Levels of pathogen virulence and host resistance both shape the antibody response to an emerging bacterial disease.

Quantifying variation in the ability to fight infection among free-living hosts is challenging and often constrained to one or a few measures of immune activity. While such measures are typically taken to reflect host resistance, they can also be shaped by pathogen effects, for example, if more virulent strains trigger more robust immune responses. Here, we test the extent to which pathogen-specific antibody levels, a commonly used measure of immunocompetence, reflect variation in host resistance versus pathogen virulence, and whether these antibodies effectively clear infection. House finches (Haemorhous mexicanus) from resistant and susceptible populations were inoculated with > 50 isolates of their novel Mycoplasma gallisepticum pathogen collected over a 20-year period during which virulence increased. Serum antibody levels were higher in finches from resistant populations and increased with year of pathogen sampling. Higher antibody levels, however, did not subsequently give rise to greater reductions in pathogen load. Our results show that antibody responses can be shaped by levels of host resistance and pathogen virulence, and do not necessarily signal immune clearance ability. While the generality of this novel finding remains unclear, particularly outside of mycoplasmas, it cautions against using antibody levels as implicit proxies for immunocompetence and/or host resistance.

Multiple differences in pathogen-host cell interactions following a bacterial host shift.

Novel disease emergence is often associated with changes in pathogen traits that enable pathogen colonisation, persistence and transmission in the novel host environment. While understanding the mechanisms underlying disease emergence is likely to have critical implications for preventing infectious outbreaks, such knowledge is often based on studies of viral pathogens, despite the fact that bacterial pathogens may exhibit very different life histories. Here, we investigate the ability of epizootic outbreak strains of the bacterial pathogen, Mycoplasma gallisepticum, which jumped from poultry into North American house finches (Haemorhous mexicanus), to interact with model avian cells. We found that house finch epizootic outbreak strains of M. gallisepticum displayed a greater ability to adhere to, invade, persist within and exit from cultured chicken embryonic fibroblasts, than the reference virulent (R_low) and attenuated (R_high) poultry strains. Furthermore, unlike the poultry strains, the house finch epizootic outbreak strain HF_1994 displayed a striking lack of cytotoxicity, even exerting a cytoprotective effect on avian cells. Our results suggest that, at epizootic outbreak in house finches, M. gallisepticum was particularly adept at using the intra-cellular environment, which may have facilitated colonisation, dissemination and immune evasion within the novel finch host. Whether this high-invasion phenotype is similarly displayed in interactions with house finch cells, and whether it contributed to the success of the host shift, remains to be determined.

Transcriptomic analysis of immune response to bacterial lipopolysaccharide in zebra finch (Taeniopygia guttata).

BACKGROUND: Despite the convergence of rapid technological advances in genomics and the maturing field of ecoimmunology, our understanding of the genes that regulate immunity in wild populations is still nascent. Previous work to assess immune function has relied upon relatively crude measures of immunocompetence. However, with next-generation RNA-sequencing, it is now possible to create a profile of gene expression in response to an immune challenge. In this study, captive zebra finch (Taeniopygia guttata; adult males) were challenged with bacterial lipopolysaccharide (LPS) or vehicle to stimulate the innate immune system. 2 hours after injection, birds were euthanized and hypothalami, spleen, and red blood cells (RBCs) were collected. Taking advantage of the fully sequenced genome of zebra finch, total RNA was isolated, sequenced, and partially annotated in these tissue/cells. RESULTS: In hypothalamus, there were 707 significantly upregulated transcripts, as well as 564 and 144 in the spleen and RBCs, respectively, relative to controls. Also, 155 transcripts in the hypothalamus, 606 in the spleen, and 61 in the RBCs were significantly downregulated. More specifically, a number of immunity-related transcripts (e.g., IL-1ß, RSAD2, SOCS3) were upregulated among tissues/cells. Additionally, transcripts involved in metabolic processes (APOD, LRAT, RBP4) were downregulated. CONCLUSIONS: These results suggest a potential trade-off in expression of genes that regulate immunity and metabolism in birds challenged with LPS. This finding is consistent with a hypothermic response to LPS treatment in small birds. Unlike mammals, birds have nucleated RBCs, and these results support a novel transcriptomic response of avian RBCs to immune challenge.

Early Life Stress Strengthens Trait Covariance: A Plastic Response That Results in Reduced Flexibility.

Stress exposure during development can impact both the expression of individual traits and associations between traits, but whether stress results in stronger or weaker associations between traits is unclear. In this study, we examined within- and among-trait associations for morphological and physiological traits in zebra finches (Taeniopygia guttata) exposed to corticosterone (CORT) during the nestling and fledging stages as well as in control birds. Birds exposed to CORT exhibited stronger within-trait correlations over time and stronger associations among traits. We found preliminary evidence that birds that died before the median age of death had stronger within- and among-trait correlations independent of treatment, and among CORT-treated birds, smaller birds were more likely to survive beyond the median age than larger birds. These findings suggest that stress hormone exposure in early life can result in reduced developmental flexibility, with potential fitness ramifications, and that these costs may be greater for larger offspring. Furthermore, our results provide experimental evidence for pleiotropic effects of hormones during development through altered patterns of phenotypic correlation.

Categorical perception of colour signals in a songbird.

In many contexts, animals assess each other using signals that vary continuously across individuals and, on average, reflect variation in the quality of the signaller1,2. It is often assumed that signal receivers perceive and respond continuously to continuous variation in the signal2. Alternatively, perception and response may be discontinuous3, owing to limitations in discrimination, categorization or both. Discrimination is the ability to tell two stimuli apart (for example, whether one can tell apart colours close to each other in hue). Categorization concerns whether stimuli are grouped based on similarities (for example, identifying colours with qualitative similarities in hue as similar even if they can be distinguished)4. Categorical perception is a mechanism by which perceptual systems categorize continuously varying stimuli, making specific predictions about discrimination relative to category boundaries. Here we show that female zebra finches (Taeniopygia guttata) categorically perceive a continuously variable assessment signal: the orange to red spectrum of male beak colour. Both predictions of categorical perception5 were supported: females (1) categorized colour stimuli that varied along a continuum and (2) showed increased discrimination between colours from opposite sides of a category boundary compared to equally different colours from within a category. To our knowledge, this is the first demonstration of categorical perception of signal-based colouration in a bird, with implications for understanding avian colour perception and signal evolution in general.

No Effect of Human Presence at Night on Disease, Body Mass, or Metabolism in Rural and Urban House Finches (Haemorhous mexicanus).

Global urban development continues to accelerate and have diverse effects on wildlife. Although most studies of anthropogenic impacts on animals have focused on indirect effects (e.g., environmental modifications like habitat change or pollution), there may also be direct effects of physical human presence and actions on wildlife stress, behavior, and persistence in cities. Most studies on how humans physically interact with wildlife have focused on the active, daytime phase of diurnal animals, rarely considering effects of our night-time activities. We hypothesized that, if night-time human presence is a stressor for wildlife that are not commonly exposed to humans, night-disturbed rural animals would show stronger physiological signs of elevated stress than would urban individuals. Specifically, we experimentally investigated the effects of human presence at night (HPAN) on disease, body mass, and mass-specific metabolic rates in urban- and rural-caught house finches (Haemorhous mexicanus) in captivity. Our HPAN treatment consisted of a human entering the housing room of the birds and briefly jostling the home cages of each finch as the person walked around the room for a 3-min period on five randomly selected nights per week. Compared with a control (night-undisturbed) group, we found that HPAN greatly increased the odds finches were awake for ca. 33 min post-disturbance, but that chronic treatment did not alter body mass, parasitic infection by coccidian endoparasites, or mass-specific basal metabolic rates. Additionally, finches caught from urban and rural sites did not differ in their response to the treatment. Overall, our results are consistent with those showing that brief but regular human disturbances can have acute negative effects on wildlife, but carry few if any long-term metabolic or disease-related costs in fast-lived birds. However, these findings contrast with the broad, chronic physiological effects of other anthropogenic changes, such as artificial light at night, and highlight the differential impacts that various human activities (which differ in sensory stimulus type, perceived threat, duration and intensity, etc.) can have on wildlife health and behavior.

Early stress priming and the effects on fitness-related traits following an adult stress exposure.

Developmental stressors can have strong effects that persist well into adulthood, and are generally seen as detrimental. However, recent work suggests that a mild developmental stressor can have beneficial effects by preparing the organism to better withstand negative impacts when exposed to high levels of the stressor later in life, also known as a conditioning hormesis. Still, little is known about the influence of such hormetic effects on fitness-related measures. We hypothesized that exposure to a mild stressor during development will protect individuals later in life from the negative effects of a high heat stressor on immune function and reproduction. To test this hypothesis, we subjected zebra finches (Taeniopygia guttata) to a repeated mild heat stressor (38°C) as juveniles for 28 days. As adults, the birds were then exposed to a high heat stressor (42°C) for 3 consecutive days and we examined the effects on immune function via wound healing, and on female reproductive output. We found that females given the mild heat stressor as juveniles healed wounds marginally slower, but also had higher clutch viability than controls. For the adult treatment, we saw that high heat had a stimulatory effect on clutch viability as well. Our findings point toward the occurrence of trade-offs between immune function and reproduction due to a cost of hormetic priming when the adult environment does not match that of early life.

Feeder density enhances house finch disease transmission in experimental epidemics.

Anthropogenic food provisioning of wildlife can alter the frequency of contacts among hosts and between hosts and environmental sources of pathogens. Despite the popularity of garden bird feeding, few studies have addressed how feeders influence host contact rates and disease dynamics. We experimentally manipulated feeder density in replicate aviaries containing captive, pathogen-naive, groups of house finches (Haemorhous mexicanus) and continuously tracked behaviours at feeders using radio-frequency identification devices. We then inoculated one bird per group with Mycoplasma gallisepticum (Mg), a common bacterial pathogen for which feeders are fomites of transmission, and assessed effects of feeder density on house finch behaviour and pathogen transmission. We found that pathogen transmission was significantly higher in groups with the highest density of bird feeders, despite a significantly lower rate of intraspecific aggressive interactions relative to the low feeder density groups. Conversely, among naive group members that never showed signs of disease, we saw significantly higher concentrations of Mg-specific antibodies in low feeder density groups, suggesting that birds in low feeder density treatments had exposure to subclinical doses of Mg. We discuss ways in which the density of garden bird feeders could play an important role in mediating the intensity of Mg epidemics.This article is part of the theme issue 'Anthropogenic resource subsidies and host-parasite dynamics in wildlife'.

Differing House Finch Cytokine Expression Responses to Original and Evolved Isolates of Mycoplasma gallisepticum.

The recent emergence of the poultry bacterial pathogen Mycoplasma gallisepticum (MG) in free-living house finches (Haemorhous mexicanus), which causes mycoplasmal conjunctivitis in this passerine bird species, resulted in a rapid coevolutionary arms-race between MG and its novel avian host. Despite extensive research on the ecological and evolutionary dynamics of this host-pathogen system over the past two decades, the immunological responses of house finches to MG infection remain poorly understood. We developed seven new probe-based one-step quantitative reverse transcription polymerase chain reaction assays to investigate mRNA expression of house finch cytokine genes (IL1B, IL6, IL10, IL18, TGFB2, TNFSF15, and CXCLi2, syn. IL8L). These assays were then used to describe cytokine transcription profiles in a panel of 15 house finch tissues collected at three distinct time points during MG infection. Based on initial screening that indicated strong pro-inflammatory cytokine expression during MG infection at the periorbital sites in particular, we selected two key house finch tissues for further characterization: the nictitating membrane, i.e., the internal eyelid in direct contact with MG, and the Harderian gland, the secondary lymphoid tissue responsible for regulation of periorbital immunity. We characterized cytokine responses in these two tissues for 60 house finches experimentally inoculated either with media alone (sham) or one of two MG isolates: the earliest known pathogen isolate from house finches (VA1994) or an evolutionarily more derived isolate collected in 2006 (NC2006), which is known to be more virulent. We show that the more derived and virulent isolate NC2006, relative to VA1994, triggers stronger local inflammatory cytokine signaling, with peak cytokine expression generally occurring 3-6 days following MG inoculation. We also found that the extent of pro-inflammatory interleukin 1 beta signaling was correlated with conjunctival MG loads and the extent of clinical signs of conjunctivitis, the main pathological effect of MG in house finches. These results suggest that the pathogenicity caused by MG infection in house finches is largely mediated by host pro-inflammatory immune responses, with important implications for the dynamics of host-pathogen coevolution.

Developmental corticosterone treatment does not program immune responses in zebra finches (Taeniopygia guttata).

Developmental conditions may impact the expression of immune traits throughout an individual's life. Early-life challenges may lead to immunological constraints that are mediated by endocrine-immune interactions. In particular, individual differences in the ability to mount immune responses may be programmed by exposure to stressors or glucocorticoid hormones during development. To test this hypothesis, we experimentally elevated levels of the glucocorticoid hormone corticosterone during the nestling and fledgling periods in captive zebra finches (Taeniopygia guttata). We subsequently challenged birds with the antigen lipopolysaccharide (LPS) on days 60 and 100 post-hatch to determine if developmental exposure to elevated corticosterone impacted the later response to LPS. As measures of immune function, we quantified bacteria killing ability, haptoglobin concentrations, and LPS-specific antibody responses at multiple time points. We also measured circulating corticosterone concentrations during the experimental period and on day 60 before and after endotoxin challenge. During the experimental period, corticosterone treatment elevated corticosterone levels. Corticosterone treatment did not induce programming effects on immune function or corticosterone production. Independent of treatment, individuals with higher corticosterone concentrations during the nestling period had lower bacteria killing ability on day 36 and higher baseline corticosterone concentrations on day 60 post-hatch. These results suggest a limited role for corticosterone exposure during early life to mediate immunological constraints later in life. Manipulation of cortisol may be necessary to conclusively determine if developmental glucocorticoid exposure can program immune function in birds. To determine if developmental stress can program the immune response, exposure to environmentally relevant stressors should also be manipulated.

Development and validation of a house finch interleukin-1ß (HfIL-1ß) ELISA system.

BACKGROUND: A unique clade of the bacterium Mycoplasma gallisepticum (MG), which causes chronic respiratory disease in poultry, has resulted in annual epidemics of conjunctivitis in North American house finches since the 1990s. Currently, few immunological tools have been validated for this songbird species. Interleukin-1ß (IL-1ß) is a prototypic multifunctional cytokine and can affect almost every cell type during Mycoplasma infection. The overall goal of this study was to develop and validate a direct ELISA assay for house finch IL-1ß (HfIL-1ß) using a cross-reactive chicken antibody. METHODS: A direct ELISA approach was used to develop this system using two different coating methods, carbonate and dehydration. In both methods, antigens (recombinant HfIL-1b or house finch plasma) were serially diluted in carbonate-bicarbonate coating buffer and either incubated at 4 °C overnight or at 60 °C on a heating block for 2 hr. To generate the standard curve, rHfIL-1b protein was serially diluted at 0, 3, 6, 9, 12, 15, 18, 21, and 24 ng/mL. Following blocking and washing, anti-chicken IL-1b polyclonal antibody was added, plates were later incubated with detecting antibodies, and reactions developed with tetramethylbenzidine solution. RESULTS: A commercially available anti-chicken IL-1ß (ChIL-1ß) polyclonal antibody (pAb) cross-reacted with house finch plasma IL-1ß as well as bacterially expressed recombinant house finch IL-1ß (rHfIL-1ß) in immunoblotting assays. In a direct ELISA system, rHfIL-1ß could not be detected by an anti-ChIL-1ß pAb when the antigen was coated with carbonate-bicarbonate buffer at 4°C overnight. However, rHfIL-1ß was detected by the anti-ChIL-1ß pAb when the antigen was coated using a dehydration method by heat (60°C). Using the developed direct ELISA for HfIL-1ß with commercial anti-ChIL-1ß pAb, we were able to measure plasma IL-1ß levels from house finches. CONCLUSIONS: Based on high amino acid sequence homology, we hypothesized and demonstrated cross-reactivity of anti-ChIL-1ß pAb and HfIL-1ß. Then, we developed and validated a direct ELISA system for HfIL-1ß using a commercial anti-ChIL-1ß pAb by measuring plasma HfIL-1ß in house finches.

Activation of the peripheral immune system regulates neuronal aromatase in the adult zebra finch brain.

Estradiol provision via neural aromatization decreases neuro-inflammation and -degeneration, but almost nothing is known about the interactions between the peripheral immune system and brain aromatase. Given the vulnerability of the CNS we reasoned that brain aromatization may protect circuits from the threats of peripheral infection; perhaps shielding cells that are less resilient from the degeneration associated with peripheral infection or trauma. Lipopolysaccharide (LPS) or vehicle was administered peripherally to adult zebra finches and sickness behavior was recorded 2 or 24 hours later. The central transcription of cytokines and aromatase was measured, as were telencephalic aromatase activity and immunoreactive aromatase (24 hour time point only). Two hours post LPS, sickness-like behaviors increased, the transcription of IL-1ß was higher in both sexes, and TNFα was elevated in females. 24 hours post-LPS, the behavior of LPS birds was similar to controls, and cytokines had returned to baseline, but aromatase mRNA and activity were elevated in both sexes. Immunocytochemistry revealed greater numbers of aromatase-expressing neurons in LPS birds. These data suggest that the activation of the immune system via peripheral endotoxin increases neuronal aromatase; a mechanism that may rapidly generate a potent anti-neuroinflammatory steroid in response to peripheral activation of the immune system.

Host Responses to Pathogen Priming in a Natural Songbird Host.

Hosts in free-living populations can experience substantial variation in the frequency and dose of pathogen exposure, which can alter disease progression and protection from future exposures. In the house finch-Mycoplasma gallisepticum (MG) system, the pathogen is primarily transmitted via bird feeders, and some birds may be exposed to frequent low doses of MG while foraging. Here we experimentally determined how low dose, repeated exposures of house finches to MG influence host responses and protection from secondary high-dose challenge. MG-naive house finches were given priming exposures that varied in dose and total number. After quantifying host responses to priming exposures, all birds were given a secondary high-dose challenge to assess immunological protection. Dose, but not the number of exposures, significantly predicted both infection and disease severity following priming exposure. Furthermore, individuals given higher priming doses showed stronger protection upon secondary, high-dose challenge. However, even single low-dose exposures to MG, a proxy for what some birds likely experience in the wild while feeding, provided significant protection against a high-dose challenge. Our results suggest that bird feeders, which serve as sources of infection in the wild, may in some cases act as "immunizers," with important consequences for disease dynamics.

Identification and functional characterization of the house finch interleukin-1ß.

Interleukin-1ß (IL-1ß), an inflammatory cytokine of the IL-1 family, is primarily produced as a precursor protein by monocytes and macrophages, then matures and becomes activated through proteolytic catalysis. Although the biological characteristics of avian IL-1ß are well known, little information is available about its biological role in songbird species such as house finches that are vulnerable to naturally-occurring inflammatory diseases. In this study, house finch IL-1ß (HfIL-1ß) was cloned, expressed, and its biological function examined. Both precursor and mature forms of HfIL-1ß consisting of 269 and 162 amino acids, respectively, were amplified from total RNA of spleen and cloned into expression vectors. HfIL-1ß showed high sequential and tertiary structural similarity to chicken homologue that allowed detection of the expressed mature recombinant HfIL-1ß (rHfIL-1ß) with anti-ChIL-1ß antibody by immunoblot analysis. For further characterization, we used primary splenocytes and hepatocytes that are predominant sources of IL-1ß upon stimulation, as well as suitable targets to stimulation by IL-1ß. Isolated house finch splenocytes were stimulated with rHfIL-1ß in the presence and absence of concanavalin A (Con A), RNA was extracted and transcript levels of Th1/Th2 cytokines and a chemokine were measured by qRT-PCR. The addition of rHfIL-1ß induced significant enhancement of IL-2 transcript, a Th1 cytokine, while transcription of IL-1ß and the Th2 cytokine IL-10 was slightly enhanced by rHfIL-1ß treatment. rHfIL-1ß also led to elevated levels of the chemokine CXCL1 and nitric oxide production regardless of co-stimulation with Con A. In addition, the production of the acute phase protein serum amyloid A and the antimicrobial peptide LEAP2 was observed in HfIL-1ß-stimulated hepatocytes. Taken together, these observations revealed the basic functions of HfIL-1ß including the stimulatory effect on cell proliferation, production of Th1/Th2 cytokines and acute phase proteins by immune cells, thus providing valuable insight into how HfIL-1ß is involved in regulating inflammatory response.

Impact of nest sanitation on the immune system of parents and nestlings in a passerine bird.

Bacterial communities are thought to have fundamental effects on the growth and development of nestling birds. The antigen exposure hypothesis suggests that, for both nestlings and adult birds, exposure to a diverse range of bacteria would select for stronger immune defences. However, there are relatively few studies that have tested the immune/bacterial relationships outside of domestic poultry. We therefore sought to examine indices of immunity (microbial killing ability in naive birds, which is a measure of innate immunity, and the antibody response to sheep red blood cells, which measures adaptive immunity) in both adult and nestling zebra finches (Taeniopygia guttata). We did this throughout breeding and between reproductive attempts in nests that were experimentally manipulated to change the intensity of bacterial exposure. Our results suggest that nest sanitation and bacterial load affected measures of the adaptive immune system, but not the innate immune parameters tested. Adult finches breeding in clean nests had a lower primary antibody response to sheep red blood cells, particularly males, and a greater difference between primary and secondary responses. Adult microbial killing of Escherichia coli decreased as parents moved from incubation to nestling rearing for both nest treatments; however, killing of Candida albicans remained consistent throughout. In nestlings, both innate microbial killing and the adaptive antibody response did not differ between nest environments. Together, these results suggest that exposure to microorganisms in the environment affects the adaptive immune system in nesting birds, with exposure upregulating the antibody response in adult birds.

Locally elevated cortisol in lymphoid organs of the developing zebra finch but not Japanese quail or chicken.

Glucocorticoids are important for production of functional lymphocytes and immunity. In altricial neonates, adrenal glands are unresponsive and local glucocorticoid synthesis in lymphoid organs may be necessary to support lymphocyte development. Precocial neonates, in contrast, have fully responsive adrenal glucocorticoid production, and lymphoid glucocorticoid synthesis may not be necessary. Here, we found that in altricial zebra finch hatchlings, lymphoid organs had dramatically elevated endogenous glucocorticoid (and precursor) levels compared to levels in circulating blood. Furthermore, while avian adrenals produce corticosterone, finch lymphoid organs had much higher levels of cortisol, an unexpected glucocorticoid in birds. In contrast, precocial Japanese quail and chicken offspring did not have locally elevated lymphoid glucocorticoid levels, nor did their lymphoid organs contain high proportions of cortisol. These results show that lymphoid glucocorticoids differ in identity, concentration, and possibly source, in hatchlings of three different bird species. Locally-regulated glucocorticoids might have species-specific roles in immune development.

High major histocompatibility complex class I polymorphism despite bottlenecks in wild and domesticated populations of the zebra finch (Taeniopygia guttata).

BACKGROUND: Two subspecies of zebra finch, Taeniopygia guttata castanotis and T. g. guttata are native to Australia and the Lesser Sunda Islands, respectively. The Australian subspecies has been domesticated and is now an important model system for research. Both the Lesser Sundan subspecies and domesticated Australian zebra finches have undergone population bottlenecks in their history, and previous analyses using neutral markers have reported reduced neutral genetic diversity in these populations. Here we characterize patterns of variation in the third exon of the highly variable major histocompatibility complex (MHC) class I α chain. As a benchmark for neutral divergence, we also report the first mitochondrial NADH dehydrogenase 2 (ND2) sequences in this important model system. RESULTS: Despite natural and human-mediated population bottlenecks, we find that high MHC class I polymorphism persists across all populations. As expected, we find higher levels of nucleotide diversity in the MHC locus relative to neutral loci, and strong evidence of positive selection acting on important residues forming the peptide-binding region (PBR). Clear population differentiation of MHC allele frequencies is also evident, and this may be due to adaptation to new habitats and associated pathogens and/or genetic drift. Whereas the MHC Class I locus shows broad haplotype sharing across populations, ND2 is the first locus surveyed to date to show reciprocal monophyly of the two subspecies. CONCLUSIONS: Despite genetic bottlenecks and genetic drift, all surveyed zebra finch populations have maintained high MHC Class I diversity. The diversity at the MHC Class I locus in the Lesser Sundan subspecies contrasts sharply with the lack of diversity in previously examined neutral loci, and may thus be a result of selection acting to maintain polymorphism. Given uncertainty in historical population demography, however, it is difficult to rule out neutral processes in maintaining the observed diversity. The surveyed populations also differ in MHC Class I allele frequencies, and future studies are needed to assess whether these changes result in functional immune differences.

Transient and permanent effects of suboptimal incubation temperatures on growth, metabolic rate, immune function and adrenocortical responses in zebra finches.

In birds, incubation temperature can vary by several degrees Celsius among nests of a given species. Parents may alter incubation temperature to cope with environmental conditions and/or to manipulate embryonic development, and such changes in incubation behavior could have long-lasting effects on offspring phenotype. To investigate short- and long-term effects of suboptimal incubation temperatures on survival and physiological functions in zebra finches, eggs were incubated at 36.2, 37.4 or 38.4 °C for the entire incubation period. The post-hatch environment was identical among the treatment groups. We found that hatching success was lowest in the 38.4 °C group, while post-hatch survival was lowest in the 36.2 °C group. Incubation temperature had sex-specific effects on offspring phenotype: incubation temperatures affected body mass (Mb) but not physiological parameters of males and conversely, the physiological parameters but not Mb of females. Specifically, males from the 38.4 °C group weighed significantly less than males from the 36.2 °C group from the nestling period to adulthood, whereas females from different incubation temperature groups did not differ in Mb. In contrast, females incubated at 36.2 °C had transient but significantly elevated basal metabolic rate and adrenocortical responses during the nestling and fledgling periods, whereas no treatment effect was observed in males. Innate immunity was not affected by incubation temperature in either sex. These results suggest that a 1 °C deviation from what is considered an optimal incubation temperature can lower offspring performance and offspring survival.

Response of black-capped chickadees to house finch Mycoplasma gallisepticum.

Tests for the presence of pathogen DNA or antibodies are routinely used to survey for current or past infections. In diseases that emerge following a host jump estimates of infection rate might be under- or overestimated. We here examine whether observed rates of infection are biased for a non-focal host species in a model system. The bacterium Mycoplasma gallisepticum is a widespread pathogen in house finches (Haemorhous mexicanus), a fringillid finch, but an unknown proportion of individuals of other songbird species are also infected. Our goal is to determine the extent to which detection of M. gallisepticum DNA or antibodies against the bacteria in a non-fringillid bird species is over- or underestimated using black-capped chickadees Poecile atricapillus, a species in which antibodies against M. gallisepticum are frequently detected in free-living individuals. After keeping black-capped chickadees in captivity for 12 weeks, during which period the birds remained negative for M. gallisepticum, four were inoculated with M. gallisepticum and four were sham inoculated in both eyes to serve as negative controls. Simultaneously we inoculated six house finches with the same isolate of M. gallisepticum as a positive control. All inoculated birds of both species developed infections detectable by qPCR in the conjunctiva. For the 6 weeks following inoculation we detected antibodies in all M. gallisepticum-inoculated house finches but in only three of the four M. gallisepticum-inoculated black-capped chickadees. All house finches developed severe eye lesions but none of the black-capped chickadees did. Modeling the Rapid Plate Agglutination test results of black-capped chickadees shows that the rate of false-positive tests would be not more than 3.2%, while the estimated rate of false negatives is 55%. We conclude that the proportion of wild-caught individuals in which we detect M. gallisepticum-specific antibodies using Rapid Plate Agglutination is, if anything, substantially underestimated.

PHA-stimulated immune-responsiveness in mercury-dosed zebra finches does not match results from environmentally exposed songbirds.

Dietary mercury exposure is associated with suppressed immune responsiveness in birds. This study examined the immune-responsiveness of domestic zebra finches (Taeniopygia guttata) experimentally exposed to mercury through their diet. We used the phytohemagglutinin (PHA) skin-swelling test to assay the effect of two modes of mercury exposure. Some finches received exposure to mercury only after reaching sexual maturity, while others were maintained on a mercury-dosed diet throughout life, including development. Each bird received one of five dietary concentrations of methylmercury cysteine (0.0, 0.3, 0.6, 1.2 or 2.4 ppm). In contrast to a study on wild songbirds at a mercury-contaminated site, we detected no relationship between mercury level and immunological response to PHA, regardless of mode of exposure. This result represents the first major difference found by our laboratory between wild birds exposed to environmental mercury and captive birds experimentally exposed to mercury.