Experimental infection of Artibeus lituratus bats and no detection of Zika virus in neotropical bats from French Guiana, Peru, and Costa Rica suggests a limited role of bats in Zika transmission.

Bats are important natural reservoir hosts of a diverse range of viruses that can be transmitted to humans and have been suggested to play an important role in the Zika virus (ZIKV) transmission cycle. However, the exact role of these animals as reservoirs for flaviviruses is still controversial. To further expand our understanding of the role of bats in the ZIKV transmission cycle in Latin America, we carried out an experimental infection in wild-caught Artibeus lituratus bats and sampled several free-living neotropical bats across three countries of the region. Experimental ZIKV infection was performed in wild-caught adult bats (4 females and 5 males). The most relevant findings were hemorrhages in the bladder, stomach and patagium. Significant histological findings included inflammatory infiltrate consisting of a predominance of neutrophils and lymphocytes, in addition to degeneration in the reproductive tract of males and females. This suggests that bat reproduction might be at some level affected by ZIKV. Leukopenia was also observed in some inoculated animals. Hemorrhages, genital alterations, and leukopenia are suggested to be caused by ZIKV; however, since these were wild-caught bats, we cannot exclude other agents. Detection of ZIKV by qPCR was observed at low concentrations in only two urine samples in two inoculated animals. All other animals and tissues tested were negative. Finally, no virus-neutralizing antibodies were found in any animal. To determine ZIKV infection in nature, the blood of a total of 2056 bats was sampled for ZIKV detection by qPCR. Most of the sampled individuals belonged to the genus Pteronotus sp. (23%), followed by the species Carollia sp. (17%), Anoura sp. (14%), and Molossus sp. (13.7%). No sample of any tested species was positive for ZIKV by qPCR. These results together suggest that bats are not efficient amplifiers or reservoirs of ZIKV and may not have an important role in ZIKV transmission dynamics.

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.

Adaptive duplication and genetic diversification of protein kinase R contribute to the specificity of bat-virus interactions.

Several bat species act as asymptomatic reservoirs for many viruses that are highly pathogenic in other mammals. Here, we have characterized the functional diversification of the protein kinase R (PKR), a major antiviral innate defense system. Our data indicate that PKR has evolved under positive selection and has undergone repeated genomic duplications in bats in contrast to all studied mammals that have a single copy of the gene. Functional testing of the relationship between PKR and poxvirus antagonists revealed how an evolutionary conflict with ancient pathogenic poxviruses has shaped a specific bat host-virus interface. We determined that duplicated PKRs of the Myotis species have undergone genetic diversification, allowing them to collectively escape from and enhance the control of DNA and RNA viruses. These findings suggest that viral-driven adaptations in PKR contribute to modern virus-bat interactions and may account for bat-specific immunity.

Estimation of Bait Uptake by Badgers, Using Non-invasive Methods, in the Perspective of Oral Vaccination Against Bovine Tuberculosis in a French Infected Area.

Although France is officially declared free of bovine tuberculosis (TB), Mycobacterium bovis infection is still observed in several regions in cattle and wildlife, including badgers (Meles meles). In this context, vaccinating badgers should be considered as a promising strategy for the reduction in M. bovis transmission between badgers and other species, and cattle in particular. An oral vaccine consisting of live Bacille Calmette-Guérin (BCG) contained in bait is currently under assessment for badgers, for which testing bait deployment in the field and assessing bait uptake by badgers are required. This study aimed to evaluate the bait uptake by badgers and determine the main factors influencing uptake in a TB-infected area in Burgundy, north-eastern France. The baits were delivered at 15 different setts located in the vicinity of 13 pastures within a TB-infected area, which has been subject to intense badger culling over the last decade. Pre-baits followed by baits containing a biomarker (Rhodamine B; no BCG vaccine) were delivered down sett entrances in the spring (8 days of pre-baiting and 4 days of baiting) and summer (2 days of pre-baiting and 2 days of baiting) of 2018. The consumption of the marked baits was assessed by detecting fluorescence, produced by Rhodamine B, in hair collected in hair traps positioned at the setts and on the margins of the targeted pastures. Collected hairs were also genotyped to differentiate individuals using 24 microsatellites markers and one sex marker. Bait uptake was estimated as the proportion of badgers consuming baits marked by the biomarker over all the sampled animals (individual level), per badger social group, and per targeted pasture. We found a bait uptake of 52.4% (43 marked individuals of 82 genetically identified) at the individual level and a mean of 48.9 and 50.6% at the social group and pasture levels, respectively. The bait uptake was positively associated with the presence of cubs (social group level) and negatively influenced by the intensity of previous trapping (social group and pasture levels). This study is the first conducted in France on bait deployment in a badger population of intermediate density after several years of intensive culling. The results are expected to provide valuable information toward a realistic deployment of oral vaccine baits to control TB in badger populations.

DNA methylation as a tool to explore ageing in wild roe deer populations.

DNA methylation-based biomarkers of ageing (epigenetic clocks) promise to lead to new insights into evolutionary biology of ageing. Relatively little is known about how the natural environment affects epigenetic ageing effects in wild species. In this study, we took advantage of a unique long-term (>40 years) longitudinal monitoring of individual roe deer (Capreolus capreolus) living in two wild populations (Chizé and Trois-Fontaines, France) facing different ecological contexts, to investigate the relationship between chronological age and levels of DNA methylation (DNAm). We generated novel DNA methylation data from n = 94 blood samples, from which we extracted leucocyte DNA, using a custom methylation array (HorvathMammalMethylChip40). We present three DNA methylation-based estimators of age (DNAm or epigenetic age), which were trained in males, females, and both sexes combined. We investigated how sex differences influenced the relationship between DNAm age and chronological age using sex-specific epigenetic clocks. Our results highlight that old females may display a lower degree of biological ageing than males. Further, we identify the main sites of epigenetic alteration that have distinct ageing patterns between the two sexes. These findings open the door to promising avenues of research at the crossroads of evolutionary biology and biogerontology.

Metabarcoding in two isolated populations of wild roe deer (Capreolus capreolus) reveals variation in gastrointestinal nematode community composition between regions and among age classes.

BACKGROUND: Gastrointestinal nematodes are ubiquitous for both domestic and wild ungulates and have varying consequences for health and fitness. They exist as complex communities of multiple co-infecting species, and we have a limited understanding of how these communities vary in different hosts, regions and circumstances or of how this affects their impacts. METHODS: We have undertaken ITS2 rDNA nemabiome metabarcoding with next-generation sequencing on populations of nematode larvae isolated from 149 fecal samples of roe deer of different sex and age classes in the two isolated populations of Chizé and Trois Fontaines in France not co-grazing with any domestic ungulate species. RESULTS: We identified 100 amplified sequence variants (ASVs) that were assigned to 14 gastrointestinal nematode taxa overall at either genus (29%) or species (71%) level. These taxa were dominated by parasites classically found in cervids-e.g. Ostertagia leptospicularis, Spiculopteragia spp. Higher parasite species diversity was present in the Trois Fontaines population than in the Chizé population including the presence of species more typically seen in domestic livestock (Haemonchus contortus, Bunostomum sp., Cooperia punctata, Teladorsagia circumcincta). No differences in parasite species diversity or community composition were seen in the samples collected from three zones of differing habitat quality within the Chizé study area. Young roe deer hosted the highest diversity of gastrointestinal nematodes, with more pronounced effects of age apparent in Trois Fontaines. The effect of host age differed between gastrointestinal nematode species, e.g. there was little effect on O. leptospicularis but a large effect on Trichostrongylus spp. No effect of host sex was detected in either site. CONCLUSIONS: The presence of some livestock parasite species in the Trois Fontaines roe deer population was unexpected given the isolation of this population away from grazing domestic livestock since decades. Overall, our results illustrate the influence of host traits and the local environment on roe deer nemabiome and demonstrate the power of the nemabiome metabarcoding approach to elucidate the composition of gastrointestinal nematode communities in wildlife.

Breaking down population density into different components to better understand its spatial variation.

BACKGROUND: Population size and densities are key parameters in both fundamental and applied ecology, as they affect population resilience to density-dependent processes, habitat changes and stochastic events. Efficient management measures or species conservation programs thus require accurate estimates of local population densities across time and space, especially for continuously distributed species. For social species living in groups, population density depends on different components, namely the number of groups and the group size, for which relative variations in space may originate from different environmental factors. Whether resulting spatial variations in density are mostly triggered by one component or the other remains poorly known. Here, we aimed at determining the magnitude of the spatial variation in population densities of a social, group-living species, i.e. the European badger Meles meles, in 13 different sites of around 50 km2 across France, to decipher whether sett density, group size or proportion of occupied sett variation is the main factor explaining density variation. Besides the intrinsic factors of density variation, we also assessed whether habitat characteristics such as habitat fragmentation, urbanisation, and resource availability, drove both the spatial variation of density components and local population densities. RESULTS: We proposed a new standardised approach combining use of multiple methods, namely distance sampling for estimating the density of occupied sett clusters, i.e. group density, and camera and hair trapping for genetic identification to determine the mean social group size. The density of adult badgers was on average 3.8 per km2 (range 1.7-7.9 per km2) and was positively correlated with the density of sett clusters. The density of adult badgers per site was less related to the social group size or to the proportion of occupied sett clusters. Landscape fragmentation also explained the spatial variation of adult badger density, with highly fragmented landscapes supporting lower adult densities. Density components were linked differently to environmental variables. CONCLUSIONS: These results underline the need to break down population density estimates into several components in group-living species to better understand the pattern of temporal and spatial variation in population density, as different components may vary due to different ecological factors.

Venezuelan Equine Encephalitis Complex Alphavirus in Bats, French Guiana.

Although essential for control strategies, knowledge about transmission cycles is limited for Venezuelan equine encephalitis complex alphaviruses (VEEVs). After testing 1,398 bats from French Guiana for alphaviruses, we identified and isolated a new strain of the encephalitogenic VEEV species Tonate virus (TONV). Bats may contribute to TONV spread in Latin America.

Reproductive females and young mouflon (Ovis gmelini musimon × Ovis sp.) in poor body condition are the main spreaders of gastrointestinal parasites.

Several individual, environmental and parasitic factors can influence the impacts of parasites on host's fitness and on host's ability to transmit these parasites to new hosts. Identifying these factors and the individuals who play a greater role in parasite transmission is of main concern for the development of parasite control strategies. In the present study, we aimed to describe the diversity of gastrointestinal parasites and to identify the individual factors influencing the faecal spreading of parasites in a free-ranging population of Mediterranean mouflon. From the analysis of 433 faecal samples, we found Eimeria spp. and gastrointestinal strongyles (GIS) were the most common parasites (>94%). The faecal oocyst counts of Eimeria spp. were the highest during the first years of life. It was 1.6 times higher in females than in males and 2.5 times higher in individuals in poor than in good body condition. Similarly, the faecal egg count of GIS was higher in females and decreased with age, but only in males. Finally, reproductive females had GIS faecal egg count values 2.6 times higher than non-reproductive females. Management strategies of parasites should thus primarily focus on reproductive females and young individuals in poor body condition as they represent the main contamination source of the environment.

Maternal effects shape offspring physiological condition but do not senesce in a wild mammal.

In vertebrates, offspring survival often decreases with increasing maternal age. While many studies have reported a decline in fitness-related traits of offspring with increasing maternal age, the study of senescence in maternal effect through age-specific changes in offspring physiological condition is still at its infancy. We assessed the influence of maternal age and body mass on offspring physiological condition in two populations of roe deer (Capreolus capreolus) subjected to markedly different environmental conditions. We measured seven markers to index body condition and characterize the immune profile in 86 fawns which became recently independent of their known-aged mothers. We did not find striking effects of maternal age on offspring physiological condition measured at 8 months of age. This absence of evidence for senescence in maternal effects is likely due to the strong viability selection observed in the very first months of life in this species. Offspring physiological condition was, on the other hand, positively influenced by maternal body mass. Between-population differences in environmental conditions experienced by fawns also influenced their average body condition and immune phenotype. Fawns facing food limitation displayed lower values in some markers of body condition (body mass and haemoglobin levels) than those living in good quality habitat. They also allocated preferentially to humoral immunity, contrary to those living in good conditions, which allocated more to cellular response. These results shed a new light on the eco-physiological pathways mediating the relationship between mother's mass and offspring condition.

Short-term telomere dynamics is associated with glucocorticoid levels in wild populations of roe deer.

While evidence that telomere length is associated with health and mortality in humans and birds is accumulating, a large body of research is currently seeking to identify factors that modulate telomere dynamics. We tested the hypothesis that high levels of glucocorticoids in individuals under environmental stress should accelerate telomere shortening in two wild populations of roe deer (Capreolus capreolus) living in different ecological contexts. From two consecutive annual sampling sessions, we found that individuals with faster rates of telomere shortening had higher concentrations of fecal glucocorticoid metabolites, suggesting a functional link between glucocorticoid levels and telomere attrition rate. This relationship was consistent for both sexes and populations. This finding paves the way for further studies of the fitness consequences of exposure to environmental stressors in wild vertebrates.

Integrating population genetics to define conservation units from the core to the edge of Rhinolophus ferrumequinum western range.

The greater horseshoe bat (Rhinolophus ferrumequinum) is among the most widespread bat species in Europe but it has experienced severe declines, especially in Northern Europe. This species is listed Near Threatened in the European IUCN Red List of Threatened Animals, and it is considered to be highly sensitive to human activities and particularly to habitat fragmentation. Therefore, understanding the population boundaries and demographic history of populations of this species is of primary importance to assess relevant conservation strategies. In this study, we used 17 microsatellite markers to assess the genetic diversity, the genetic structure, and the demographic history of R. ferrumequinum colonies in the western part of its distribution. We identified one large population showing high levels of genetic diversity and large population size. Lower estimates were found in England and northern France. Analyses of clustering and isolation by distance suggested that the Channel and the Mediterranean seas could impede R. ferrumequinum gene flow. These results provide important information to improve the delineation of R. ferrumequinum management units. We suggest that a large management unit corresponding to the population ranging from Spanish Basque Country to northern France must be considered. Particular attention should be given to mating territories as they seem to play a key role in maintaining high levels of genetic mixing between colonies. Smaller management units corresponding to English and northern France colonies must also be implemented. These insular or peripheral colonies could be at higher risk of extinction in the near future.

Does host socio-spatial behavior lead to a fine-scale spatial genetic structure in its associated parasites?

Gastro-intestinal nematodes, especially Haemonchus contortus, are widespread pathogenic parasites of small ruminants. Studying their spatial genetic structure is as important as studying host genetic structure to fully understand host-parasite interactions and transmission patterns. For parasites having a simple life cycle (e.g., monoxenous parasites), gene flow and spatial genetic structure are expected to strongly rely on the socio-spatial behavior of their hosts. Based on five microsatellite loci, we tested this hypothesis for H. contortus sampled in a wild Mediterranean mouflon population (Ovis gmelini musimon × Ovis sp.) in which species- and environment-related characteristics have been found to generate socio-spatial units. We nevertheless found that their parasites had no spatial genetic structure, suggesting that mouflon behavior was not enough to limit parasite dispersal in this study area and/or that other ecological and biological factors were involved in this process, for example other hosts, the parasite life cycle, or the study area history.

TITLE: Le comportement socio-spatial de l'hôte conduit-il à une structure génétique à fine échelle de ses parasites ? ABSTRACT: Les nématodes gastro-intestinaux, et plus particulièrement Haemonchus contortus, sont cosmopolites et pathogènes chez les petits ruminants. Étudier leur structure génétique spatiale est aussi important que d'étudier celle des hôtes pour pleinement comprendre les interactions hôtes-parasites et les processus de transmission. Pour les parasites ayant des cycles de vie simples (par exemple, les parasites monoxènes), on s'attend à ce que les flux de gènes et la structure génétique spatiale dépendent fortement du comportement socio-spatial de leurs hôtes. En utilisant cinq loci microsatellites, nous avons testé cette hypothèse pour des H. contortus échantillonnés dans une population sauvage de mouflons méditerranéens (Ovis gmelini musimon × Ovis sp.) dans laquelle les caractéristiques de l'espèce et de l'environnement génèrent des unités socio-spatiales. Nous avons néanmoins mis en évidence que leurs parasites ne présentent pas de structure génétique spatiale, ce qui suggère que le comportement des mouflons ne restreint pas la dispersion des parasites dans cette aire d'étude et/ou que d'autres facteurs biologiques et écologiques tels que d'autres hôtes, le cycle de vie du parasite, ou l'histoire de l'aire d'étude jouent un rôle dans ce processus.

Both candidate gene and neutral genetic diversity correlate with parasite resistance in female Mediterranean mouflon.

BACKGROUND: Parasite infections can have substantial impacts on population dynamics and are accordingly a key challenge for wild population management. Here we studied genetic mechanisms driving parasite resistance in a large herbivore through a comprehensive approach combining measurements of neutral (16 microsatellites) and adaptive (MHC DRB1 exon 2) genetic diversity and two types of gastrointestinal parasites (nematodes and coccidia). RESULTS: While accounting for other extrinsic and intrinsic predictors known to impact parasite load, we show that both neutral genetic diversity and DRB1 are associated with resistance to gastrointestinal nematodes. Intermediate levels of multi-locus heterozygosity maximized nematodes resistance, suggesting that both in- and outbreeding depression might occur in the population. DRB1 heterozygosity and specific alleles effects were detected, suggesting the occurrence of heterozygote advantage, rare-allele effects and/or fluctuating selection. On the contrary, no association was detected between genetic diversity and resistance to coccidia, indicating that different parasite classes are impacted by different genetic drivers. CONCLUSIONS: This study provides important insights for large herbivores and wild sheep pathogen management, and in particular suggests that factors likely to impact genetic diversity and allelic frequencies, including global changes, are also expected to impact parasite resistance.