Age and spatio-temporal variations in food resources modulate stress-immunity relationships in three populations of wild roe deer.

Living in variable and unpredictable environments, organisms face recurrent stressful situations. The endocrine stress response, which includes the secretion of glucocorticoids, helps organisms to cope with these perturbations. Although short-term elevations of glucocorticoid levels are often associated with immediate beneficial consequences for individuals, long-term glucocorticoid elevation can compromise key physiological functions such as immunity. While laboratory works highlighted the immunosuppressive effect of long-term elevated glucocorticoids, it remains largely unknown, especially in wild animals, whether this relationship is modulated by individual and environmental characteristics. In this study, we explored the co-variation between integrated cortisol levels, assessed non-invasively using faecal cortisol metabolites (FCMs), and 12 constitutive indices of innate, inflammatory, and adaptive immune functions, in wild roe deer living in three populations with previously known contrasting environmental conditions. Using longitudinal data on 564 individuals, we further investigated whether age and spatio-temporal variations in the quantity and quality of food resources modulate the relationship between FCMs and immunity. Negative covariation with glucocorticoids was evident only for innate and inflammatory markers of immunity, while adaptive immunity appeared to be positively or not linked to glucocorticoids. In addition, the negative covariations were generally stronger in individuals facing harsh environmental constraints and in old individuals. Therefore, our results highlight the importance of measuring multiple immune markers of immunity in individuals from contrasted environments to unravel the complex relationships between glucocorticoids and immunity in wild animals. Our results also help explain conflicting results found in the literature and could improve our understanding of the link between elevated glucocorticoid levels and disease spread, and its consequences on population dynamics.

Quantifying heritability and estimating evolutionary potential in the wild when individuals that share genes also share environments.

Accurate heritability estimates for fitness-related traits are required to predict an organism's ability to respond to global change. Heritability estimates are theoretically expected to be inflated if, due to limited dispersal, individuals that share genes are also likely to share similar environments. However, if relatives occupy similar environments due, at least partly, to genetic variation for habitat selection, then accounting for environmental similarity in quantitative genetic models may result in diminished heritability estimates in wild populations. This potential issue has been pointed out in the literature, but has not been evaluated by empirical studies. Here, we investigate whether environmental similarity among individuals can be partly explained by genetic variation for habitat selection, and how this link potentially blurs estimates for heritability in fitness-related traits. Using intensive GPS monitoring, we quantified home-range habitat composition for 293 roe deer inhabiting a heterogeneous landscape to assess environmental similarity. To investigate if environmental similarity might harbour genetic variation, we combined genome-wide data in a quantitative genetic framework to evaluate genetic variation for home-range habitat composition, which is partly the result of habitat selection at settlement. Finally, we explored how environmental similarity affects heritability estimates for behaviours related to the risk avoidance-resource acquisition trade-off (i.e. being in open habitat and distance to roads) and proxies of individual performance (i.e. body mass and hind foot length). We found substantial heritability for home-range habitat composition, with estimates ranging from 0.40 (proportion of meadows) to 0.85 (proportion of refuge habitat). Accounting for similarity in habitat composition between relatives decreased the heritability estimates for both behavioural and morphological traits (reduction ranging from 55% to 100% and from 22% to 41% respectively). As a consequence, only half of these heritability estimates remained significantly different from zero. Our results show that similar genotypes occupy similar environments, which could lead to heritable variation being incorrectly attributed to environmental effects. To accurately distinguish the sources of phenotypic variation and predict the ability of organisms to respond to global change, it is necessary to develop quantitative genetic studies investigating the mechanisms underpinning environmental similarity among relatives.

Pathogen-mediated selection favours the maintenance of innate immunity gene polymorphism in a widespread wild ungulate.

Toll-like receptors (TLR) play a central role in recognition and host frontline defence against a wide range of pathogens. A number of recent studies have shown that TLR genes (Tlrs) often exhibit large polymorphism in natural populations. Yet, there is little knowledge on how this polymorphism is maintained and how it influences disease susceptibility in the wild. In previous work, we showed that some Tlrs exhibit similarly high levels of genetic diversity as genes of the Major Histocompatibility Complex (MHC), and signatures of contemporary balancing selection in roe deer (Capreolus capreolus), the most abundant cervid species in Europe. Here, we investigated the evolutionary mechanisms by which pathogen-mediated selection could shape this innate immunity genetic diversity by examining the relationships between Tlr (Tlr2, Tlr4 and Tlr5) genotypes (heterozygosity status and presence of specific alleles) and infections with Toxoplasma and Chlamydia, two widespread intracellular pathogens known to cause reproductive failure in ungulates. We showed that Toxoplasma and Chlamydia exposures vary significantly across years and landscape features with few co-infection events detected and that the two pathogens exert antagonistic selection on Tlr2 polymorphism. By contrast, we found limited support for Tlr heterozygote advantage. Our study confirmed the importance of looking beyond Mhc genes in wildlife immunogenetic studies. It also emphasized the necessity to consider multiple pathogen challenges and their spatiotemporal variation to improve our understanding of vertebrate defence evolution against pathogens.

Distribution of ticks, tick-borne pathogens and the associated local environmental factors including small mammals and livestock, in two French agricultural sites: the OSCAR database.

BACKGROUND: In Europe, ticks are major vectors of both human and livestock pathogens (e.g. Lyme disease, granulocytic anaplasmosis, bovine babesiosis). Agricultural landscapes, where animal breeding is a major activity, constitute a mosaic of habitat types of various quality for tick survival and are used at different frequencies by wild and domestic hosts across seasons. This habitat heterogeneity, in time and space, conditions the dynamics of these host-vector-pathogen systems and thus drives acarological risk (defined as the density of infected ticks). The principal objective of the OSCAR project (2011-2016) was to examine the links between this heterogeneity and acarological risk for humans and their domestic animals. Here, we present the data associated with this project. NEW INFORMATION: This paper reports a database on the distribution and densities of I. ricinus ticks - the most common tick species in French agricultural landscapes - and the prevalence of three tick-borne pathogens (Anaplasma phagocytophilum, Borrelia spp. and Babesia spp.) in two sites in north-western ("Zone Atelier Armorique": ZA site) and south-western ("Vallées et Coteaux de Gascogne": VG site) France. The distribution and density of ticks along a gradient of wooded habitats, as well as biotic variables, such as the presence and abundance of their principal domestic (livestock) and wild hosts (small mammals), were measured from forest cores and edges to more or less isolated hedges, all bordering meadows. Ticks, small mammals and information on local environmental conditions were collected along 90 transects in each of the two sites in spring and autumn 2012 and 2013 and in spring 2014, corresponding to the main periods of tick activity. Local environmental conditions were recorded along each tick and small mammal transect: habitat type, vegetation type and characteristics, slope and traces of livestock presence. Samples consisted of questing ticks collected on the vegetation (mainly I. ricinus nymphs), biopsies of captured small mammals and ticks fixed on small mammals. In the VG site, livestock occurrence and abundance were recorded each week along each tick transect.A total of 29004 questing ticks and 1230 small mammals were captured during the study across the two sites and over the five field campaigns. All questing nymphs (N = 12287) and questing adults (N = 646) were identified to species. Ticks from small mammals (N = 1359) were also identified to life stage. Questing nymphs (N = 4518 I. ricinus) and trapped small mammals (N = 908) were analysed for three pathogenic agents: A. phagocytophilum, Borrelia spp. and Babesia spp.In the VG site, the average prevalence in I. ricinus nymphs for A. phagocytophilum, Borrelia spp. and Babesia spp. were, respectively 1.9% [95% CI: 1.2-2.5], 2.5% [95% CI: 1.8-3.2] and 2.7% [95% CI: 2.0-3.4]. In small mammals, no A. phagocytophilum was detected, but the prevalence for Borrelia spp. was 4.2% [95% CI: 0.9-7.5]. On this site, there was no screening of small mammals for Babesia spp. In ZA site, the average prevalence in nymphs for A. phagocytophilum, Borrelia spp. and Babesia were, respectively 2.2% [95% CI: 1.6-2.7], 3.0% [95% CI: 2.3-3.6] and 3.1% [95% CI: 2.5-3.8]. In small mammals, the prevalence of A. phagocytophilum and Borrelia spp. were, respectively 6.9% [95% CI: 4.9-8.9] and 4.1% [95% CI: 2.7-5.9]. A single animal was found positive for Babesia microti at this site amongst the 597 tested.

Genetic epidemiology of the Alpine ibex reservoir of persistent and virulent brucellosis outbreak.

While it is now broadly accepted that inter-individual variation in the outcomes of host-pathogen interactions is at least partially genetically controlled, host immunogenetic characteristics are rarely investigated in wildlife epidemiological studies. Furthermore, most immunogenetic studies in the wild focused solely on the major histocompatibility complex (MHC) diversity despite it accounts for only a fraction of the genetic variation in pathogen resistance. Here, we investigated immunogenetic diversity of the Alpine ibex (Capra ibex) population of the Bargy massif, reservoir of a virulent outbreak of brucellosis. We analysed the polymorphism and associations with disease resistance of the MHC Class II Drb gene and several non-MHC genes (Toll-like receptor genes, Slc11A1) involved in the innate immune response to Brucella in domestic ungulates. We found a very low neutral genetic diversity and a unique MHC Drb haplotype in this population founded few decades ago from a small number of individuals. By contrast, other immunity-related genes have maintained polymorphism and some showed significant associations with the brucellosis infection status hence suggesting a predominant role of pathogen-mediated selection in their recent evolutionary trajectory. Our results highlight the need to monitor immunogenetic variation in wildlife epidemiological studies and to look beyond the MHC.

Pedigree-free quantitative genetic approach provides evidence for heritability of movement tactics in wild roe deer.

Assessing the evolutionary potential of animal populations in the wild is crucial to understanding how they may respond to selection mediated by rapid environmental change (e.g. habitat loss and fragmentation). A growing number of studies have investigated the adaptive role of behaviour, but assessments of its genetic basis in a natural setting remain scarce. We combined intensive biologging technology with genome-wide data and a pedigree-free quantitative genetic approach to quantify repeatability, heritability and evolvability for a suite of behaviours related to the risk avoidance-resource acquisition trade-off in a wild roe deer (Capreolus capreolus) population inhabiting a heterogeneous, human-dominated landscape. These traits, linked to the stress response, movement and space-use behaviour, were all moderately to highly repeatable. Furthermore, the repeatable among-individual component of variation in these traits was partly due to additive genetic variance, with heritability estimates ranging from 0.21 ± 0.08 to 0.70 ± 0.11 and evolvability ranging from 1.1% to 4.3%. Changes in the trait mean can therefore occur under hypothetical directional selection over just a few generations. To the best of our knowledge, this is the first empirical demonstration of additive genetic variation in space-use behaviour in a free-ranging population based on genomic relatedness data. We conclude that wild animal populations may have the potential to adjust their spatial behaviour to human-driven environmental modifications through microevolutionary change.

RAD-sequencing for estimating genomic relatedness matrix-based heritability in the wild: A case study in roe deer.

Estimating the evolutionary potential of quantitative traits and reliably predicting responses to selection in wild populations are important challenges in evolutionary biology. The genomic revolution has opened up opportunities for measuring relatedness among individuals with precision, enabling pedigree-free estimation of trait heritabilities in wild populations. However, until now, most quantitative genetic studies based on a genomic relatedness matrix (GRM) have focused on long-term monitored populations for which traditional pedigrees were also available, and have often had access to knowledge of genome sequence and variability. Here, we investigated the potential of RAD-sequencing for estimating heritability in a free-ranging roe deer (Capreolous capreolus) population for which no prior genomic resources were available. We propose a step-by-step analytical framework to optimize the quality and quantity of the genomic data and explore the impact of the single nucleotide polymorphism (SNP) calling and filtering processes on the GRM structure and GRM-based heritability estimates. As expected, our results show that sequence coverage strongly affects the number of recovered loci, the genotyping error rate and the amount of missing data. Ultimately, this had little effect on heritability estimates and their standard errors, provided that the GRM was built from a minimum number of loci (above 7,000). Genomic relatedness matrix-based heritability estimates thus appear robust to a moderate level of genotyping errors in the SNP data set. We also showed that quality filters, such as the removal of low-frequency variants, affect the relatedness structure of the GRM, generating lower h2 estimates. Our work illustrates the huge potential of RAD-sequencing for estimating GRM-based heritability in virtually any natural population.

Individual variation in an acute stress response reflects divergent coping strategies in a large herbivore.

Individuals differ in the manner that they cope with risk. When these behavioral differences are manifested in risky or challenging environments (i.e. stressful situations), they are generally interpreted within the "coping style" framework. As studying inter-individual variability in behavior is particularly challenging in the wild, we used a captive facility to explore consistency in the individual behavioral response to an acute stress in roe deer (Capreolus capreolus). Using behavioral and physiological parameters measured six times across a calendar year, we first quantified individual repeatability and, second, explored the correlations among these parameters that might indicate a coherent stress response. Finally, we analyzed the link between the stress response and individual body mass, a reliable indicator of phenotypic quality in roe deer. We found that the measured parameters were highly repeatable across seasons, indicating that the individual stress response is consistent over time. Furthermore, there was considerable covariation among the stress response parameters, describing a proactivity-reactivity gradient at the individual level. Finally, proactive individuals had higher body mass than reactive individuals. We suggest that consistent individual differences in energy metabolism and physiology may promote consistent individual differences in behavioral traits, providing a mechanistic link between food acquisition tactics and demographic performance.

Immunogenetic heterogeneity in a widespread ungulate: the European roe deer (Capreolus capreolus).

Understanding how immune genetic variation is shaped by selective and neutral processes in wild populations is of prime importance in both evolutionary biology and epidemiology. The European roe deer (Capreolus capreolus) has considerably expanded its distribution range these last decades, notably by colonizing agricultural landscapes. This range shift is likely to have led to bottlenecks and increased roe deer exposure to a new range of pathogens that until recently predominantly infected humans and domestic fauna. We therefore investigated the historical and contemporary forces that have shaped variability in a panel of genes involved in innate and acquired immunity in roe deer, including Mhc-Drb and genes encoding cytokines or toll-like receptors (TLRs). Together, our results suggest that genetic drift is the main contemporary evolutionary force shaping immunogenetic variation within populations. However, in contrast to the classical view, we found that some innate immune genes involved in micropathogen recognition (e.g. Tlrs) continue to evolve dynamically in roe deer in response to pathogen-mediated positive selection. Most studied Tlrs (Tlr2, Tlr4 and Tlr5) had similarly high levels of amino acid diversity in the three studied populations including one recently established in southwestern France that showed a clear signature of genetic bottleneck. Tlr2 implicated in the recognition of Gram-positive bacteria in domestic ungulates, showed strong evidence of balancing selection. The high immunogenetic variation revealed here implies that roe deer are able to cope with a wide spectrum of pathogens and to respond rapidly to emerging infectious diseases.

Reduced microsatellite heterozygosity does not affect natal dispersal in three contrasting roe deer populations.

Although theoretical studies have predicted a link between individual multilocus heterozygosity and dispersal, few empirical studies have investigated the effect of individual heterozygosity on dispersal propensity or distance. We investigated this link using measures of heterozygosity at 12 putatively neutral microsatellite markers and natal dispersal behaviour in three contrasting populations of European roe deer (Capreolus capreolus), a species displaying pre-saturation condition-dependent natal dispersal. We found no effect of individual heterozygosity on either dispersal propensity or dispersal distance. Average heterozygosity was similar across the three studied populations, but dispersal propensity and distance differed markedly among them. In Aurignac, dispersal propensity and distance were positively related to individual body mass, whereas there was no detectable effect of body mass on dispersal behaviour in Chizé and Trois Fontaines. We suggest that we should expect both dispersal propensity and distance to be greater when heterozygosity is lower only in those species where dispersal behaviour is driven by density-dependent competition for resources.

Landscape fragmentation generates spatial variation of diet composition and quality in a generalist herbivore.

Forest fragmentation may benefit generalist herbivores by increasing access to various substitutable food resources, with potential consequences for their population dynamics. We studied a European roe deer (Capreolus capreolus) population living in an agricultural mosaic of forest, woodlots, meadows and cultivated crops. We tested whether diet composition and quality varied spatially across the landscape using botanical analyses of rumen contents and chemical analyses of the plants consumed in relation to landscape metrics. In summer and non-mast winters, roe deer ate more cultivated seeds and less native forest browse with increasing availability of crops in the local landscape. This spatial variation resulted in contrasting diet quality, with more cell content and lower lignin and hemicellulose content (high quality) for individuals living in more open habitats. The pattern was less marked in the other seasons when diet composition, but not diet quality, was only weakly related to landscape structure. In mast autumns and winters, the consumption of acorns across the entire landscape resulted in a low level of differentiation in diet composition and quality. Our results reflect the ability of generalist species, such as roe deer, to adapt to the fragmentation of their forest habitat by exhibiting a plastic feeding behavior, enabling them to use supplementary resources available in the agricultural matrix. This flexibility confers nutritional advantages to individuals with access to cultivated fields when their native food resources are depleted or decline in quality (e.g. during non-mast years) and may explain local heterogeneities in individual phenotypic quality.