytokines in the blood. These data indicate that social cues of infection can alter immune responses in healthy individuals and suggest that public information about the disease can shape how individuals respond to infection.

Effects of sublethal application of Deepwater Horizon oil to bird eggs on embryonic heart and metabolic rate.

Following large crude oil spills, oil from feathers of brooding birds and oiled nesting material can transfer to eggs, resulting in reduced embryonic viability for heavily oiled eggs. Eggs may also be subjected to trace or light oiling, but functional teratogenic effects from sublethal crude oil exposure have not been examined. We assessed whether sublethal application of weathered Deepwater Horizon crude oil to the eggshell surface alters heart rate and metabolic rate in Zebra Finch (Taeniopygia guttata) embryos. We first determined sublethal applications with a dosing experiment. Embryo viability for eggs exposed to 5 µL or more of crude oil decreased significantly. We conducted a second experiment to measure heart rate and metabolic rate (CO2 production) 5 and 9 d after 1 sublethal application of crude oil to eggshells on day 3 of incubation. One application of 1.0 or 2.5 µL of crude oil reduced embryonic heart rate and metabolic rate on day 12 of incubation. Using unfertilized eggs, we measured the transfer of polycyclic aromatic hydrocarbons (PAHs) from the eggshell surface to egg contents 9 d after a single application of sublethal crude oil. Our results suggest avian eggs externally exposed to small amounts of crude oil may exhibit protracted embryonic development and impaired postnatal cardiac performance.

Aplicaciones Subletales de Petróleo de la Plataforma Deepwater Horizon en los Huevos de Aves y sus Efectos sobre la Frecuencia Cardíaca y la Tasa Metabólica de Embriones Resumen Después de grandes derrames de crudo, el petróleo que se encuentra en las plumas de las aves incubadoras y el petróleo adherido al material para nidos puede transferirse a los huevos, lo que resulta en la reducción de la viabilidad embrionaria en el caso de los huevos con un contacto alto con el petróleo. Los huevos también pueden estar sujetos a manchas de petróleo ligeras o por contacto, pero no se han examinado los efectos teratogénicos funcionales de la exposición subletal al crudo. Evaluamos si la aplicación subletal de petróleo crudo desgastado del derrame de la plataforma Deepwater Horizon a la superficie de los cascarones altera la frecuencia cardíaca y la tasa metabólica de los embriones de gorrión cebra (Taeniopygia guttata). Primero determinamos las aplicaciones subletales con un experimento de dosificación. La viabilidad de los embriones en huevos expuestos a 5 µL o más de crudo disminuyó significativamente. Realizamos un segundo experimento para medir la frecuencia cardíaca y la tasa metabólica (producción de CO2 ) cinco y nueve días después de una aplicación subletal de crudo a los cascarones durante el tercer día de incubación. La aplicación de 1.0 o de 2.5 µL de crudo redujo la frecuencia cardíaca y la tasa metabólica al décimo segundo día de incubación. También medimos la transferencia de hidrocarburos aromáticos policíclicos (PAHs) en huevos sin fertilizar desde la superficie del cascarón hasta el contenido del huevo nueve días después de una aplicación subletal única de crudo. Nuestros resultados sugieren que los huevos de aves expuestos externamente a pequeñas cantidades de crudo pueden tener un desarrollo embrionario prolongado y un desempeño cardíaco postnatal deteriorado.

Cortisol is the predominant glucocorticoid in the giant paedomorphic hellbender salamander (Cryptobranchus alleganiensis).

Corticosterone is widely regarded to be the predominant glucocorticoid produced in amphibians. However, we recently described unusually low baseline and stress-induced corticosterone profiles in eastern hellbenders (Cryptobranchus alleganiensis alleganiensis), a giant, fully aquatic salamander. Here, we hypothesized that hellbenders might also produce cortisol, the predominant glucocorticoid used by fishes and non-rodent mammals. To test our hypothesis, we collected plasma samples in two field experiments and analyzed them using multiple analytical techniques to determine how plasma concentrations of cortisol and corticosterone co-varied after 1) physical restraint and 2) injection with adrenocorticotropic hormone (ACTH), the pituitary hormone responsible for triggering the release of glucocorticoids from amphibian interrenal glands. Using liquid chromatography-mass spectrometry, we found that baseline and restraint-induced plasma concentrations of cortisol were more than five times those of corticosterone. We then demonstrated that plasma concentrations of both glucocorticoids increased in response to ACTH in a dose-dependent manner, but cortisol concentrations were consistently higher (up to 10-fold) than corticosterone. Cortisol and corticosterone concentrations were not correlated with one another at basal or induced conditions. The extremely low plasma concentrations of corticosterone in hellbenders suggests that corticosterone could simply be a byproduct of cortisol production, and raises questions as to whether corticosterone has any distinct physiological function in hellbenders. Our results indicate that hellbenders produce cortisol as their predominant glucocorticoid, supporting a small and inconclusive body of literature indicating that some other amphibians may produce appreciable quantities of cortisol. We hypothesize that the use of cortisol by hellbenders could be an adaptation to their fully aquatic life history due to cortisol's ability to fulfill both mineralocorticoid and glucocorticoid functions, similar to its functions in fishes. Given the large number of amphibian species that are fully aquatic or have aquatic life stages, we suggest that the broadly held assumption that corticosterone is the predominant glucocorticoid in all amphibians requires further scrutiny. Ultimately, multi-species tests of this assumption will reveal the ecological factors that influenced the evolution of endocrine adaptations among amphibian lineages, and may provide insight into convergent evolution of endocrine traits in paedomorphic species.

Prolactin is related to incubation constancy and egg temperature following a disturbance in a precocial bird.

To maximize fitness, parents may trade-off time and energy between parental care and self-maintenance. In vertebrates, prolactin and corticosterone are two important hormones that regulate parental investment because they stimulate parental care and mobilize energy, respectively. Further, concentrations of both hormones change in response to disturbances. One of the most important parental care behaviors in birds is incubation, since small changes in egg temperature have large effects on offspring. We investigated how prolactin and corticosterone may mediate parental incubation constancy (i.e., the daily amount of time spent incubating eggs) and regulation of egg temperature. We collected blood samples from female wood ducks (Aix sponsa) near the start and end of the incubation period to measure baseline and stress-induced (30 min after capture and restraint) hormone concentrations. We also quantified incubation constancy and egg temperature using artificial egg temperature loggers. As expected, prolactin decreased and corticosterone increased after 30 min of capture and restraint. Corticosterone concentrations (baseline and stress-induced) were negatively related to body mass, but were not related to incubation constancy. In contrast, prolactin concentrations (baseline and stress-induced) were higher at the end than the start of the incubation period, and stress-induced prolactin concentrations were positively related to incubation constancy following a nest disturbance (i.e., capture). Further, prolactin (baseline and stress-induced) concentrations were positively related to egg temperatures, but only after the disturbance. These results suggest that prolactin may be associated with the regulation of parental incubation constancy and resulting heat-transfer after a disturbance, which may ultimately affect offspring development.

Avian parental behavior and nest success influenced by temperature fluctuations.

Behavioral adjustments and parental decisions during reproduction can influence the thermal environment at nests, yet our understanding into how environmental factors (i.e., temperature and precipitation) constrain an adult's ability to balance self-maintenance and incubation demands is limited. To expand our understanding of how species respond to environmental factors, we investigated the reproductive ecology of two ground-nesting species (northern bobwhite [Colinus virginianus] and scaled quail [Callipepla squamata]) in a region (i.e., the Southern Great Plains) prone to thermal variability (i.e., extreme hot and cold temperatures). Specifically, our objective was to examine how temperature and precipitation directly influenced behavioral adjustments (i.e., off-bout duration, frequency, and nest attentiveness) and parental decisions (i.e., nest site selection), and indirectly influenced nest fate. Overall, we found that parents chose to nest in sites that were significantly cooler in temperature than randomly selected sites, and parents further altered the thermal environment experienced by embryos through incubation behavior. Daily precipitation and average ambient temperature and/or their interaction best predicted incubation behaviors, yet each species differed in the timing (i.e., morning vs. evening), frequency, and duration of off-bouts. Furthermore, successful nests were associated with cooler nest site temperatures for bobwhite and warmer nest site temperatures for scaled quail. Our finding of relatively stable (35.5 °C) incubation temperature for developing embryos of both species suggests that ground-nesting birds are able to regulate microclimate through behavioral adjustments and parental decisions even under extreme temperature fluctuations. Nevertheless, the ability for a ground-nesting species to effectively modify behavioral adjustments and decisions may be altered during long periods of enhanced physiological and environmental stress.

Captivity influences immune responses, stress endocrinology, and organ size in house sparrows (Passer domesticus).

Studies using wild animals in laboratory-based research require bringing wild-captured organisms into a novel setting, which can have long-lasting impacts on physiology and behavior. In several species, captivity stimulates stress hormone production and can alter immune function. Despite this, there is little consensus on how captivity influences stress hormone regulation, or if captivity-induced changes in stress hormone production and regulation mediate changes in immune function. In this study, we investigate the influence of captivity on the physiology of a wild bird commonly-used in laboratory-based research, the house sparrow (Passer domesticus). We tested how captivity influences stress endocrinology, immune responses, and organ mass, and also investigated if the production or regulation of corticosterone, the main stress hormone in birds, correlated with changes in immunity. We found that baseline corticosterone concentrations and maximum capacity of the adrenals to secrete corticosterone increase following captivity and remain elevated after 9weeks of captivity. A measure of innate immune function, the bactericidal ability of plasma, also increased with time spent in captivity. Wound healing was also influenced by time spent in captivity, with birds taking almost 2days longer to heal if they were wounded after 3weeks in captivity when compared with birds that were wounded immediately upon capture. Additionally, captivity caused notable reductions in spleen and liver mass. Together, these results imply that captivity can have long-lasting effects on house sparrow corticosterone release and immune function, and suggest that even after 9weeks house sparrows do not acclimate physiologically to life in captivity.

Incubation temperature causes skewed sex ratios in a precocial bird.

Many animals with genetic sex determination are nonetheless capable of manipulating sex ratios via behavioral and physiological means, which can sometimes result in fitness benefits to the parent. Sex ratio manipulation in birds is not widely documented, and revealing the mechanisms for altered sex ratios in vertebrates remains a compelling area of research. Incubation temperature is a key component of the developmental environment for birds, but despite its well-documented effects on offspring phenotype it has rarely been considered as a factor in avian sex ratios. Using ecologically relevant manipulations of incubation temperature within the range 35.0-37.0°C, we found greater mortality of female embryos during incubation than males regardless of incubation temperature, and evidence that more female than male embryos die at the lowest incubation temperature (35.0°C). Our findings in conjunction with previous work in brush turkeys suggest incubation temperature is an important determinant of avian secondary sex ratios that requires additional study, and should be considered when estimating the impact of climate change on avian populations.

Evidence of ectoparasite-induced endocrine disruption in an imperiled giant salamander, the eastern hellbender (Cryptobranchus alleganiensis).

Parasitic leeches and trypanosomes release chemical signals into their hosts to evade immuno-detection, but it is unknown whether these compounds manipulate host behavior or endocrine physiology. We determined whether parasitic infections with leeches and/or trypanosomes affected the immune and stress response of an imperiled giant species of amphibian, the eastern hellbender (Cryptobranchus alleganiensis Daudin). We monitored corticosterone and white blood cell counts in response to restraint and injection with adrenocorticotropin hormone (ACTH) or saline for up to 50 h. The presence of leeches dampened hellbender corticosterone responses to restraint and reduced diel patterns of plasma corticosterone. Injection with ACTH restored the normal inter-renal responses of hellbenders, suggesting that leeches, possibly through neurotransmitters in leech saliva, cause down-regulation of corticosterone release at the level of the pituitary or hypothalamus. Infection with leeches also increased the relative abundance of eosinophils, white blood cells often recruited into circulation in response to parasitic infection. Lastly, neutrophil to lymphocyte (N:L) ratios increased in all animals after 24 h of capture and remained elevated for up to 50 h, but these temporal dynamics did not differ with parasite infection. Trypanosome infection did not affect any aspect of hellbender physiology that we measured. Our findings reveal a previously undocumented host-parasite dynamic. While the functional significance to the parasite is unclear, the physiological and behavioral implications for the host are great, given the important role of glucocorticoids in regulating physiology and behavior.

Energetic constraints and parental care: is corticosterone indicative of energetic costs of incubation in a precocial bird?

Suppression of the adrenocortical response (e.g., corticosterone release) to an acute stressor is a physiological adjustment thought to decrease the likelihood of avian parents abandoning their nests. However, some periods of parental care, like incubation, are energetically costly, thus corticosterone could increase during these stages to allow incubating parents to utilize energy reserves. Wood ducks (Aix sponsa) have ~30 day incubation periods and only the female incubates the eggs. We hypothesized that corticosterone would be important in regulating energy availability during incubation in this species. Because resources invested in reproduction increase with clutch size, we also hypothesized that clutch size would influence plasma corticosterone during incubation. We measured baseline and stress-induced corticosterone in incubating females during early and late stages of incubation. At both stages of incubation all hens had low baseline corticosterone levels. However, we found that stress-induced corticosterone was 105% greater late in incubation than early in incubation. We also detected a significant negative correlation between female body mass and stress-induced corticosterone late in incubation, but not during the early stages of incubation. Furthermore, we found a significant positive relationship between stress-induced corticosterone and clutch size. These lines of evidence support the hypothesis that incubation in wood ducks is energetically costly and corticosterone is important in supporting the energetic demands of incubating hens. Our findings suggest that corticosterone's role in supporting parental care behaviors are dynamic and are influenced by several factors and that there is a greater physiological cost associated with incubating larger clutches.

Incubation temperature affects multiple measures of immunocompetence in young wood ducks (Aix Sponsa).

Parental effects play a vital role in shaping offspring phenotype. In birds, incubation behaviour is a critical parental effect because it influences the early developmental environment and can therefore have lifelong consequences for offspring phenotype. Recent studies that manipulated incubation temperature found effects on hatchling body composition, condition and growth, suggesting that incubation temperature could also affect energetically costly physiological processes of young birds that are important to survival (e.g. immune responses). We artificially incubated wood duck (Aix sponsa) eggs at three biologically relevant temperatures. Following incubation, we used two immunoassays to measure acquired immune responses of ducklings. Ducklings incubated at the lowest temperature had reduced growth, body condition and responses to both of our immune challenges, compared with those from the higher temperatures. Our results show that incubation temperatures can be an important driver of phenotypic variation in avian populations.

Elevated plasma corticosterone increases metabolic rate in a terrestrial salamander.

Plasma glucocorticoid hormones (GCs) increase intermediary metabolism, which may be reflected in whole-animal metabolic rate. Studies in fish, birds, and reptiles have shown that GCs may alter whole-animal energy expenditure, but results are conflicting and often involve GC levels that are not physiologically relevant. A previous study in red-legged salamanders found that male courtship pheromone increased plasma corticosterone (CORT; the primary GC in amphibians) concentrations in males, which could elevate metabolic processes to sustain courtship behaviors. To understand the possible metabolic effect of elevated plasma CORT, we measured the effects of male courtship pheromone and exogenous application of CORT on oxygen consumption in male red-legged salamanders (Plethodon shermani). Exogenous application of CORT elevated plasma CORT to physiologically relevant levels. Compared to treatment with male courtship pheromone and vehicle, treatment with CORT increased oxygen consumption rates for several hours after treatment, resulting in 12% more oxygen consumed (equivalent to 0.33 J) during our first 2h sampling period. Contrary to our previous work, treatment with pheromone did not increase plasma CORT, perhaps because subjects used in this study were not in breeding condition. Pheromone application did not affect respiration rates. Our study is one of the few to evaluate the influence of physiologically relevant elevations in CORT on whole-animal metabolism in vertebrates, and the first to show that elevated plasma CORT increases metabolism in an amphibian.

Innate immunity and stress physiology of eastern hellbenders (Cryptobranchus alleganiensis) from two stream reaches with differing habitat quality.

In addition to depriving amphibians of physical habitat requirements (e.g., shelter, moisture, and food), habitat modification may also have subtle effects on the health of amphibians and potentially precipitate interactions with other deleterious factors such as pathogens, contaminants, and invasive species. The current study was designed to evaluate the physiological state of imperiled giant salamanders, the eastern hellbender (Cryptobranchus alleganiensis), experiencing different surrounding land use that influences in-stream habitat quality. When we compared hellbenders from a stream reach with greater anthropogenic disturbance to a more forested site, we found that baseline and stress-induced plasma levels of corticosterone were similar in the two areas, but were very low compared to other amphibians. Males consistently had higher plasma corticosterone levels than females, a finding congruent with the known territorial activities of males early in the breeding season. Innate immune responsiveness (measured as bactericidal ability of blood; BKA) was also similar at the two sites, but juveniles had less robust BKA than adults. We found a positive relationship between restraint time and BKA, suggesting that the bactericidal ability of hellbenders may improve following acute stress. Finally, there was a tendency for hellbenders with skin abnormalities to have higher BKA compared to individuals with normal integument, an observation consistent with patterns observed in other animals actively responding to pathogens. Our study provides foundational physiological information on an imperiled amphibian species and reveals important knowledge gaps that will be important for understanding the ecology, evolution, and conservation of hellbenders.

Bold Behavior Is Associated with Genes That Regulate Energy Use but Does Not Covary with Body Condition in Food-Restricted Snails.

AbstractTheoretical models about the relationship between food restriction and individual differences in risk-taking behavior (i.e., boldness) have led to conflicting predictions: some models predict that food restriction increases boldness, while other models predict that food restriction decreases boldness. This discrepancy may be partially attributable to an underappreciation for animals' complex physiological responses to food restriction. To understand the proximate mechanisms mediating state-dependent boldness, we used freshwater snails (Helisoma trivolvis) to examine the relationships among food availability, body condition, boldness (latency to reemerge from shell and exploration), and mRNA expression of three genes (adenosine monophosphate-activated protein kinase [AMPK], molluscan insulin-like peptide [MIP], and serotonin receptor [5-HT]) involved in maintaining energy homeostasis during periods of moderate food restriction. Latency to reemerge and exploratory behavior decreased over time, but fed snails were bolder than fasted snails, suggesting that food restriction reduces bold behavior. Although food restriction decreased body condition, there was not a relationship between body condition and latency to reemerge from shell. However, expression of MIP was positively correlated with latency to reemerge from shell. Furthermore, AMPK was positively correlated with MIP and negatively correlated with body condition and 5-HT. Therefore, individual differences in physiological responses to food restriction, not overall body condition per se, appear to be more closely associated with state-dependent bold behavior. Finally, snails that experienced a novel assay environment returned to their initial "shy" behavior, suggesting that habituation to the assay environment may contribute to snails expressing bolder behavior over time.

Hematological and histological changes from ingestion of Deepwater Horizon crude oil in zebra finches (Taeniopygia guttata).

Exposure to crude oil during spill events causes a variety of pathologic effects in birds, including oxidative injury to erythrocytes, which is characterized in some species by the formation of Heinz bodies and subsequent anemia. However, not all species appear to develop Heinz bodies or anemia when exposed to oil, and there are limited controlled experiments that use both light and electron microscopy to evaluate structural changes within erythrocytes following oil exposure. In this study, we orally dosed zebra finches (Taeniopygia guttata) with 3.3 or 10 mL/kg of artificially weathered Deepwater Horizon crude oil or 10 mL/kg of peanut oil (vehicle control) daily for 15 days. We found that birds receiving the highest dosage experienced a significant increase in reticulocyte percentage, mean corpuscular hemoglobin concentration, and liver mass, as well as inflammation of the gastrointestinal tract and lymphocyte proliferation in the spleen. However, we found no evidence of Heinz body formation based on both light and transmission electron microscopy. Although there was a tendency for packed cell volume and hemoglobin to decrease in birds from the high dose group compared to control and low dose groups, the changes were not statistically significant. Our results indicate that additional experimental dosing studies are needed to understand factors (e.g., dose- and species-specific sensitivity) and confounding variables (e.g., dispersants) that contribute to the presence and severity of anemia resulting from oil exposure in birds.

Applications of machine learning in behavioral ecology: Quantifying avian incubation behavior and nest conditions in relation to environmental temperature.

In the age of machine learning, building programs that take advantage of the speed and specificity of algorithm development can greatly aid efforts to quantify and interpret changes in animal behavior in response to abiotic environmental factors, like temperature. For both endotherms and ectotherms, temperature can affect everything from daily energy budgets to nesting behaviors. For instance, in birds environmental temperature plays a key role in shaping parental incubation behavior and temperatures experienced by embryos. Recent research indicates that temperatures experienced by embryos affect viability and are important in shaping fitness-related traits in young birds, sparking renewed interest in relationships among environmental factors, parental incubation behavior, and incubation temperature. Incubation behavior of birds can be monitored non-invasively by placing thermal probes into the nest and analyzing temperature fluctuations that occur as parents attend and leave the nest (on- and off-bouts, respectively). When other measures of temperature (e.g., ambient air or operative temperature) are collected simultaneously with incubation temperature it is possible to compare shifts in behavior with environmental changes. To improve analysis of incubation behavior using these large thermal data sets we developed a program, NestIQ, that uses machine learning to guide parameter optimization allowing it to track the behavior of diverse species. NestIQ's algorithm was tested using six species incubating in lab or field scenarios, that exhibit unique incubation patterns. This stand-alone and open source software is operated through a graphical user interface (i.e., no user programming is required) that provides important behavioral and thermal output statistics. Further, measures of environmental temperature can be imported alongside nest temperature into the program, which then reports various attributes of environmental temperature during shifts in parental behavior. This program will improve the ability of avian ecologists to interpret a critical parental care behavior that can be used across diverse incubation scenarios and species. Although specifically designed for quantifying avian incubation, NestIQ has the potential for broader applications, including basking and nesting behaviors of non-avian reptiles in relation to ambient temperature.

Fluorescent Heme Degradation Products Are Biomarkers of Oxidative Stress and Linked to Impaired Membrane Integrity in Avian Red Blood Cells.

Oxidative stress is generally understood to be an important mediator of life-history traits, yet the specific relationships between oxidative stress and life-history traits have been difficult to describe because there is often a lack of covariation among biomarkers of oxidative stress. For instance, although oxidative damage to red blood cell (RBC) membranes can lead to pathological conditions (i.e., anemia), in some cases there is not a clear relationship between lipid oxidation and RBC membrane resistance to pro-oxidants. Alternatively, oxidative damage to hemoglobin may be an indirect mechanism contributing to RBC membrane damage. To better understand the mechanisms contributing to oxidative damage and probe new approaches to measuring oxidative stress, we used a series of in vitro and in vivo procedures in zebra finches (Taeniopygia guttata) to explore (1) whether avian RBCs exposed to a pro-oxidant generate fluorescent heme degradation products (HDPs), (2) whether HDPs interact with RBC membranes, and (3) whether HDPs are linked to impaired RBC integrity. We found that finch RBCs exposed in vitro to hydrogen peroxide produced fluorescent HDPs and HDPs associated with RBC membranes. Exposure to hydrogen peroxide also caused a reduction in hemoglobin and an increase in percent methemoglobin (a hemoglobin oxidation product), further indicating hemoglobin degradation. Moreover, HDP fluorescence correlated with impaired membrane integrity and erythrocyte osmotic fragility in vivo. This study suggests that reactive oxygen species may indirectly impair RBC membrane integrity via hemoglobin degradation products that associate with RBC membranes and that HDPs may be an inexpensive and logistically simple tool for measuring oxidative stress.

Weathered Mississippi Canyon 252 crude oil ingestion alters cytokine signaling, lowers heterophil:lymphocyte ratio, and induces sickness behavior in zebra finches (Taeniopygia guttata).

The Deepwater Horizon (DWH) oil spill caused an estimated 100,000 bird mortalities. However, mortality estimates are often based on the number of visibly oiled birds and likely underestimate the true damage to avian populations as they do not include toxic effects from crude oil ingestion. Elevated susceptibility to disease has been postulated to be a significant barrier to recovery for birds that have ingested crude oil. Effective defense against pathogens involves integration of physiological and behavioral traits, which are regulated in-part by cytokine signaling pathways. In this study, we tested whether crude oil ingestion altered behavioral and physiological aspects of disease defense in birds. To do so, we used artificially weathered Mississippi Canyon 242 crude oil to orally dose zebra finches (Taeniopygia guttata) with 3.3 mL/kg or 10 mL/kg of crude oil or a control (peanut oil) for 14 days. We measured expression of cytokines (interleukin [IL]-1ß, IL-6, IL-10) and proinflammatory pathways (NF-κB, COX-2) in the intestine, liver, and spleen (tissues that exhibit pathology in oil-exposed birds). We also measured heterophil:lymphocyte (H:L) ratio and complement system activity, and video-recorded birds to analyze sickness behavior. Finches that ingested crude oil exhibited tissue-specific changes in cytokine mRNA expression. Proinflammatory cytokine expression decreased in the intestine but increased in the liver and spleen. Birds exposed to crude oil had lower H:L ratios compared to the control on day 14, but there were no differences in complement activity among treatments. Additionally, birds exposed to 10 mL/kg crude oil had reduced activity, indicative of sickness behavior. Our results suggest cytokines play a role in mediating physiological and behavioral responses to crude oil ingestion. Although most avian population damage assessments focus on mortality caused by external oiling, crude oil ingestion may also indirectly affect survival by altering physiological and behavioral traits important for disease defense.

Parental Effects and Climate Change: Will Avian Incubation Behavior Shield Embryos from Increasing Environmental Temperatures?

A major driver of wildlife responses to climate change will include non-genomic effects, like those mediated through parental behavior and physiology (i.e., parental effects). Parental effects can influence lifetime reproductive success and survival, and thus population-level processes. However, the extent to which parental effects will contribute to population persistence or declines in response to climate change is not well understood. These effects may be substantial for species that exhibit extensive parental care behaviors, like birds. Environmental temperature is important in shaping avian incubation behavior, and these factors interact to determine the thermal conditions embryos are exposed to during development, and subsequently avian phenotypes and secondary sex ratios. In this article, we argue that incubation behavior may be an important mediator of avian responses to climate change, we compare incubation strategies of two species adapted to different thermal environments nesting in extreme heat, and we present a simple model that estimates changes in egg temperature based on these incubation patterns and predicted increases in maximum daily air temperature. We demonstrate that the predicted increase in air temperature by 2100 in the central USA will increase temperatures that eggs experience during afternoon off-bouts and the proportion of nests exposed to lethal temperatures. To better understand how species and local adaptations and behavioral-plasticity of incubation behavior will contribute to population responses to climate change comparisons are needed across more avian populations, species, and thermal landscapes.