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

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

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

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

Shifts in bacterial communities of eggshells and antimicrobial activities in eggs during incubation in a ground-nesting passerine

Microbial invasion of egg contents is a cause of embryonic death. To counter infection risks, the embryo is protected physically by the eggshell and chemically by antimicrobial proteins. If microbial pressure drives embryo mortality, then females may have evolved, through natural selection, to adapt their immune investment into eggs. Although frequently hypothesized, this match between immune allocation and microorganisms has not been explored yet. To examine if correlations between microbes on eggs and immunity in eggs exist, we collected eggs from red-capped larks (Calandrella cinerea) and simultaneously examined their bacterial communities and antimicrobial components--pH, lysozyme and ovotransferrin--during natural incubation. Using molecular techniques, we find that bacterial communities are highly dynamic: bacterial abundance increases from the onset to late incubation, Shannon's α-diversity index increases during early incubation stages, and β-diversity analysis shows that communities from 1 day-old clutches are phylogenetically more similar to each other than the older ones. Regarding the antimicrobials, we notice a decrease of pH and lysozyme concentration, while ovotransferrin concentration increases during incubation. Interestingly, we show that two eggs of the same clutch share equivalent immune protection, independent of clutch age. Lastly, our results provide limited evidence of significant correlation between antimicrobial compounds and bacterial communities. Our study examined simultaneously, for the first time in a wild bird, the dynamics of bacterial communities present on eggshells and of albumen-associated antimicrobial components during incubation and investigated their relationship. However, the link between microorganisms and immunity of eggs remains to be elucidated further. Identifying invading microbes and their roles in embryo mortality, as well as understanding the role of the eggshell microbiome, might be key to better understand avian strategies of immune maternal investment

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

Data from: Nest survival in year-round breeding tropical Red-capped Larks (Calandrella cinerea) increases with higher nest abundance but decreases with higher invertebrate availability and rainfall

Nest survival is critical to breeding in birds and plays an important role in life-history evolution and population dynamics. Studies evaluating the proximate factors involved in explaining nest survival and the resulting temporal patterns are biased in favor of temperate regions. Yet, such studies are especially pertinent to the tropics, where nest predation rates are typically high and environmental conditions often allow for year-round breeding. To tease apart the effects of calendar month and year, population-level breeding activity and environmental conditions, we studied nest survival over a 64-month period in equatorial, year-round breeding red-capped larks Calandrella cinerea in Kenya. We show that daily nest survival rates varied with time, but not in a predictable seasonal fashion among months or consistently among years. We found negative influences of flying invertebrate biomass and rain on nest survival and higher survival of nests when nests were more abundant, which suggests that nest predation resulted from incidental predation. Although an increase in nest predation is often attributed to an increase in nest predators, we suggest that in our study, it may be caused by altered predator activity resulting from increased activity of the primary prey, invertebrates, rather than activity of the red-capped larks. Our results emphasize the need to conduct more studies in Afro-tropical regions because proximate mechanisms explaining nest predation can be different in the unpredictable and highly variable environments of the tropics compared with the relatively predictable seasonal changes found in temperate regions. Such studies will aid in better understanding of the environmental influences on life-history variation and population dynamics in birds

Year-round breeding equatorial Larks from three climatically-distinct populations do not use rainfall, temperature or invertebrate biomass to time reproduction

Timing of reproduction in birds is important for reproductive success and is known to depend on environmental cues such as day length and food availability. However, in equatorial regions, where day length is nearly constant, other factors such as rainfall and temperature are thought to determine timing of reproduction. Rainfall can vary at small spatial and temporal scales, providing a highly fluctuating and unpredictable environmental cue. In this study we investigated the extent to which spatio-temporal variation in environmental conditions can explain the timing of breeding of Red-capped Lark, Calandrella cinerea, a species that is capable of reproducing during every month of the year in our equatorial east African study locations. For 39 months in three climatically-distinct locations, we monitored nesting activities, sampled ground and flying invertebrates, and quantified rainfall, maximum (Tmax) and minimum (Tmin) temperatures. Among locations we found that lower rainfall and higher temperatures did not coincide with lower invertebrate biomasses and decreased nesting activities, as predicted. Within locations, we found that rainfall, Tmax, and Tmin varied unpredictably among months and years. The only consistent annually recurring observations in all locations were that January and February had low rainfall, high Tmax, and low Tmin. Ground and flying invertebrate biomasses varied unpredictably among months and years, but invertebrates were captured in all months in all locations. Red-capped Larks bred in all calendar months overall but not in every month in every year in every location. Using model selection, we found no clear support for any relationship between the environmental variables and breeding in any of the three locations. Contrary to popular understanding, this study suggests that rainfall and invertebrate biomass as proxy for food do not influence breeding in equatorial Larks. Instead, we propose that factors such as nest predation, female protein reserves, and competition are more important in environments where weather and food meet minimum requirements for breeding during most of the year.</p

Linear mixed-effect models examining the relationship between antimicrobial compounds and bacterial communities.

<p>^a OTUs affiliated to the <i>Proteobacteria</i> phylum are used as representative of Gram-negative bacteria.</p><p>^b OTUs affiliated to the <i>Actinobacteria</i> phylum are used as representative of Gram-positive bacteria.</p><p>Several bacterial community characteristics are examined such as the taxonomical composition at the phylum or class levels, and the four α-diversity indices. Sample sizes are such as 17 eggs for pH, 16 eggs for lysozyme or ovotransferrin concentrations. Lines separate each new model.</p><p>Linear mixed-effect models examining the relationship between antimicrobial compounds and bacterial communities.</p

Phylogenetic β-diversity of eggshell bacterial communities at different clutch ages.

<p>Dots are plotted based on the weighted UniFrac distances among bacterial communities and visualized on a Principal Coordinates Analysis (PCoA) plot. The variability of those communities is based on the two first axes of the PCoA. The percentage of variation explained per axis is mentioned on the graph: PC1 explained 68.93% of variation among communities and PC2 explained 11.92%. Egg age is symbolized by: day 1 (<i>red</i>), 2 (<i>orange</i>), 3 (<i>yellow</i>), 5 (<i>green</i>), 8 (<i>blue</i>) and 11 (<i>purple</i>). Each dot represents the bacterial community associated with one eggshell.</p

Egg-related bacteria and antimicrobials associated with clutch age.

<p>The clutch age is the number of days that the completed clutch spent in the nest; day 1 is the day of clutch completion. Incubation starts between day 1 and day 2 (<i>arrow</i>). ‘r²’ corresponds to the coefficient of determination between clutch age and bacterial/antimicrobial data. (A) Bacterial abundance is examined from day 1 to day 11 (<i>full line</i>) and from day 2 to day 11 (<i>dashed line</i>). (B) Shannon‘s diversity index. (C) Relative abundance of Operational Taxonomic Units (OTUs) of three main classes of <i>Proteobacteria</i>: <i>Alphaproteobacteria</i> (Alpha<i>-</i>) (<i>dotted line</i>), <i>Betaproteobacteria</i> (Beta<i>-</i>) (<i>dashed line</i>) and <i>Gammaproteobacteria</i> (Gamma<i>-</i>) (<i>full line</i>). (D) Albumen pH. (E) Lysozyme (<i>dashed line</i>) and ovotransferrin (<i>full line</i>) concentrations.</p

Linear mixed-effect models examining variations in bacterial abundance.

<p>Two models including different clutch ages are examined: (i) from day 1 to day 11 (n = 29 eggs) and (ii) from day 2 to day 11 (n = 18 eggs). Log copy numbers of 16S rRNA gene are analyzed as estimator of bacterial abundance. Models are based on backward elimination procedure. P-values are marked up in bold when significant (P<0.05).</p><p>Linear mixed-effect models examining variations in bacterial abundance.</p