Immune challenge reduces daily activity period in free-living birds for three weeks.

Non-lethal infections are common in free-living animals and the associated sickness behaviours can impact crucial life-history trade-offs. However, little is known about the duration and extent of such sickness behaviours in free-living animals, and consequently how they affect life-history decisions. Here, free-living Eurasian blackbirds, Turdus merula, were immune-challenged with lipopolysaccharide (LPS) to mimic a bacterial infection and their behaviour was monitored for up to 48 days using accelerometers. As expected, immune-challenged birds were less active than controls within the first 24 h. Unexpectedly, this reduced activity remained detectable for 20 days, before both groups returned to similar activity levels. Furthermore, activity was positively correlated with a pre-experimental index of complement activity, but only in immune-challenged birds, suggesting that sickness behaviours are modulated by constitutive immune function. Differences in daily activity levels stemmed from immune-challenged birds resting earlier at dusk than control birds, while activity levels between groups were similar during core daytime hours. Overall, activity was reduced by 19% in immune-challenged birds and they were on average almost 1 h less active per day for 20 days. This unexpected longevity in sickness behaviour may have severe implications during energy-intense annual-cycle stages (e.g. breeding, migration, winter). Thus, our data help to understand the consequences of non-lethal infections on free-living animals.

Cloacal microbiota are biogeographically structured in larks from desert, tropical and temperate areas.

BACKGROUND: In contrast with macroorganisms, that show well-documented biogeographical patterns in distribution associated with local adaptation of physiology, behavior and life history, strong biogeographical patterns have not been found for microorganisms, raising questions about what determines their biogeography. Thus far, large-scale biogeographical studies have focused on free-living microbes, paying little attention to host-associated microbes, which play essential roles in physiology, behavior and life history of their hosts. Investigating cloacal gut microbiota of closely-related, ecologically similar free-living songbird species (Alaudidae, larks) inhabiting desert, temperate and tropical regions, we explored influences of geographical location and host species on α-diversity, co-occurrence of amplicon sequence variants (ASVs) and genera, differentially abundant and dominant bacterial taxa, and community composition. We also investigated how geographical distance explained differences in gut microbial community composition among larks. RESULTS: Geographic location did not explain variation in richness and Shannon diversity of cloacal microbiota in larks. Out of 3798 ASVs and 799 bacterial genera identified, 17 ASVs (< 0.5%) and 43 genera (5%) were shared by larks from all locations. Desert larks held fewer unique ASVs (25%) than temperate zone (31%) and tropical larks (34%). Five out of 33 detected bacterial phyla dominated lark cloacal gut microbiomes. In tropical larks three bacterial classes were overrepresented. Highlighting the distinctiveness of desert lark microbiota, the relative abundances of 52 ASVs differed among locations, which classified within three dominant and 11 low-abundance phyla. Clear and significant phylogenetic clustering in cloacal microbiota community composition (unweighted UniFrac) showed segregation with geography and host species, where microbiota of desert larks were distinct from those of tropical and temperate regions. Geographic distance was nonlinearly associated with pairwise unweighted UniFrac distances. CONCLUSIONS: We conclude that host-associated microbiota are geographically structured in a group of widespread but closely-related bird species, following large-scale macro-ecological patterns and contrasting with previous findings for free-living microbes. Future work should further explore if and to what extent geographic variation in host-associated microbiota can be explained as result of co-evolution between gut microbes and host adaptive traits, and if and how acquisition from the environmental pool of bacteria contributes to explaining host-associated communities.

Interactive effects of rising temperatures and urbanisation on birds across different climate zones: A mechanistic perspective.

Climate change and urbanisation are among the most pervasive and rapidly growing threats to biodiversity worldwide. However, their impacts are usually considered in isolation, and interactions are rarely examined. Predicting species' responses to the combined effects of climate change and urbanisation, therefore, represents a pressing challenge in global change biology. Birds are important model taxa for exploring the impacts of both climate change and urbanisation, and their behaviour and physiology have been well studied in urban and non-urban systems. This understanding should allow interactive effects of rising temperatures and urbanisation to be inferred, yet considerations of these interactions are almost entirely lacking from empirical research. Here, we synthesise our current understanding of the potential mechanisms that could affect how species respond to the combined effects of rising temperatures and urbanisation, with a focus on avian taxa. We discuss potential interactive effects to motivate future in-depth research on this critically important, yet overlooked, aspect of global change biology. Increased temperatures are a pronounced consequence of both urbanisation (through the urban heat island effect) and climate change. The biological impact of this warming in urban and non-urban systems will likely differ in magnitude and direction when interacting with other factors that typically vary between these habitats, such as resource availability (e.g. water, food and microsites) and pollution levels. Furthermore, the nature of such interactions may differ for cities situated in different climate types, for example, tropical, arid, temperate, continental and polar. Within this article, we highlight the potential for interactive effects of climate and urban drivers on the mechanistic responses of birds, identify knowledge gaps and propose promising future research avenues. A deeper understanding of the behavioural and physiological mechanisms mediating species' responses to urbanisation and rising temperatures will provide novel insights into ecology and evolution under global change and may help better predict future population responses.

Innate immune function and antioxidant capacity of nestlings of an African raptor covary with the level of urbanisation around breeding territories.

Urban areas provide breeding habitats for many species. However, animals raised in urban environments face challenges such as altered food availability and quality, pollution and pathogen assemblages. These challenges can affect physiological processes such as immune function and antioxidant defences which are important for fitness. Here, we explore how levels of urbanisation influence innate immune function, immune response to a mimicked bacterial infection and antioxidant capacity of nestling Black Sparrowhawks Accipiter melanoleucus in South Africa. We also explore the effect of timing of breeding and rainfall on physiology since both can influence the environmental condition under which nestlings are raised. Finally, because urbanisation can influence immune function indirectly, we use path analyses to explore direct and indirect associations between urbanisation, immune function and oxidative stress. We obtained measures of innate immunity (haptoglobin, lysis, agglutination, bactericidal capacity), indices of antioxidant capacity (total non-enzymatic antioxidant capacity (tAOX) and total glutathione from nestlings from 2015 to 2019. In addition, in 2018 and 2019, we mimicked a bacterial infection by injecting nestlings with lipopolysaccharide and quantified their immune response. Increased urban cover was associated with an increase in lysis and a decrease in tAOX, but not with any of the other physiological parameters. Furthermore, except for agglutination, no physiological parameters were associated with the timing of breeding. Lysis and bactericidal capacity, however, varied consistently with the annual rainfall pattern. Immune response to a mimicked a bacterial infection decreased with urban cover but not with the timing of breeding nor rainfall. Our path analyses suggested indirect associations between urban cover and some immune indices via tAOX but not via the timing of breeding. Our results show that early-life development in an urban environment is associated with variation in immune and antioxidant functions. The direct association between urbanisation and antioxidant capacity and their impact on immune function is likely an important factor mediating the impact of urbanisation on urban-dwelling animals. Future studies should explore how these results are linked to fitness and whether the responses are adaptive for urban-dwelling species.

Recovery of constitutive immune function after migratory endurance flight in free-living birds.

Strenuous physical activity can negatively affect constitutive innate immune function (CIF), the always present first line of defence against pathogens. CIF is non-specific, and thus vital when encountering novel pathogens. A lowered CIF likely increases the risk of infection and disease. Migratory birds engage in truly extreme physical activity during their endurance flights, however, little is known about how they deal with the negative impact this has on their immune function. By collecting both between- and within-individual data we show, for the first time, that free-flying migratory birds can recover several parameters of CIF during stopovers, which are stationary periods in between migratory flights. With this, we provide an important piece of the puzzle on how migrating birds cope with the physiological challenges they face on their biannual journeys. Furthermore, our study stresses the importance of migratory stopovers beyond fuel accumulation.

Telomeres in ecology and evolution: A review and classification of hypotheses.

Research on telomeres in the fields of ecology and evolution has been rapidly expanding over the last two decades. This has resulted in the formulation of a multitude of, often name-given, hypotheses related to the associations between telomeres and life-history traits or fitness-facilitating processes (and the mechanisms underlying them). However, the differences (or similarities) between the various hypotheses, which can originate from different research fields, are often not obvious. Our aim here is therefore to give an overview of the hypotheses that are of interest in ecology and evolution and to provide two frameworks that help discriminate among them. We group the hypotheses (i) based on their association with different research questions, and (ii) using a hierarchical approach that builds on the assumptions they make, such as about causality of telomere length/shortening and/or the proposed functional consequences of telomere shortening on organism performance. Both our frameworks show that there exist parallel lines of thoughts in different research fields. Moreover, they also clearly illustrate that there are in many cases competing hypotheses within clusters, and that some of these even have contradictory assumptions and/or predictions. We also touch upon two topics in telomere research that would benefit from further conceptualization. This review should help researchers, both those familiar with and those new to the subject, to identify future avenues of research.

Exposure to artificial light at night alters innate immune response in wild great tit nestlings.

The large-scale impact of urbanization on wildlife is rather well documented; however, the mechanisms underlying the effects of urban environments on animal physiology and behaviour are still poorly understood. Here, we focused on one major urban pollutant - artificial light at night (ALAN) - and its effects on the capacity to mount an innate immune response in wild great tit (Parus major) nestlings. Exposure to ALAN alters circadian rhythms of physiological processes, by disrupting the nocturnal production of the hormone melatonin. Nestlings were exposed to a light source emitting 3 lx for seven consecutive nights. Subsequently, nestlings were immune challenged with a lipopolysaccharide injection, and we measured haptoglobin and nitric oxide levels pre- and post-injection. Both haptoglobin and nitric oxide are important markers for innate immune function. We found that ALAN exposure altered the innate immune response, with nestlings exposed to ALAN having lower haptoglobin and higher nitric oxide levels after the immune challenge compared with dark-night nestlings. Unexpectedly, nitric oxide levels were overall lower after the immune challenge than before. These effects were probably mediated by melatonin, as ALAN-treated birds had on average 49% lower melatonin levels than the dark-night birds. ALAN exposure did not have any clear effects on nestling growth. This study provides a potential physiological mechanism underlying the documented differences in immune function between urban and rural birds observed in other studies. Moreover, it gives evidence that ALAN exposure affects nestling physiology, potentially causing long-term effects on physiology and behaviour, which ultimately can affect their fitness.

Reduced ectoparasite load, body mass and blood haemolysis in Eurasian kestrels (Falco tinnunculus) along an urban-rural gradient.

Urbanisation is proceeding at an alarming rate which forces wildlife to either retreat from urban areas or cope with novel stressors linked to human presence and activities. For example, urban stressors like anthropogenic noise, artificial light at night and chemical pollution can have severe impacts on the physiology of wildlife (and humans), in particular the immune system and antioxidant defences. These physiological systems are important to combat and reduce the severity of parasitic infections, which are common among wild animals. One question that then arises is whether urban-dwelling animals, whose immune and antioxidant system are already challenged by the urban stressors, are more susceptible to parasitic infections. To assess this, we studied nestlings of Eurasian kestrels (Falco tinnunculus) in Vienna, Austria, during 2015 and 2017. We measured biomarkers of innate immune function, oxidative stress and body mass index and ectoparasite infection intensity in 143 nestlings (from 56 nests) along an urban gradient. Nestlings in more urbanised areas had overall fewer ectoparasites, lower haemolysis (complement activity) and lower body mass index compared to nestlings in less urbanised areas. None of the other immune or oxidative stress markers were associated with the urban gradient. Despite some non-significant results, our data still suggest that kestrel nestlings experience some level of reduced physiological health, perhaps as a consequence of exposure to more urban stressors or altered prey availability in inner-city districts even though they had an overall lower ectoparasite burden in these heavily urbanised areas.

Reduced immune responsiveness contributes to winter energy conservation in an Arctic bird.

Animals in seasonal environments must prudently manage energy expenditure to survive the winter. This may be achieved through reductions in the allocation of energy for various purposes (e.g. thermoregulation, locomotion, etc.). We studied whether such trade-offs also include suppression of the innate immune response, by subjecting captive male Svalbard ptarmigan (Lagopus muta hyperborea) to bacterial lipopolysaccharide (LPS) during exposure to either mild temperature (0°C) or cold snaps (acute exposure to -20°C), in constant winter darkness when birds were in energy-conserving mode, and in constant daylight in spring. The innate immune response was mostly unaffected by temperature. However, energy expenditure was below baseline when birds were immune challenged in winter, but significantly above baseline in spring. This suggests that the energetic component of the innate immune response was reduced in winter, possibly contributing to energy conservation. Immunological parameters decreased (agglutination, lysis, bacteriostatic capacity) or did not change (haptoglobin/PIT54) after the challenge, and behavioural modifications (anorexia, mass loss) were lengthy (9 days). While we did not study the mechanisms explaining these weak, or slow, responses, it is tempting to speculate they may reflect the consequences of having evolved in an environment where pathogen transmission rate is presumably low for most of the year. This is an important consideration if climate change and increased exploitation of the Arctic would alter pathogen communities at a pace outwith counter-adaption in wildlife.

Experimental facilitation of heat loss affects work rate and innate immune function in a breeding passerine bird.

The capacity to get rid of excess heat produced during hard work is a possible constraint on parental effort during reproduction [heat dissipation limit (HDL) theory]. We released hard-working blue tits (Cyanistes caeruleus) from this constraint by experimentally removing ventral plumage. We then assessed whether this changed their reproductive effort (feeding rate and nestling size) and levels of self-maintenance (change in body mass and innate immune function). Feather-clipped females reduced the number of feeding visits and increased levels of constitutive innate immunity compared with unclipped females but did not fledge smaller nestlings. Thus, they increased self-maintenance without compromising current reproductive output. In contrast, feather clipping did not affect the number of feeding visits or innate immune function in males, despite increased heat loss rate. Our results show that analyses of physiological parameters, such as constitutive innate immune function, can be important when trying to understand sources of variation in investment in self-maintenance versus reproductive effort and that risk of overheating can influence innate immune function during reproduction.

Defence versus defence: Are crucian carp trading off immune function against predator-induced morphology?

Numerous species adopt inducible defence strategies; that is, they have phenotypically plastic traits that decrease the risk of capture and consumption by potential predators. The benefits of expressing alternative phenotypes in high- vs. low-risk environments are well documented. However, inducible anti-predator traits are also expected to incur costs, as they are not expressed when predators are absent, yet empirical evidence of such costs remains scarce. Virtually, all animals in nature are simultaneously under strong selection to evade both capture by predators and infection by parasites or pathogens and, hence, display a diverse arsenal of defences to combat these threats, raising the possibility of trade-offs between defences. A classic example of a predator-induced morphological defence is the deep-bodied shape of crucian carp that reduces risk of predation from gape-size-limited predators. The goal of this study was to examine whether predator exposure affects also immune function in crucian carp, and whether the degree of expressed morphological defence is traded off against immune function in individuals. Following exposure to manipulations of perceived risk (predator presence/absence) in a long-term experiment (8 months), key aspects of innate immune function and individual differences in the expression of inducible morphological defence were quantified. Predator-exposed individuals showed lower haptoglobin levels and complement activity, but higher natural antibody titres than fish from predator-free conditions. When experimentally challenged with a mimicked bacterial infection (LPS injection), fish reared in the presence of a natural predator showed a weaker immune response. Moreover, among predator-exposed individuals, the magnitude of morphological defence expression correlated with both baseline immune function and the ability to mount an immune response. However, these relationships were not consistently supportive of a general trade-off among defences. Our results suggest that fish exposed to predators on average reduce investment in immune function, and, further, the observed relationships among defences in predator-exposed individuals can best be explained from individual fitness and pace-of-life perspectives.

A mimicked bacterial infection prolongs stopover duration in songbirds-but more pronounced in short- than long-distance migrants.

Migration usually consists of intermittent travel and stopovers, the latter being crucially important for individuals to recover and refuel to successfully complete migration. Quantifying how sickness behaviours influence stopovers is crucial for our understanding of migration ecology and how diseases spread. However, little is known about infections in songbirds, which constitute the majority of avian migrants. We experimentally immune-challenged autumn migrating passerines (both short- and long-distance migrating species) with a simulated bacterial infection. Using an automated radiotelemetry system in the stopover area, we subsequently quantified stopover duration, "bush-level" activity patterns (0.1-30 m) and landscape movements (30-6,000 m). We show that compared to controls, immune-challenged birds prolonged their stopover duration by on average 1.2 days in long-distance and 2.9 days in short-distance migrants, respectively (100%-126% longer than controls, respectively). During the prolonged stopover, the immune-challenged birds kept a high "bush-level" activity (which was unexpected) but reduced their local movements, independent of migration strategy. Baseline immune function, but not blood parasite infections prior to the immune challenge, had a prolonging effect on stopover duration, particularly in long-distance migrants. We conclude that a mimicked bacterial infection does not cause lethargy, per se, but restricts landscape movements and prolongs stopover duration, and that this behavioural response also depends on the status of baseline immune function and migration strategy. This adds a new level to the understanding of how acute inflammation affect migration behaviour and hence the ecology and evolution of migration. Accounting for these effects of bacterial infections will also enable us to fine-tune and apply optimal migration theory. Finally, it will help us predicting how migrating animals may respond to increased pathogen pressure caused by global change.

Immune function and blood parasite infections impact stopover ecology in passerine birds.

Stopovers play a crucial role for the success of migrating animals and are key to optimal migration theory. Variation in refuelling rates, stopover duration and departure decisions among individuals has been related to several external factors. The physiological mechanisms shaping stopover ecology are, however, less well understood. Here, we explore how immune function and blood parasite infections relate to several aspects of stopover behaviour in autumn migrating short- and long-distance migrating songbirds. We blood sampled individuals of six species and used an automated radio-telemetry system in the stopover area to subsequently quantify stopover duration, 'bush-level' activity patterns (~ 0.1-30 m), landscape movements (~ 30-6000 m), departure direction and departure time. We show that complement activity, the acute phase protein haptoglobin and blood parasite infections were related to prolonged stopover duration. Complement activity (i.e., lysis) and total immunoglobulins were negatively correlated with bush-level activity patterns. The differences partly depended on whether birds were long-distance or short-distance migrants. Birds infected with avian malaria-like parasites showed longer landscape movements during the stopover than uninfected individuals, and birds with double blood parasite infections departed more than 2.5 h later after sunset/sunrise suggesting shorter flight bouts. We conclude that variation in baseline immune function and blood parasite infection status affects stopover ecology and helps explain individual variation in stopover behaviour. These differences affect overall migration speed, and thus can have significant impact on migration success and induce carry-over effects on other annual-cycle stages. Immune function and blood parasites should, therefore, be considered as important factors when applying optimal migration theory.

Indices of immune function used by ecologists are mostly unaffected by repeated freeze-thaw cycles and methodological deviations.

BACKGROUND: Over the past couple of decades, measuring immunological parameters has become widespread in studies of ecology and evolution. A combination of different immunological indices is useful for quantifying different parts of the immune system and comprehensively assessing immune function. Running multiple immune assays usually requires samples to be repeatedly thawed and re-frozen. There is some evidence that repeated freezing and thawing can affect assay results, but this has never been comprehensively studied in some common ecological immunology assays. We tested the effect of multiple (1, 2, 3, 4, 5, 10) freeze-thaw cycles on the results of four commonly used immunological assays: haemolysis-haemagglutination titres, haptoglobin concentration, bacterial killing capacity and total immunoglobulins (IgY). We tested five different bird species from four different bird orders (Passeriformes, Columbiformes, Charadriiformes and Galliformes), and we included both captive and free-living individuals. In addition, we tested for haptoglobin concentrations and the haemolysis-haemagglutination assay if re-analysing samples 1 year apart led to different results. For the haemolysis-haemagglutination assay we also tested two different sources of rabbit blood, and we compared untreated microtitre plates with plates that were "blocked" to prevent nonspecific interactions between the plate and assay reagents. RESULTS: Repeated freezing and thawing of plasma had no effect on lysis titres, haptoglobin concentrations, bacterial killing capacity, or total immunoglobulin levels. Agglutination titres were unaffected by up to five cycles but were lower after ten freeze-thaw cycles. For the haemolysis-haemagglutination assay and haptoglobin concentrations, re-analysing samples 1 year apart yielded highly correlated data. For the haemolysis-haemagglutination assay, the source of rabbit blood did not influence the results, and the untreated vs. blocked plates differed slightly overall, but at the individual level assay results were highly correlated. Using different rabbit blood sources or different types of microtitre plates yielded highly correlated data. CONCLUSIONS: Our data suggest that repeated freeze-thaw cycles do not impair assay results to the point of influencing ecological or evolutionary conclusions. Plasma samples can be safely stored in one tube and thawed repeatedly for different assays. Nevertheless, we recommend consistent treatment of samples in terms of freeze-thaw cycles or other laboratory treatments to minimize the potential for introducing a systematic bias.

Migratory common blackbirds have lower innate immune function during autumn migration than resident conspecifics.

Animals need a well-functioning immune system to protect themselves against pathogens. The immune system, however, is costly and resource trade-offs with other demands exist. For migratory animals several (not mutually exclusive) hypotheses exist. First, migrants reduce immune function to be able to allocate resources to migration. Second, migrants boost immune function to cope with more and/or novel pathogens encountered during migration. Third, migrants reallocate resources within the immune system. We tested these hypotheses by comparing baseline immune function in resident and migratory common blackbirds (Turdus merula), both caught during the autumn migration season on the island of Helgoland, Germany. Indices of baseline innate immune function (microbial killing capacity and haptoglobin-like activity) were lower in migrants than in residents. There was no difference between the groups in total immunoglobulins, a measure of baseline acquired immune function. Our study on a short-distance avian migrant supports the hypothesis that innate immune function is compromised during migration.

Causes and Consequences of Partial Migration in a Passerine Bird.

Many animal species have populations in which some individuals migrate and others remain on the breeding grounds. This phenomenon is called partial migration. Despite substantial theoretical work, empirical data on causes and consequences of partial migration remain scarce, mainly because of difficulties associated with tracking individuals over large spatial scales. We used stable hydrogen isotopes in claw material to determine whether skylarks Alauda arvensis from a single breeding population in the Netherlands had migrated or remained resident in the previous winter and investigated whether there were causes or consequences of either strategy. Age and sex had no influence on the propensity to migrate, but larger individuals were more likely to be residents. The wintering strategy was not fixed within individuals. Up to 45% of individuals measured in multiple years switched strategies. Reproductive parameters were not related to the wintering strategy, but individuals that wintered locally experienced lower future return rates, and this was directly correlated with two independent measures of immune function. Our results suggest that partial migration in skylarks is based neither on genetic dimorphism nor on an age- and sex-dependent condition. Instead, the wintering strategy is related to structural size and immune function. These new insights on causes and consequences of partial migration advance our understanding of the ecology, evolution, and coexistence of different life-history strategies.

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.

Are antimicrobial defences in bird eggs related to climatic conditions associated with risk of trans-shell microbial infection?

INTRODUCTION: All bird eggs are exposed to microbes in the environment, which if transmitted to the developing embryo, could cause hatching failure. However, the risk of trans-shell infection varies with environmental conditions and is higher for eggs laid in wetter environments. This might relate to generally higher microbial abundances and diversity in more humid environments, including on the surface of eggshells, as well as the need for moisture to facilitate microbial penetration of the eggshell. To protect against microbial infection, the albumen of avian eggs contains antimicrobial proteins, including lysozyme and ovotransferrin. We tested whether lysozyme and ovotransferrin activities varied in eggs of larks (Alaudidae) living along an arid-mesic gradient of environmental aridity, which we used as a proxy for risk of trans-shell infection. RESULTS: Contrary to expectations, lysozyme activity was highest in eggs from hotter, more arid locations, where we predicted the risk of trans-shell infection would be lower. Ovotransferrin concentrations did not vary with climatic factors. Temperature was a much better predictor of antimicrobial protein activity than precipitation, a result inconsistent with studies stressing the importance of moisture for trans-shell infection. CONCLUSIONS: Our study raises interesting questions about the links between temperature and lysozyme activity in eggs, but we find no support for the hypothesis that antimicrobial protein deposition is higher in eggs laid in wetter environments.

Constitutive immune function is not associated with fuel stores in spring migrating passerine birds.

Migratory birds may either upregulate their immune system during migration as they might encounter novel pathogens or downregulate their immune system as a consequence of trade-offs with the resource costs of migration. Support for the latter comes not least from a study that reports a positive correlation in autumn migrating birds between fuel stores and parameters of innate and acquired immune function, that is, energy-exhausted migrants appear to have lowered immune function. However, to our knowledge, no study has tested whether this pattern exists in spring migrating birds, which may face other trade-offs than autumn migrants. Here, we investigate if in spring there is a relationship between fuel stores and microbial-killing ability, a measure of innate immune function, and total immunoglobulin (IgY), a measure of acquired immune function, in four migrating bird species: chaffinches (Fringilla coelebs), dunnocks (Prunella modularis), song thrushes (Turdus philomelos) and northern wheatears (Oenanthe oenanthe). Our findings indicate no significant correlation between fuel stores and either microbial killing ability or IgY levels when considering all species collectively. When analysing species separately, we found a significant negative correlation between fuel stores and microbial-killing ability in chaffinches and a positive correlation between fuel stores and IgY levels in wheatears. In song thrushes, but not in any of the other species, there was a significant negative correlation between relative arrival date and microbial-killing ability and between arrival date and IgY levels. Sex did not affect immune function in any of the species. Our study suggests that the relationship between immune function and fuel stores may be different during spring migration compared to autumn migration. Differences in the speed of migration or pathogen pressure may result in different outcomes of the resource trade-off between investment in immune function and migration among the seasons.

Urbanisation-associated shifts in the avian metabolome within the annual cycle.

While organisms have evolved to cope with predictable changes in the environment, the rapid rate of current global change presents numerous novel and unpredictable stressors to which organisms have had less time to adapt. To persist in the urban environment, organisms must modify their physiology, morphology and behaviour accordingly. Metabolomics offers great potential for characterising organismal responses to natural and anthropogenic stressors at the systems level and can be applied to any species, even without genomic knowledge. Using metabolomic profiling of blood, we investigated how two closely related species of passerine bird respond to the urban environment. Great tits Parus major and blue tits Cyanistes caeruleus residing in urban and forest habitats were sampled during the breeding (spring) and non-breeding (winter) seasons across replicated sites in southern Sweden. During breeding, differences in the plasma metabolome between urban and forest birds were characterised by higher levels of amino acids in urban-dwelling tits and higher levels of fatty acyls in forest-dwelling tits. The suggested higher rates of fatty acid oxidation in forest tits could be driven by habitat-associated differences in diet and could explain the higher reproductive investment and success of forest tits. High levels of amino acids in breeding urban tits could reflect the lack of lipid-rich caterpillars in the urban environment and a dietary switch to protein-rich spiders, which could be of benefit for tackling inflammation and oxidative stress associated with pollution. In winter, metabolomic profiles indicated lower overall levels of amino acids and fatty acyls in urban tits, which could reflect relaxed energetic demands in the urban environment. Our metabolomic profiling of two urban-adapted species suggests that their metabolism is modified by urban living, though whether these changes represent adaptative or non-adaptive mechanisms to cope with anthropogenic challenges remains to be determined.