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.

Gut microbiota of homing pigeons shows summer-winter variation under constant diet indicating a substantial effect of temperature.

BACKGROUND: Gut microbiotas play a pivotal role in host physiology and behaviour, and may affect host life-history traits such as seasonal variation in host phenotypic state. Generally, seasonal gut microbiota variation is attributed to seasonal diet variation. However, seasonal temperature and day length variation may also drive gut microbiota variation. We investigated summer-winter differences in the gut bacterial community (GBC) in 14 homing pigeons living outdoors under a constant diet by collecting cloacal swabs in both seasons during two years. Because temperature effects may be mediated by host metabolism, we determined basal metabolic rate (BMR) and body mass. Immune competence is influenced by day length and has a close relationship with the GBC, and it may thus be a link between day length and gut microbiota. Therefore, we measured seven innate immune indices. We expected the GBC to show summer-winter differences and to correlate with metabolism and immune indices. RESULTS: BMR, body mass, and two immune indices varied seasonally, other host factors did not. The GBC showed differences between seasons and sexes, and correlated with metabolism and immune indices. The most abundant genus (Lachnoclostridium 12, 12%) and associated higher taxa, were more abundant in winter, though not significantly at the phylum level, Firmicutes. Bacteroidetes were more abundant in summer. The Firmicutes:Bacteroidetes ratio tended to be higher in winter. The KEGG ortholog functions for fatty acid biosynthesis and linoleic acid metabolism (PICRUSt2) had increased abundances in winter. CONCLUSIONS: The GBC of homing pigeons varied seasonally, even under a constant diet. The correlations between immune indices and the GBC did not involve consistently specific immune indices and included only one of the two immune indices that showed seasonal differences, suggesting that immune competence may be an unlikely link between day length and the GBC. The correlations between the GBC and metabolism indices, the higher Firmicutes:Bacteroidetes ratio in winter, and the resemblance of the summer-winter differences in the GBC with the general temperature effects on the GBC in the literature, suggest that temperature partly drove the summer-winter differences in the GBC in homing pigeons.

Effects of manipulated food availability and seasonality on innate immune function in a passerine.

The innate immune system is essential for survival, yet many immune traits are highly variable between and within individuals. In recent years, attention has shifted to the role of environmental factors in modulating this variation. A key environmental factor is food availability, which plays a major role in shaping life histories, and may affect resource allocation to immune function through its effect on nutritional state. We developed a technique to permanently increase foraging costs in seed-eating birds, and leveraged this technique to study the effects of food availability on the innate immune system over a 3-year period in 230 zebra finches housed in outdoor aviaries. The immune components we studied were haptoglobin, ovotransferrin, nitric oxide, natural antibodies through agglutination, complement-mediated lysis, and killing capacity of Escherichia coli and Candida albicans, covering a broad spectrum of the innate immune system. We explored the effects of food availability in conjunction with other potentially important variables: season, age, sex and manipulated natal brood size. Increased foraging costs affected multiple components of the immune system, albeit in a variable way. Nitric oxide and agglutination levels were lower under harsh foraging conditions, while Escherichia coli killing capacity was increased. Agglutination levels also varied seasonally, but only at low foraging costs. C. albicans killing capacity was lower in winter, and even more so for animals in harsh foraging conditions that were raised in large broods. Effects of food availability on ovotransferrin were also seasonal, and only apparent in males. Haptoglobin levels were independent of foraging costs and season. Males had higher levels of immune function than females for three of the measured immune traits. Innate immune function was independent of age and manipulated natal brood size. Our finding that food availability affects innate immune function suggests that fitness effects of food availability may at least partially be mediated by effects on the immune system. However, food availability effects on innate immunity varied in direction between traits, illustrating the complexity of the immune system and precluding conclusions on the level of disease resistance.

The microbial environment modulates non-genetic maternal effects on egg immunity.

BACKGROUND: In a diverse microbial world immune function of animals is essential. Diverse microbial environments may contribute to extensive variation in immunological phenotypes of vertebrates, among and within species and individuals. As maternal effects benefit offspring development and survival, whether females use cues about their microbial environment to prime offspring immune function is unclear. To provide microbial environmental context to maternal effects, we asked if the bacterial diversity of the living environment of female zebra finches Taeniopygia guttata shapes maternal effects on egg immune function. We manipulated environmental bacterial diversity of birds and tested if females increased immunological investment in eggs in an environment with high bacterial diversity (untreated soil) versus low (gamma-sterilized soil). We quantified lysozyme and ovotransferrin in egg albumen and IgY in egg yolk and in female blood, and we used 16S rRNA gene sequencing to profile maternal cloacal and eggshell microbiotas. RESULTS: We found a maternal effect on egg IgY concentration that reflected environmental microbial diversity: females who experienced high diversity deposited more IgY in their eggs, but only if maternal plasma IgY levels were relatively high. We found no effects on lysozyme and ovotransferrin concentrations in albumen. Moreover, we uncovered that variation in egg immune traits could be significantly attributed to differences among females: for IgY concentration in yolk repeatability R = 0.80; for lysozyme concentration in albumen R = 0.27. Furthermore, a partial least squares path model (PLS-PM) linking immune parameters of females and eggs, which included maternal and eggshell microbiota structures and female body condition, recapitulated the treatment-dependent yolk IgY response. The PLS-PM additionally suggested that the microbiota and physical condition of females contributed to shaping maternal effects on egg immune function, and that (non-specific) innate egg immunity was prioritized in the environment with low bacterial diversity. CONCLUSIONS: The microbial environment of birds can shape maternal effects on egg immune function. Since immunological priming of eggs benefits offspring, we highlight that non-genetic maternal effects on yolk IgY levels based on cues from the parental microbial environment may prove important for offspring to thrive in the microbial environment that they are expected to face.

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.

Effects of early-life conditions on innate immune function in adult zebra finches.

Early life conditions can affect individuals for life, with harsh developmental conditions resulting in lower fitness, but the underlying mechanisms are not well understood. We hypothesized that immune function may be part of the underlying mechanism, when harsh developmental conditions result in less effective immune function. We tested this hypothesis by comparing innate immune function between zebra finches (Taeniopygia guttata) in adulthood (n=230; age 108-749 days) that were reared in either small or large broods. We used this experimental background to follow up our earlier finding that finches reared in large broods have a shorter lifespan. To render a broad overview of innate immune function, we used an array of six measures: bacterial killing capacity, hemagglutination, hemolysis, haptoglobin, nitric oxide and ovotransferrin. We found no convincing evidence for effects of natal brood size on any of the six measures of innate immune function. This raised the question whether the origin of variation in immune function was genetic, and we therefore estimated heritabilities using animal models. However, we found heritability estimates to be low (range 0.04-0.11) for all measured immune variables, suggesting variation in innate immune function can largely be attributed to environmental effects independent of early-life conditions as modified by natal brood size.

Costs of reproduction and migration are paid in later return to the colony, not in physical condition, in a long-lived seabird.

Life history theory suggests a trade-off between costly activities such as breeding and migration and somatic self-maintenance. However, how the short-term cost of parental effort is expressed in species with a slow pace-of-life is not well understood. Also, investigating carry-over effects of migration is most meaningful when comparing migratory strategies within the same population, but this has rarely been done. We explore this hypothesis in a long-lived, pelagic seabird, the Cory's Shearwater, Calonectris borealis, where males display partial migration. By manipulating reproductive effort and taking advantage of the natural variation in migratory strategy, we investigate whether early reproductive failure and migratory strategy had implications on the physical condition of males on return to the colony the following year. We experimentally induced breeding failure from mid-incubation, tracked the over-winter movements of these males and of males that invested in parental effort, and assessed innate immunity, stress, and residual body mass the following year. Early breeding failure resulted in earlier return to the colony among all males, associated with greater probability of reproductive success. Residents had a lower tail feather fault bar intensity, an indicator of stress during the non-breeding period, compared to migrants. Reproductive effort and migratory strategy had no impact on physiological condition otherwise. Our results provide evidence that in species with a slow-pace of life, such as the Cory's Shearwater, somatic maintenance is prioritised, with the costs of reproduction and migration paid in delayed arrival date.

Geographic variation in baseline innate immune function does not follow variation in aridity along a tropical environmental gradient.

Geographic variation in aridity determines environmental productivity patterns, including large-scale variability in pathogens, vectors and associated diseases. If disease risk decreases with increasing aridity and is matched by immune defense, we predict a decrease in innate immune function along a gradient of increasing aridity from the cool-wet forest to the hot-dry Sahel, from south to north in Nigeria. We sampled blood and measured five innate immune indices from 286 Common Bulbuls Pycnonotus barbatus between 6 and 13°N. We sampled in the dry season; we resampled the first location (Jos) also as the last sample location to test temporal change in immune function. Immune indices did not decrease with aridity. One immune index, nitric oxide concentration showed a weak quadratic pattern. In Jos, ovotransferrin concentration, haemagglutination and haemolysis titres increased 12 weeks into the dry season, contrary to expectations that immune indices should decrease with increased dryness. In this tropical system, innate immune function does not decrease with increasing aridity but temporal factors within a location may influence immune function more strongly than spatial variation in aridity, suggesting that immune variation does not follow a simple environmental productivity pattern. Consequently, caution should probably be exercised in predicting effects of climate variability on immune function or disease risk.

Physiological stress does not increase with urbanization in European blackbirds: Evidence from hormonal, immunological and cellular indicators.

Urbanization changes the landscape structure and ecological processes of natural habitats. While urban areas expose animal communities to novel challenges, they may also provide more stable environments in which environmental fluctuations are buffered. Species´ ecology and physiology may determine their capacity to cope with the city life. However, the physiological mechanisms underlying organismal responses to urbanization, and whether different physiological systems are equally affected by urban environments remain poorly understood. This severely limits our capacity to predict the impact of anthropogenic habitats on wild populations. In this study, we measured indicators of physiological stress at the endocrine, immune and cellular level (feather corticosterone levels, heterophil to lymphocyte ratio, and heat-shock proteins) in urban and non-urban European blackbirds (Turdus merula) across 10 European populations. Among the three variables, we found consistent differences in feather corticosterone, which was higher in non-urban habitats. This effect seems to be dependent on sex, being greater in males. In contrast, we found no significant differences between urban and non-urban habitats in the two other physiological indicators. The discrepancy between these different measurements of physiological stress highlights the importance of including multiple physiological variables to understand the impact of urbanization on species' physiology. Overall, our findings suggest that adult European blackbirds living in urban and non-urban habitats do not differ in terms of physiological stress at an organismal level. Furthermore, we found large differences among populations on the strength and direction of the urbanization effect, which illustrates the relevance of spatial replication when investigating urban-induced physiological responses.

Microbial environment shapes immune function and cloacal microbiota dynamics in zebra finches Taeniopygia guttata.

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.

A fruit diet rather than invertebrate diet maintains a robust innate immunity in an omnivorous tropical songbird.

Diet alteration may lead to nutrient limitations even in the absence of food limitation, and this may affect physiological functions, including immunity. Nutrient limitations may also affect the maintenance of body mass and key life-history events that may affect immune function. Yet, variation in immune function is largely attributed to energetic trade-offs rather than specific nutrient constraints. To test the effect of diet on life-history traits, we tested how diet composition affects innate immune function, body mass and moult separately and in combination with each other, and then used path analyses to generate hypotheses about the mechanistic connections between immunity and body mass under different diet compositions. We performed a balanced parallel and crossover design experiment with omnivorous common bulbuls Pycnonotus barbatus in out-door aviaries in Nigeria. We fed 40 wild-caught bulbuls ad libitum on fruits or invertebrates for 24 weeks, switching half of each group between treatments after 12 weeks. We assessed innate immune indices (haptoglobin, nitric oxide and ovotransferrin concentrations, and haemagglutination and haemolysis titres), body mass and primary moult, fortnightly. We simplified immune indices into three principal components (PCs), but we explored mechanistic connections between diet, body mass and each immune index separately. Fruit-fed bulbuls had higher body mass, earlier moult and showed higher values for two of the three immune PCs compared to invertebrate-fed bulbuls. These effects were reversed when we switched bulbuls between treatments after 12 weeks. Exploring the correlations between immune function, body mass and moult, showed that an increase in immune function was associated with a decrease in body mass and delayed moult in invertebrate-fed bulbuls, while fruit-fed bulbuls maintained body mass despite variation in immune function. Path analyses indicated that diet composition was most likely to affect body mass and immune indices directly and independently from each other. Only haptoglobin concentration was indirectly linked to diet composition via body mass. We demonstrated a causal effect of diet composition on innate immune function, body mass and moult: bulbuls were in a better condition when fed on fruits than invertebrates, confirming that innate immunity is nutrient specific. Our results are unique because they show a reversible effect of diet composition on wild adult birds whose immune systems are presumably fully developed and adapted to wild conditions-demonstrating a short-term consequence of diet alteration on life-history traits.

Seasonal differences in baseline innate immune function are better explained by environment than annual cycle stage in a year-round breeding tropical songbird.

Seasonal variation in innate immunity is often attributed to either temporal environmental variation or to life-history trade-offs that arise from specific annual cycle stages but decoupling them is difficult in natural populations. Here, we effectively decouple seasonal environmental variation from annual cycle stage effects by exploiting cross-seasonal breeding and moult in the tropical Common Bulbul Pycnonotus barbatus. We test how annual cycle stage interacts with a key seasonal environmental variable, rainfall, to determine immunity at population and individual level. If immune challenge varies with precipitation, we might expect immune function to be higher in the wet season due to increased environmental productivity. If breeding or moult imposes resource constraints on birds, depending on or independent of precipitation, we might expect lower immune indices during breeding or moult. We sampled blood from 818 birds in four annual cycle stage categories: breeding, moult, simultaneous breeding and moulting, or neither. We quantified indices of innate immunity (haptoglobin, nitric oxide (NOx ) and ovotransferrin concentrations, and haemagglutination and haemolysis titres) over two annual cycles of wet and dry seasons. Environment (but not annual cycle stage or interactions between both) explained variation in all immune indices, except NOx . NOx concentration differed between annual cycle stages but not between seasons. However, within the wet season, haptoglobin, NOx , ovotransferrin and haemolysis differed significantly between breeding and non-breeding females. Aside from some recorded inconsistencies, population level results were largely similar to results within individuals that were measured repeatedly. Unexpectedly, most immune indices were higher in the dry season and during breeding. Higher immune indices may be explained if fewer or poorer quality resources force birds to increase social contact, thereby exposing individuals to novel antigens and increased infection risk, independently of environmental productivity. Breeding birds may also show higher immunity if less immune-competent and/or infected females omit breeding. We conclude that seasonal environmental variation impacts immunity more directly in natural animal populations than via resource trade-offs. In addition, immune indices were more often variable within than among individuals, but some indices are characteristic of individuals, and so may offer selective advantages if heritable.

Prenatal Transfer of Gut Bacteria in Rock Pigeon.

Vertebrates evolved in concert with bacteria and have developed essential mutualistic relationships. Gut bacteria are vital for the postnatal development of most organs and the immune and metabolic systems and may likewise play a role during prenatal development. Prenatal transfer of gut bacteria is shown in four mammalian species, including humans. For the 92% of the vertebrates that are oviparous, prenatal transfer is debated, but it has been demonstrated in domestic chicken. We hypothesize that also non-domestic birds can prenatally transmit gut bacteria. We investigated this in medium-sized Rock pigeon (Columba livia), ensuring neonates producing fair-sized first faeces. The first faeces of 21 neonate rock pigeons hatched in an incubator, contained a microbiome (bacterial community) the composition of which resembled the cloacal microbiome of females sampled from the same population (N = 5) as indicated by multiple shared phyla, orders, families, and genera. Neonates and females shared 16.1% of the total number of OTUs present (2881), and neonates shared 45.5% of their core microbiome with females. In contrast, the five females shared only 0.3% of the 1030 female OTUs present. These findings suggest that prenatal gut bacterial transfer may occur in birds. Our results support the hypothesis that gut bacteria may be important for prenatal development and present a heritability pathway of gut bacteria in vertebrates.

Nest predation risk modifies nestlings' immune function depending on the level of threat.

Predation risk is thought to modify the physiology of prey mainly through the stress response. However, little is known about its potential effects on the immunity of animals, particularly in young individuals, despite the importance of overcoming wounding and pathogen aggression following a predator attack. We investigated the effect of four progressive levels of nest predation risk on several components of the immune system in common blackbird (Turdus merula) nestlings by presenting them with four different calls during 1 h: non-predator calls, predator calls, parental alarm calls and conspecific distress calls to induce a null, moderate, high and extreme level of risk, respectively. Nest predation risk induced an increase in ovotransferrin, immunoglobulin and the number of lymphocytes and eosinophils. Thus, the perception of a potential predator per se could stimulate the mobilization of a nestling's immune function and enable the organism to rapidly respond to the immune stimuli imposed by a predator attack. Interestingly, only high and extreme levels of risk caused immunological changes, suggesting that different immunological parameters are modulated according to the perceived level of threat. We also found a mediator role of parasites (i.e. Leucocytozoon) and the current health status of the individual, as only nestlings not parasitized or in good body condition were able to modify their immune system. This study highlights a previously unknown link between predation risk and immunity, emphasizing the complex relationship among different selective pressures (predation, parasitism) in developing organisms and accentuating the importance of studying predation from a physiological point of view.

Understanding immune function as a pace of life trait requires environmental context.

This article provides a brief historical perspective on the integration of physiology into the concept of the pace of life of birds, evaluates the fit of immune function into this framework, and asks what it will take to fruitfully understand immune functioning of birds in pace of life studies in the future. In the late 1970s, physiology started to seriously enter avian life history ecology, with energy as the main currency of interest, inspired by David Lack's work in the preceding decades emphasizing how food availability explained life history variation. In an effort to understand the trade-off between survival and reproduction, and specifically the mortality costs associated with hard work, in the 1980s and 1990s, other physiological phenomena entered the realm of animal ecologists, including endocrinology, oxidative stress, and immunology. Reviewing studies thus far to evaluate the role of immune function in a life history context and particularly to address the questions whether immune function (1) consistently varies with life history variation among free-living bird species and (2) mediates life history trade-offs in experiments with free-living bird species; I conclude that, unlike energy metabolism, the immune system does not closely covary with life history among species nor mediates the classical trade-offs within individuals. Instead, I propose that understanding the tremendous immunological variation uncovered among free-living birds over the past 25 years requires a paradigm shift. The paradigm should shift from viewing immune function as a costly trait involved in life history trade-offs to explicitly including the benefits of the immune system and placing it firmly in an environmental and ecological context. A first step forward will be to quantify the immunobiotic pressures presented by diverse environmental circumstances that both shape and challenge the immune system of free-living animals. Current developments in the fields of infectious wildlife diseases and host-microbe interactions provide promising steps in this direction.

Microbiome assembly of avian eggshells and their potential as transgenerational carriers of maternal microbiota.

The microbiome is essential for development, health and homeostasis throughout an animal's life. Yet, the origins and transmission processes governing animal microbiomes remain elusive for non-human vertebrates, oviparous vertebrates in particular. Eggs may function as transgenerational carriers of the maternal microbiome, warranting characterisation of egg microbiome assembly. Here, we investigated maternal and environmental contributions to avian eggshell microbiota in wild passerine birds: woodlark Lullula arborea and skylark Alauda arvensis. Using 16S rRNA gene sequencing, we demonstrated in both lark species, at the population and within-nest levels, that bacterial communities of freshly laid eggs were distinct from the female cloacal microbiome. Instead, soil-borne bacteria appeared to thrive on freshly laid eggs, and eggshell microbiota composition strongly resembled maternal skin, body feather and nest material communities, sources in direct contact with laid eggs. Finally, phylogenetic structure analysis and microbial source tracking underscored species sorting from directly contacting sources rather than in vivo-transferred symbionts. The female-egg-nest system allowed an integrative assessment of avian egg microbiome assembly, revealing mixed modes of symbiont acquisition not previously documented for vertebrate eggs. Our findings illuminated egg microbiome origins, which suggested a limited potential of eggshells for transgenerational transmission, encouraging further investigation of eggshell microbiome functions in vertebrates.

Temperature and aridity determine body size conformity to Bergmann's rule independent of latitudinal differences in a tropical environment.

Bergmann's rule, defined as the tendency for endotherms to be larger in colder environments, is a biophysical generalization of body size variation that is frequently tested along latitudinal gradients, even though latitude is only a proxy for temperature variation. We test whether variation in temperature and aridity determine avian body size conformity to Bergmann's rule independent of latitude differences, using the ubiquitous Common Bulbul Pycnonotus barbatus, along a West African environmental gradient. We trapped 538 birds in 22 locations between latitudes 6 and 13°N in Nigeria, and estimated average body surface area to mass ratio per location. We then modelled body surface to mass ratio using general linear models, with latitude, altitude and one of 19 bioclimatic variables extracted from http://www.worldclim.org/bioclim as predictors. We sequentially dropped latitude and altitude from each model to obtain the R 2 of the resultant models. Finally, we compared the R 2 of univariate models, where bioclimatic variables predicted body surface area to mass ratio significantly (14 out of 19), to multivariate models including latitude, altitude and a bioclimatic variable, using the Wilcoxon matched pairs test. We found that multivariate models did not perform better than univariate models with only bioclimatic variables. Six temperature and eight precipitation variables significantly predicted variation in body surface area to mass ratio between locations; in fact, 50% (seven out of 14) of these better explained variation in body surface area to mass ratio than the multivariate models. Birds showed a larger body surface area relative to body mass ratio in hotter environments independent of latitude or altitude, which conforms to Bergmann's rule. Yet, a combination of morphometric analyses and controlled temperature-exposure experiments is required to prove the proposed relationship between relative body surface area and thermoregulation in endotherms.

Fecal sacs do not increase nest predation in a ground nester.

Most altricial birds remove their nestlings' feces from the nest, but the evolutionary forces driving this behavior are poorly understood. A possible adaptive explanation for this could be that birds avoid the attraction of nest predators to their nests due to the visual or olfactory cues produced by feces (nest predation hypothesis). This hypothesis has received contrasting support indicating that additional experimental studies are needed, particularly with respect to the visual component of fecal sacs. To test this hypothesis, we conducted an experiment manipulating the presence of fecal sacs on inactive Woodlark (Lullula arborea) nests. This ground nester has highly cryptic nests that are mainly depredated by visually oriented nest predators (i.e., corvids) in our study population, making it an excellent system to test for the nest predation hypothesis. Our results showed that the presence of fecal sacs in the nest does not seem to be an important factor explaining nest predation. Interestingly, the effect of nest concealment, the most important factor explaining nest predation in Woodlark nests, depended on whether the nest was depredated the previous year or not, supporting the importance of using different nesting sites between years. Our findings indicate that this important nest sanitation behavior is not likely motivated by nest predation and highlight the need to explore alternative selective pressures in this context.

Multi-level comparisons of cloacal, skin, feather and nest-associated microbiota suggest considerable influence of horizontal acquisition on the microbiota assembly of sympatric woodlarks and skylarks.

BACKGROUND: Working toward a general framework to understand the role of microbiota in animal biology requires the characterisation of animal-associated microbial communities and identification of the evolutionary and ecological factors shaping their variation. In this study, we described the microbiota in the cloaca, brood patch skin and feathers of two species of birds and the microbial communities in their nest environment. We compared patterns of resemblance between these microbial communities at different levels of biological organisation (species, individual, body part) and investigated the phylogenetic structure to deduce potential microbial community assembly processes. RESULTS: Using 16S rRNA gene amplicon data of woodlarks (Lullula arborea) and skylarks (Alauda arvensis), we demonstrated that bird- and nest-associated microbiota showed substantial OTU co-occurrences and shared dominant taxonomic groups, despite variation in OTU richness, diversity and composition. Comparing host species, we uncovered that sympatric woodlarks and skylarks harboured similar microbiota, dominated by Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes and Acidobacteria. Yet, compared with the nest microbiota that showed little variation, each species' bird-associated microbiota displayed substantial variation. The latter could be partly (~ 20%) explained by significant inter-individual differences. The various communities of the bird's body (cloaca, brood patch skin and feathers) appeared connected with each other and with the nest microbiota (nest lining material and surface soil). Communities were more similar when the contact between niches was frequent or intense. Finally, bird microbiota showed significant phylogenetic clustering at the tips, but not at deeper branches of the phylogeny. CONCLUSIONS: Our interspecific comparison suggested that the environment is more important than phylogeny in shaping the bird-associated microbiotas. In addition, variation among individuals and among body parts suggested that intrinsic or behavioural differences among females and spatial heterogeneity among territories contributed to the microbiome variation of larks. Modest but significant phylogenetic clustering of cloacal, skin and feather microbiotas suggested weak habitat filtering in these niches. We propose that lark microbiota may be primarily, but not exclusively, shaped by horizontal acquisition from the regional bacterial pool at the breeding site. More generally, we hypothesise that the extent of ecological niche-sharing by avian (or other vertebrate) hosts may predict the convergence of their microbiota.

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.