Comparative Immune Function in Wild Birds

Over the last several decades, interest in quantifying immune function in comparative studies of wild animals has grown appreciably. Now, the field of ecological immunology is undergoing a transition, and ¿second generation¿ studies are being designed and carried out. With a greater appreciation of the complexity of immune systems, these second generation studies are commonly distinguished from their antecedents by making comparisons using multiple assays and including multiple species. I worked to advance this transition by developing novel approaches to comparative immunology, exploring the interrelationships among indices of immune function, and applying multiple indices to a question of comparative avian evolution. First, I worked to develop individual methodologies that would be broadly applicable given the numerous limitations of field-based immunology. I present methodological details on two assays¿a hemolysis-hemagglutination assay and a bacteria killing assay, and I report on intra- and inter-specific comparisons using both. Relatedly, using ten species of waterfowl, I examine how these and other indices correlate at both the individual and species levels. Next, with an interest in developing a better understanding of the evolutionary forces molding immune function, I set out to broadly compare immune function in 15 phylogenetically matched pairs of bird populations from North America and from the islands of Hawaii, Bermuda, and the Gal¿pagos. If immune defenses were costly, populations from relatively disease-free, oceanic islands are expected to exhibit attenuated immune function in response to reduced pathogen and parasite pressure. In fact, many island animals exhibit this postulated ¿island syndrome,¿ one facet of which is increased susceptibility to disease. After employing three protocols to measure eight indices of immune function, I found no support for my hypothesis. Rather than evidence of depauperate parasite communities and inherent costs of immune defenses selecting for reduced immune function, I found that several indices were elevated in island birds. I suggest that life on islands is accompanied by an apparent reorganization of the relative importance of various immune components. Finally, in collaborative efforts with investigators here and at other institutions, I apply the hemolysis-hemagglutination assay to address a variety of questions across three diverse avian study systems

Histology of Dichromatic and Seasonal Color Change of the Cranial Region of Callagur borneoensis

Male Callagur borneoensis exihibit sexual dichromatism and seasonal coloration which is rare among turtles. In the breeding season male\u27s heads are colored white with a scarlet stripe from the occiput to the tip of the snout. After the breeding season they change to drab charcoal-gray to black with a dull orange-yellow stripe. Females and juveniles are a drab brown throughout the year. This study was performed to determine the mechanisms of this color change at the histological level. Histological preparation of the head revealed a significant increase in vascular tissue just below the epidermis of the stripe area with increased red color. The white color of the side of the head was attributed to no epidermal melanosomes and thickening of the epidermis. Dermal melanin was found to play a lesser role in determining the darkness of the skin. Dermal melanophores appeared to have a cycle during the color change. In dark phase I, they were the most prominant donating melanosomes to the epidermis. In dark phase II they were the least prominent appearing to donate melanosomes to the epidermis and blood vessels along which they were aggregated. The melanophores of the light phase were slightly more prominent and were not donating any melanosome to the epidermis. The intermediate had more prominent melanophores that were donating melanosomes to the epidermis

Histology of Dichromatic and Seasonal Color Change of the Cranial Region of Callagur borneoensis

Male Callagur borneoensis exihibit sexual dichromatism and seasonal coloration which is rare among turtles. In the breeding season male\u27s heads are colored white with a scarlet stripe from the occiput to the tip of the snout. After the breeding season they change to drab charcoal-gray to black with a dull orange-yellow stripe. Females and juveniles are a drab brown throughout the year. This study was performed to determine the mechanisms of this color change at the histological level. Histological preparation of the head revealed a significant increase in vascular tissue just below the epidermis of the stripe area with increased red color. The white color of the side of the head was attributed to no epidermal melanosomes and thickening of the epidermis. Dermal melanin was found to play a lesser role in determining the darkness of the skin. Dermal melanophores appeared to have a cycle during the color change. In dark phase I, they were the most prominant donating melanosomes to the epidermis. In dark phase II they were the least prominent appearing to donate melanosomes to the epidermis and blood vessels along which they were aggregated. The melanophores of the light phase were slightly more prominent and were not donating any melanosome to the epidermis. The intermediate had more prominent melanophores that were donating melanosomes to the epidermis

Offspring pay sooner, parents pay later:Experimental manipulation of body mass reveals trade-offs between immune function, reproduction and survival

Introduction: Life-history theory predicts that organisms trade off survival against reproduction. However, the time scales on which various consequences become evident and the physiology mediating the cost of reproduction remain poorly understood. Yet, explaining not only which mechanisms mediate this trade-off, but also how fast or slow the mechanisms act, is crucial for an improved understanding of life-history evolution. We investigated three time scales on which an experimental increase in body mass could affect this trade-off: within broods, within season and between years. We handicapped adult skylarks (Alauda arvensis) by attaching extra weight during first broods to both adults of a pair. We measured body mass, immune function and return rates in these birds. We also measured nest success, feeding rates, diet composition, nestling size, nestling immune function and recruitment rates.Results: When nestlings of first broods fledged, parent body condition had not changed, but experimental birds experienced higher nest failure. Depending on the year, immune parameters of nestlings from experimental parents were either higher or lower than of control nestlings. Later, when parents were feeding their second brood, the balance between self-maintenance and nest success had shifted. Control and experimental adults differed in immune function, while mass and immune function of their nestlings did not differ. Although weights were removed after breeding, immune measurements during the second brood had the capacity to predict return rates to the next breeding season. Among birds that returned the next year, body condition and reproductive performance a year after the experiment did not differ between treatment groups.Conclusions: We conclude that the balance between current reproduction and survival shifts from affecting nestlings to affecting parents as the reproductive season progresses. Furthermore, immune function is apparently one physiological mechanism involved in this trade-off. By unravelling a physiological mechanism underlying the trade-offs between current and future reproduction and by demonstrating the different time scales on which it acts, our study represents an important step in understanding a central theory of life-history evolution.</p

Immune function differs among tropical environments but is not downregulated during reproduction in three year-round breeding equatorial lark populations

Seasonal variation in immune function can be attributed to life history trade-offs, and to variation in environmental conditions. However, because phenological stages and environmental conditions co-vary in temperate and arctic zones, their separate contributions have not been determined. We compared immune function and body mass of incubating (female only), chick-feeding (female and male), and non-breeding (female and male) red-capped larks Calandrella cinerea breeding year-round in three tropical equatorial (Kenya) environments with distinct climates. We measured four immune indices: haptoglobin, nitric oxide, agglutination, and lysis. To confirm that variation in immune function between breeding (i.e., incubating or chick-feeding) and non-breeding was not confounded by environmental conditions, we tested if rainfall, average minimum temperature (Tmin), and average maximum temperature (Tmax) differed during sampling times among the three breeding statuses per location. Tmin and Tmax differed between chick-feeding and non-breeding, suggesting that birds utilized environmental conditions differently in different locations for reproduction. Immune indices did not differ between incubating, chick-feeding and non-breeding birds in all three locations. There were two exceptions: nitric oxide was higher during incubation in cool and wet South Kinangop, and it was higher during chick-feeding in the cool and dry North Kinangop compared to non-breeding birds in these locations. For nitric oxide, agglutination, and lysis, we found among-location differences within breeding stage. In equatorial tropical birds, variation in immune function seems to be better explained by among-location climate-induced environmental conditions than by breeding status. Our findings raise questions about how within-location environmental variation relates to and affects immune function

Geographical and temporal variation in environmental conditions affects nestling growth but not immune function in a year-round breeding equatorial lark

Background: Variation in growth and immune function within and among populations is often associated with specific environmental conditions. We compared growth and immune function in nestlings of year-round breeding equatorial Red-capped Lark Calandrella cinerea from South Kinangop, North Kinangop and Kedong (Kenya), three locations that are geographically close but climatically distinct. In addition, we studied growth and immune function of lark nestlings as a function of year-round variation in breeding intensity and rain within one location. We monitored mass, wing, and tarsus at hatching (day 1) and at 4, 7, and 10 days post-hatch, and we quantified four indices of immune function (haptoglobin, agglutination, lysis and nitric oxide) using blood samples collected on day 10. Results: Nestling body mass and size at hatching, which presumably reflect the resources that females allocated to their eggs, were lowest in the most arid location, Kedong. Contrary to our predictions, nestlings in Kedong grew faster than nestlings in the two other cooler and wetter locations of South and North Kinangop. During periods of peak reproduction within Kedong, nestlings were heavier at hatching, but they did not grow faster over the first 10 days post-hatch. In contrast, rainfall, which did not relate to timing of breeding, had no effect on hatching mass, but more rain did coincide with faster growth post-hatch. Finally, we found no significant differences in nestling immune function, neither among locations nor with the year-round variation within Kedong. Conclusion: Based on these results, we hypothesize that female body condition determines nestling mass and size at hatching, but other independent environmental conditions subsequently shape nestling growth. Overall, our results suggest that environmental conditions related to food availability for nestlings are relatively unimportant to the timing of breeding in equatorial regions, while these same conditions do have consequences for nestling size and growth.</p

Risk factors for Lyme disease : A scale-dependent effect of host species diversity and a consistent negative effect of host phylogenetic diversity

Biodiversity can influence disease risk. One example of a diversity-disease relationship is the dilution effect, which suggests higher host species diversity (often indexed by species richness) reduces disease risk. While numerous studies support the dilution effect, its generality remains controversial. Most studies of diversity-disease relationships have overlooked the potential importance of phylogenetic diversity. Furthermore, most studies have tested diversity-disease relationships at one spatial scale, even though such relationships are likely scale dependent. Using Lyme disease as a model system, we investigated the effects of host species richness and phylogenetic relatedness on the number of reported Lyme disease cases in humans in the U.S.A. at two spatial scales (the county level and the state level) using piecewise structural equation modelling. We also accounted for relevant climatic and habitat-related factors and tested their correlations with the number of Lyme disease cases. We found that species assemblages with more related species (i.e., host species in the order Rodentia) were associated with more Lyme disease cases in humans. Host species richness correlated negatively with the number of Lyme disease cases at the state level (i.e., a dilution effect), a pattern that might be explained by the higher number of reservoir-incompetent species at high levels of species richness at this larger spatial scale. In contrast, a positive correlation was found between species richness and the number of Lyme disease cases at the county level, where a higher proportion of rodent species was associated with higher levels of species richness, potentially amplifying the disease risk. Our results highlight that analyse at a single spatial scale can miss some impacts of biodiversity on human health. Thus, multi-scale analyses with consideration of host phylogenetic diversity are critical for improving our understanding of diversity-disease relationships.Peer reviewe

Microbial environment shapes immune function and cloacal microbiota dynamics in zebra finches <i>Taeniopygia guttata</i>

BACKGROUND: The relevance of the host microbiota to host ecology and evolution is well acknowledged. However, the effect of the microbial environment on host immune function and host microbiota dynamics is understudied in terrestrial vertebrates. Using a novel experimental approach centered on the manipulation of the microbial environment of zebra finches Taeniopygia guttata, we carried out a study to investigate effects of the host's microbial environment on: 1) constitutive immune function, 2) the resilience of the host cloacal microbiota; and 3) the degree to which immune function and host microbiota covary in microbial environments that differ in diversity. RESULTS: We explored immune indices (hemagglutination, hemolysis, IgY levels and haptoglobin concentration) and host-associated microbiota (diversity and composition) in birds exposed to two experimental microbial environments differing in microbial diversity. According to our expectations, exposure to experimental microbial environments led to differences related to specific antibodies: IgY levels were elevated in the high diversity treatment, whereas we found no effects for the other immune indices. Furthermore, according to predictions, we found significantly increased richness of dominant OTUs for cloacal microbiota of birds of the high diversity compared with the low diversity group. In addition, cloacal microbiota of individual females approached their baseline state sooner in the low diversity environment than females in the high diversity environment. This result supported a direct phenotypically plastic response of host microbiota, and suggests that its resilience depends on environmental microbial diversity. Finally, immune indices and cloacal microbiota composition tend to covary within treatment groups, while at the same time, individuals exhibited consistent differences of immune indices and microbiota characteristics. CONCLUSION: We show that microbes in the surroundings of terrestrial vertebrates can influence immune function and host-associated microbiota dynamics over relatively short time scales. We suggest that covariation between immune indices and cloacal microbiota, in addition to large and consistent differences among individuals, provides potential for evolutionary adaptation. Ultimately, our study highlights that linking environmental and host microbiotas may help unravelling immunological variation within and potentially among species, and together these efforts will advance the integration of microbial ecology and ecological immunology

Environmental proxies of antigen exposure explain variation in immune investment better than indices of pace of life.

Investment in immune defences is predicted to covary with a variety of ecologically and evolutionarily relevant axes, with pace of life and environmental antigen exposure being two examples. These axes may themselves covary directly or inversely, and such relationships can lead to conflicting predictions regarding immune investment. If pace of life shapes immune investment then, following life history theory, slow-living, arid zone and tropical species should invest more in immunity than fast-living temperate species. Alternatively, if antigen exposure drives immune investment, then species in antigen-rich tropical and temperate environments are predicted to exhibit higher immune indices than species from antigen-poor arid locations. To test these contrasting predictions we investigated how variation in pace of life and antigen exposure influence immune investment in related lark species (Alaudidae) with differing life histories and predicted risks of exposure to environmental microbes and parasites. We used clutch size and total number of eggs laid per year as indicators of pace of life, and aridity, and the climatic variables that influence aridity, as correlates of antigen abundance. We quantified immune investment by measuring four indices of innate immunity. Pace of life explained little of the variation in immune investment, and only one immune measure correlated significantly with pace of life, but not in the predicted direction. Conversely, aridity, our proxy for environmental antigen exposure, was predictive of immune investment, and larks in more mesic environments had higher immune indices than those living in arid, low-risk locations. Our study suggests that abiotic environmental variables with strong ties to environmental antigen exposure can be important correlates of immunological variation.Financial support came from the Schure-Beijerinck-Poppings Fonds (to NPCH and AH), BirdLife Netherlands (to BIT), NSF grant IBN 0212587 (to JBW), and VENI and VIDI grants from the Netherlands Organisation for Scientific Research (to KDM and BIT).This is the accepted manuscript. The final publication is available at Springer via http://dx.doi.org/10.1007%2Fs00442-014-3136-y