Babesia microti is a tick-transmitted protozoan parasite of wildlife that can also cause serious disease in humans. It is now well established that B. microti represents an assemblage of different strains or species, only some of which are important zoonotic pathogens. Therefore, in order to assess the potential public health risk associated with B. microti in any given location, it is important to determine the strains that are present. This is the first study on the presence and identity of B. microti in Ireland. Overall, 314 wood mice (Apodemus sylvaticus), 243 bank voles (Myodes glareolus) and 634 questing Ixodes ricinus nymphs collected in various locations across Ireland were screened for the presence of B. microti by metabarcoding and nested PCR, respectively. Overall 8 rodent spleen samples (1.4%) were positive for B. microti, while all tick samples tested negative. Rodent isolates were identified as the 'Munich' strain which rarely causes human disease and is chiefly transmitted by the mouse tick, Ixodes trianguliceps. Together with reports from the UK these results suggest that B. microti does not represent a sig-nificant public health risk in Britain or Ireland.

With the aim to understand how next‐generation sequencing (NGS) improves both our assessment of genetic variation within populations and our knowledge on HLA molecular evolution, we sequenced and analysed 8 HLA loci in a well‐documented population from sub‐Saharan Africa (Mandenka). The results of full‐gene NGS‐MiSeq sequencing compared with those obtained by traditional typing techniques or limited sequencing strategies showed that segregating sites located outside exon 2 are crucial to describe not only class I but also class II population diversity. A comprehensive analysis of exons 2, 3, 4 and 5 nucleotide diversity at the 8 HLA loci revealed remarkable differences among these gene regions, notably a greater variation concentrated in the antigen recognition sites of class I exons 3 and some class II exons 2, likely associated with their peptide‐presentation function, a lower diversity of HLA‐C exon 3, possibly related to its role as a KIR ligand, and a peculiar molecular diversity of HLA‐A exon 2, revealing demographic signals. Based on full‐length HLA sequences, we also propose that the most frequent DRB1 allele in the studied population, DRB1*13:04, emerged from an allelic conversion involving 3 potential alleles as donors and DRB1*11:02:01 as recipient. Finally, our analysis revealed a high occurrence of the DRB1*13:04‐DQA1*05:05:01‐DQB1*03:19 haplotype, possibly resulting from a selective sweep due to protection to Onchorcerca volvulus, a prevalent pathogen in West Africa. This study unveils highly relevant information on the molecular evolution of HLA genes in relation to their immune function, calling for similar analyses in other populations living in contrasting environments.

Understanding the processes of adaptive divergence, which may ultimately lead to speciation, is a major question in evolutionary biology. Allochronic differentiation refers to a particular situation where gene flow is primarily impeded by temporal isolation between early and late reproducers. This process has been suggested to occur in a large array of organisms, even though it is still overlooked in the literature. We here focused on a well‐documented case of incipient allochronic speciation in the winter pine processionary moth Thaumetopoea pityocampa . This species typically reproduces in summer and larval development occurs throughout autumn and winter. A unique, phenologically shifted population (SP) was discovered in 1997 in Portugal. It was proved to be strongly differentiated from the sympatric “winter population” (WP), but its evolutionary history could only now be explored. We took advantage of the recent assembly of a draft genome and of the development of pan‐genomic RAD‐seq markers to decipher the demographic history of the differentiating populations and develop genome scans of adaptive differentiation. We showed that the SP diverged relatively recently, that is, few hundred years ago, and went through two successive bottlenecks followed by population size expansions, while the sympatric WP is currently experiencing a population decline. We identified outlier SNPs that were mapped onto the genome, but none were associated with the phenological shift or with subsequent adaptations. The strong genetic drift that occurred along the SP lineage certainly challenged our capacity to reveal functionally important loci.

Background Understanding the genetic and environmental mechanisms governing variation in morphology or phenology in wild populations is currently an important challenge. While there is a general consensus that selection is stronger under stressful conditions, it remains unclear whether the evolutionary potential of traits should increase or decrease with increasingly stressful conditions. Here, we investigate how contrasting environmental conditions during growth may affect the maternal and genetic components of body mass in roe deer, the most abundant and widespread wild ungulate in Western Europe. Body mass is a key life history trait that strongly influences both survival and reproductive performance in large herbivores. We used pedigrees and animal models to determine the variance components of juvenile and adult winter body mass in two populations experiencing contrasting early-life conditions. Results Our analyses showed that roe deer at Chizé, where habitat was poor and unpredictable, exhibited very low genetic variance in juvenile body mass. Instead, variance in mass was mainly driven by among-cohort differences in early-life conditions and maternal environment. In contrast, roe deer at Bogesund, where resource availability during the critical period of fawn rearing was higher, displayed a substantial level of genetic variance in body mass. We discuss the potential role of past demography and viability selection on fawn body mass on the erosion of genetic variance in the poor habitat. Conclusions Our study highlights the importance of accounting for both spatial (i.e. between-population variation) and temporal (i.e. cohort variation) heterogeneity in environmental conditions, especially in early life, to understand the potential for adaptive responses of wild populations to selection.

Several hypotheses (such as ‘enemy release’, ‘novel weapon’, ‘spillback’ and ‘dilution/density effect’) suggest changes in host-parasite ecological interactions during biological invasion events. Such changes can impact both invasion process outcome and the dynamics of exotic and/or endemic zoonotic diseases. To evaluate these predictions, we investigated the ongoing invasions of the house mouse Mus musculus domesticus , and the black rat, Rattus rattus , in Senegal (West Africa). We focused on zoonotic bacterial communities depicted using 16S rRNA amplicon sequencing approach in both invasive and native rodents sampled along two well-defined invasion routes. Overall, this study provided new ecological evidence connecting parasitism and rodent invasion process, with diverse potential roles of zoonotic bacteria in the invasion success. Our results also highlighted the main factors that lie behind bacterial community structure in commensal rodents. Further experimental studies as well as comparative spatio-temporal surveys are necessary to decipher the actual role of zoonotic bacteria in these invasions. Our data also gave new support for the difficulty to predict the direction in which the relationship between biodiversity changes and disease risk could go. These results should be used as a basis for public health prevention services to design reservoir monitoring strategies based on multiple pathogen surveillance.

Ecoevolutionary processes affecting hosts, vectors and pathogens are important drivers of zoonotic disease emergence. In this study, we focused on nephropathia epidemica (NE), which is caused by Puumala hantavirus (PUUV) whose natural reservoir is the bank vole, Myodes glareolus. We questioned the possibility of NE emergence in a French region that is considered to be NE-free but that is adjacent to a NE-endemic region. We first confirmed the epidemiology of these two regions and we demonstrated the absence of spatial barriers that could have limited dispersal, and consequently, the spread of PUUV into the NE-free region. We next tested whether regional immunoheterogeneity could impact PUUV chances to circulate and persist in the NE-free region. We showed that bank voles from the NE-free region were sensitive to experimental PUUV infection. We observed high levels of immunoheterogeneity between individuals and also between regions. Antiviral gene expression (Tnf and Mx2) reached higher levels in bank voles from the NE-free region. During experimental infections, anti-PUUV antibody production was higher in bank voles from the NE-endemic region. These results indicated a lower susceptibility to PUUV for bank voles from this NE-free region, which might limit PUUV persistence and therefore, the risk of NE.

SUMMARY High-throughput sequencing technologies now allow for rapid cost-effective surveys of multiple pathogens in many host species including rodents, but it is currently unclear if the organ chosen for screening influences the number and identity of bacteria detected. We used 16S rRNA amplicon sequencing to identify bacterial pathogens in the heart, liver, lungs, kidneys and spleen of 13 water voles (Arvicola terrestris) collected in Franche-Comté, France. We asked if bacterial pathogen assemblages within organs are similar and if all five organs are necessary to detect all of the bacteria present in an individual animal. We identified 24 bacteria representing 17 genera; average bacterial richness for each organ ranged from 1·5 ± 0·4 (mean ± standard error) to 2·5 ± 0·4 bacteria/organ and did not differ significantly between organs. The average bacterial richness when organ assemblages were pooled within animals was 4·7 ± 0·6 bacteria/animal; Operational Taxonomic Unit accumulation analysis indicates that all five organs are required to obtain this. Organ type influences bacterial assemblage composition in a systematic way (PERMANOVA, 999 permutations, pseudo-F 4,51 = 1·37, P = 0·001). Our results demonstrate that the number of organs sampled influences the ability to detect bacterial pathogens, which can inform sampling decisions in public health and wildlife ecology.

Genetic diversity may decrease from the centre to the margin of a species distribution range due to neutral stochastic processes. Selection may also alter genetic diversity in non-neutral markers, such as genes associated with the immune system. Both neutral processes and selection on the immune system are thus expected to affect the spatial distribution of such markers, but the relative strength of each has been scarcely studied. Here, we compared the diversity of a neutral marker (mitochondrial cytochrome b) and a selected marker (DRB gene from the MHC-II), in eastern-North American populations of white-footed mice (Peromyscus leucopus), a species known for its role of main reservoir of the Lyme disease. We observed distinct phylogeographic patterns with these two markers, which may be the result of selection pressure acting upon the DRB gene. As predicted by the central marginal hypothesis, we observed a loss of neutral genetic diversity toward the margin of the species distribution. A decrease in diversity was also observed for the DRB gene, likely due to genetic drift and positive selection operated by helminth parasites. Such a loss in genetic diversity at the range margin may slow down the ongoing expansion of P. leucopus, by counterbalancing the effect of global warming on the mouse survival at higher latitude.

Rodents represent one of the major sources of pathogens; most of them are vectored by ticks. Tick-borne diseases are very diverse and cause a wide range of diseases in livestock and human populations. Rodents, carrying ticks, are distributed across a vast range of natural habitats and they often live in close contact with humans and their domestic animals, exposing them to zoonoses circulating in natural ecosystems. In this study, we analyse the potential of Next-Generation Sequencing (NGS) technologies as a tool for large-scale survey of bacterial zoonotic pathogens carried by rodents. We combined two NGS approaches in order to establish a list of zoonotic bacteria and to identify their distribution in individuals of rodents in natural populations. Briefly, RNA/DNA were extracted from the spleen of 192 rodents collected in Northeast France. RNA from all samples was pooled and submitted to high throughput RNA sequencing (RNAseq). Succeeding de novo assembly, bacterial contigs were assigned to the closest already-known taxa, revealing a list of zoonotic bacteria for the whole sample. Parallel, DNA samples were submitted to meta-barcoding approach: each sample was amplified by PCR using universal primers tagged at the V4 region of the 16S rRNA. The amplified templates were multiplexed and submitted to 454-pyrosequencing. The resulting dataset was demultiplexed using a home-made pipeline that assigns each read to a sample using the tagged primers, following these were processed using Mothur pipeline to construct OTUs and classify them using the RDP database. These methods allowed listing bacteria detected in each rodent and, so derive the prevalence, coinfections and bacteria interactions. DNA/RNA of the following bacteria genera were detected by both approaches, RNAseq and DNA 16S-metabarcoding: Bartonella, Leptospira, Borrelia, Rickettsia, Treponema, Neisseria, Spiroplasma, Klebsiella, Listeria and Shigella. Some unexpected genera were detected; such as Orientia, up to now only found in Asia or Helicobacter, generally thought to be restricted to animal guts. Several bacterial pathogens explored by RNAseq passed undetected by 16S-metabarcoding: Anaplasma, (Neo)Ehrlichia, Wolbachia, Brucella, Coxiella, Campylobacter, Mycoplasma, Salmonella, Yersinia, and Francisella. Furthermore, 16S meta-barcoding allowed to specify prevalence of bacteria within our sample, and revealed high level of coinfection in wild rodents. Our data demonstrate that NGS allows having a rather complete screening of pathogenic bacteria present in animal reservoirs without any a priori on their presence, while having a price compatible with cohort studies. NGS approaches are becoming the new routine approaches in large-scale epidemiological studies.

Heterogeneity in environmental conditions helps to maintain genetic and phenotypic diversity in ecosystems. As such, it may explain why the capacity of animals to mount immune responses is highly variable. The quality of habitat patches, in terms of resources, parasitism, predation and habitat fragmentation may, for example, trigger trade-offs ultimately affecting the investment of individuals in various immunological pathways. We described spatial immunoheterogeneity in bank vole populations with respect to landscape features and co-infection. We focused on the consequences of this heterogeneity for the risk of Puumala hantavirus (PUUV) infection. We assessed the expression of the Tnf-α and Mx2 genes and demonstrated a negative correlation between PUUV load and the expression of these immune genes in bank voles. Habitat heterogeneity was partly associated with differences in the expression of these genes. Levels of Mx2 were lower in large forests than in fragmented forests, possibly due to differences in parasite communities. We previously highlighted the positive association between infection with Heligmosomum mixtum and infection with PUUV. We found that Tnf-α was more strongly expressed in voles infected with PUUV than in uninfected voles or in voles co-infected with the nematode H. mixtum and PUUV. H. mixtum may limit the capacity of the vole to develop proinflammatory responses. This effect may increase the risk of PUUV infection and replication in host cells. Overall, our results suggest that close interactions between landscape features, co-infection and immune gene expression may shape PUUV epidemiology.

Rodent host dynamics and dispersal are thought to be critical for hantavirus epidemiology as they determine pathogen persistence and transmission within and between host populations. We used landscape genetics to investigate how the population dynamics of the bank vole Myodes glareolus, the host of Puumala hantavirus (PUUV), vary with forest fragmentation and influence PUUV epidemiology. We sampled vole populations within the Ardennes, a French PUUV endemic area. We inferred demographic features such as population size, isolation and migration with regard to landscape configuration. We next analysed the influence of M. glareolus population dynamics on PUUV spatial distribution. Our results revealed that the global metapopulation dynamics of bank voles were strongly shaped by landscape features, including suitable patch size and connectivity. Large effective size in forest might therefore contribute to the higher observed levels of PUUV prevalence. By contrast, populations from hedge networks highly suffered from genetic drift and appeared strongly isolated from all other populations. This might result in high probabilities of local extinction for both M. glareolus and PUUV. Besides, we detected signatures of asymmetric bank vole migration from forests to hedges. These movements were likely to sustain PUUV in fragmented landscapes. In conclusion, our study provided arguments in favour of source-sink dynamics shaping PUUV persistence and spread in heterogeneous, Western European temperate landscapes. It illustrated the potential contribution of landscape genetics to the understanding of the epidemiological processes occurring at this local scale.

An explanation for female multiple mating when males offer no material benefits but sperm remains elusive, largely because of a lack of empirical support for the genetic benefits hypothesis. We used 21 microsatellite markers to test for multiple paternities among 88 litters of roe deer, Capreolus capreolus, and to investigate the inbreeding avoidance hypothesis as a potential mechanism for the evolution of female multiple mating. From paternity analyses, we found that 13.5% of polytocous litters were sired by more than one male. We also found that a half-sib relationship was more likely than a full-sib relationship for 20.5% of all litters. This is the first report of multiple paternities in a territorial ungulate species. In support of the inbreeding avoidance hypothesis, we found that parents who were strongly related produced offspring with lower individual heterozygosity that survived less well during their first summer than fawns with unrelated parents. In addition, fawns from multiple paternity litters survived their first summer better than fawns from single paternity litters. However, it remains unclear whether all female multiple paternity events in this species are provoked by an initial consanguineous mating. © 2009 The Linnean Society of London, Biological Journal of the Linnean Society 2009, 97, 128–139.

Trichuris arvicolae and T. muris are gastro-intestinal nematodes of respectively arvicoline and murine rodents. We aim to investigate the ecology of these Trichuris species using population genetics. We sampled nematodes from rodents trapped in the East of France. After confirming the species identification of the nematodes using ITS1–5.8S–ITS2 ribosomal DNA sequences, we isolated and characterized twelve dinucleotide microsatellite loci in T. arvicolae. A multiplex panel was developed. Application to a set of 30 individuals allowed clear and easy characterization of allele sizes. The number of alleles ranged from 2 to 6 per locus with observed heterozygosities ranging from 0 to 0.93. A test on eleven T. muris revealed that eight loci among twelve amplified, and five were polymorphic. These sets of microsatellite loci provide high throughput capacity for population genetic studies.

We investigated the factors mediating selection acting on two MHC class II genes (DQA and DRB) in water vole (Arvicola scherman) natural populations in the French Jura Mountains. Population genetics showed significant homogeneity in allelic frequencies at the DQA1 locus as opposed to neutral markers (nine microsatellites), indicating balancing selection acting on this gene. Moreover, almost exhaustive screening for parasites, including gastrointestinal helminths, brain coccidia and antibodies against viruses responsible for zoonoses, was carried out. We applied a co-inertia approach to the genetic and parasitological data sets to avoid statistical problems related to multiple testing. Two alleles, Arte-DRB-11 and Arte-DRB-15, displayed antagonistic associations with the nematode Trichuris arvicolae, revealing the potential parasite-mediated selection acting on DRB locus. Selection mechanisms acting on the two MHC class II genes thus appeared different. Moreover, overdominance as balancing selection mechanism was showed highly unlikely in this system.

Hantaviruses are among the main emerging infectious agents in Europe. Their mode of transmission in natura is still not well known. In particular, social features and behaviours could be crucial for understanding the persistence and the spread of hantaviruses in rodent populations. Here, we investigated the importance of kinclustering and dispersal in hantavirus transmission by combining a fine-scale spatiotemporal survey (4 km(2)) and a population genetics approach. Two specific host-hantavirus systems were identified and monitored: the bank vole Myodes, earlier Clethrionomys glareolus--Puumala virus and the common vole Microtus arvalis-Tula virus. Sex, age and landscape characteristics significantly influenced the spatial distribution of infections in voles. The absence of temporal stability in the spatial distributions of viruses suggested that dispersal is likely to play a role in virus propagation. Analysing vole kinship from microsatellite markers, we found that infected voles were more closely related to each other than non-infected ones. Winter kin-clustering, shared colonies within matrilineages or delayed dispersal could explain this pattern. These two last results hold, whatever the host-hantavirus system considered. This supports the roles of relatedness and dispersal as general features for hantavirus transmission.

Host-pathogen interactions are of particular interest in studies of the interplay between population dynamics and natural selection. The major histocompatibility complex (MHC) genes of demographically fluctuating species are highly suitable markers for such studies, because they are involved in initiating the immune response against pathogens and display a high level of adaptive genetic variation. We investigated whether two MHC class II genes (DQA1, DRB) were subjected to contemporary selection during increases in the density of fossorial water vole (Arvicola terrestris) populations, by comparing the neutral genetic structure of seven populations with that estimated from MHC genes. Tests for heterozygosity excess indicated that DQA1 was subject to intense balancing selection. No such selection operated on neutral markers. This pattern of selection became more marked with increasing abundance. In the low-abundance phase, when populations were geographically isolated, both overall differentiation and isolation-by-distance were more marked for MHC genes than for neutral markers. Model-based simulations identified DQA1 as an outlier (i.e. under selection) in a single population, suggesting the action of local selection in fragmented populations. The differences between MHC and neutral markers gradually disappeared with increasing effective migration between sites. In the high-abundance year, DQA1 displayed significantly lower levels of overall differentiation than the neutral markers. This gene therefore displayed stronger homogenization than observed under drift and migration alone. The observed signs of selection were much weaker for DRB. Spatial and temporal fluctuations in parasite pressure and locus-specific selection are probably the most plausible mechanisms underlying the observed changes in selection pattern during the demographic cycle.

We isolated and characterized 14 dinucleotide microsatellite loci in the common vole Microtus arvalis (Palas). Two multiplex panels both comprising seven loci were developed. Application to a set of 21 individuals allowed clear and easy characterization of allele sizes except for two loci which were then withdrawn from further analyses. The number of alleles ranged from four to 19 per locus with the observed heterozygosity ranging from 0.55 to 0.95. These sets of microsatellite loci provide high throughput capacity for population genetic studies at a minimum cost.

The Praomys complex contains some of the most important agricultural pests of Africa, including Mastomys species. We describe the development of nine supplementary microsatellite markers isolated from Mastomys huberti. We show the potential utility of M. huberti microsatellites as population markers for two other species of Mastomys, and two other species of the Praomys complex, Myomys daltoni and Praomys cf. rostratus.