Changing landscapes of Southeast Asia and rodent-borne diseases: decreased diversity but increased transmission risks.

The reduction in biodiversity from land use change due to urbanization and agricultural intensification appears to be linked to major epidemiological changes in many human diseases. Increasing disease risks and the emergence of novel pathogens result from increased contact among wildlife, domesticated animals, and humans. We investigated the relationship between human alteration of the environment and the occurrence of generalist and synanthropic rodent species in relation to the diversity and prevalence of rodent-borne pathogens in Southeast Asia, a hotspot of threatened and endangered species, and a foci of emerging infectious diseases. We used data from an extensive pathogen survey of rodents from seven sites in mainland Southeast Asia in conjunction with past and present land cover analyses. At low spatial resolutions, we found that rodent-borne pathogen richness is negatively associated with increasing urbanization, characterized by increased habitat fragmentation, agriculture cover and deforestation. However, at a finer spatial resolution, we found that some major pathogens are favored by environmental characteristics associated with human alteration including irrigation, habitat fragmentation, and increased agricultural land cover. In addition, synanthropic rodents, many of which are important pathogen reservoirs, were associated with fragmented and human-dominated landscapes, which may ultimately enhance the opportunities for zoonotic transmission and human infection by some pathogens.

[Ecology of Lyme disease]. / Écologie de la maladie de Lyme.

Diseases spread by ticks are complex and typically come under the One Health approach because the implications for human, animal and environmental health are so intricately interconnected. In Europe and North America, these diseases, particularly the emblematic case of Lyme disease, are constantly on the rise. They are associated with a very strong emotional element in Western societies, where citizens are preoccupied by this upsurge and call on governments and health services to act. There is no vaccine against Lyme disease. This is the backdrop against which scientists are looking for alternative solutions based on the identification of ecological factors that are liable to better control tick populations and the movements of pathogens within ecosystems. This article describes the main knowledge already acquired about the ecology of Lyme disease and then provides a list of a number of instruments that can be leveraged to limit the risks and improve control.

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.

Habitat fragmentation alters the properties of a host-parasite network: rodents and their helminths in South-East Asia.

1. While the effects of deforestation and habitat fragmentation on parasite prevalence or richness are well investigated, host-parasite networks are still understudied despite their importance in understanding the mechanisms of these major disturbances. Because fragmentation may negatively impact species occupancy, abundance and co-occurrence, we predict a link between spatiotemporal changes in habitat and the architecture of host-parasite networks. 2. For this, we used an extensive data set on 16 rodent species and 29 helminth species from seven localities of South-East Asia. We analysed the effects of rapid deforestation on connectance and modularity of helminth-parasite networks. We estimated both the degree of fragmentation and the rate of deforestation through the development of land uses and their changes through the last 20 to 30 years in order to take into account the dynamics of habitat fragmentation in our statistical analyses. 3. We found that rapid fragmentation does not affect helminth species richness per se but impacts host-parasite interactions as the rodent-helminth network becomes less connected and more modular. 4. Our results suggest that parasite sharing among host species may become more difficult to maintain with the increase of habitat disturbance.

Species undersampling in tropical bat surveys: effects on emerging biodiversity patterns.

Undersampling is commonplace in biodiversity surveys of species-rich tropical assemblages in which rare taxa abound, with possible repercussions for our ability to implement surveys and monitoring programmes in a cost-effective way. We investigated the consequences of information loss due to species undersampling (missing subsets of species from the full species pool) in tropical bat surveys for the emerging patterns of species richness (SR) and compositional variation across sites. For 27 bat assemblage data sets from across the tropics, we used correlations between original data sets and subsets with different numbers of species deleted either at random, or according to their rarity in the assemblage, to assess to what extent patterns in SR and composition in data subsets are congruent with those in the initial data set. We then examined to what degree high sample representativeness (r ≥ 0·8) was influenced by biogeographic region, sampling method, sampling effort or structural assemblage characteristics. For SR, correlations between random subsets and original data sets were strong (r ≥ 0·8) with moderate (ca. 20%) species loss. Bias associated with information loss was greater for species composition; on average ca. 90% of species in random subsets had to be retained to adequately capture among-site variation. For nonrandom subsets, removing only the rarest species (on average c. 10% of the full data set) yielded strong correlations (r > 0·95) for both SR and composition. Eliminating greater proportions of rare species resulted in weaker correlations and large variation in the magnitude of observed correlations among data sets. Species subsets that comprised ca. 85% of the original set can be considered reliable surrogates, capable of adequately revealing patterns of SR and temporal or spatial turnover in many tropical bat assemblages. Our analyses thus demonstrate the potential as well as limitations for reducing survey effort and streamlining sampling protocols, and consequently for increasing the cost-effectiveness in tropical bat surveys or monitoring programmes. The dependence of the performance of species subsets on structural assemblage characteristics (total assemblage abundance, proportion of rare species), however, underscores the importance of adaptive monitoring schemes and of establishing surrogate performance on a site by site basis based on pilot surveys.

Dispersal, landscape and travelling waves in cyclic vole populations.

Travelling waves (TW) are among the most striking ecological phenomena emerging in oscillating populations. Despite much theory, understanding how real-world TW arise remains a challenge for ecology. Herein, we analyse 16-year time series of cyclic vole populations collected at 314 localities covering 2500 km² in France. We found evidence for a linear front TW spreading at a speed of 7.4 km year(-1) along a north-west/south-east direction and radiating away from a major landscape discontinuity as predicted by recent theory. The spatial signature of vole dispersal was assessed using genetic data collected at 14 localities. Both data sets were handled using similar autocorrelation approaches. Our results revealed a remarkable congruence of the spatial extent and direction of anisotropy of both demographic and genetic structures. Our results constitute the first empirical evidence that effective dispersal is limited in the direction of TW while most of the individual exchanges occur along the wave front.

Short-term variations in gene flow related to cyclic density fluctuations in the common vole.

In highly fluctuating populations with complex social systems, genetic patterns are likely to vary in space and time due to demographic and behavioural processes. Cyclic rodents are extreme examples of demographically instable populations that often exhibit strong social organization. In such populations, kin structure and spacing behaviour may vary with density fluctuations and impact both the composition and spatial structure of genetic diversity. In this study, we analysed the multiannual genetic structure of a cyclic rodent, Microtus arvalis, using a sample of 875 individuals trapped over three complete cycles (from 1999 to 2007) and genotyped at 10 microsatellite loci. We tested the predictions that genetic diversity and gene flow intensity vary with density fluctuations. We found evidences for both spatial scale-dependant variations in genetic diversity and higher gene flow during high density. Moreover, investigation of sex-specific relatedness patterns revealed that, although dispersal is biased toward males in this species, distances moved by both sexes were lengthened during high density. Altogether, these results suggest that an increase in migration with density allows to restore the local loss of genetic diversity occurring during low density. We then postulate that this change in migration results from local competition, which enhances female colonization of empty spaces and male dispersal among colonies.

Contrasted evolutionary histories of two Toll-like receptors (Tlr4 and Tlr7) in wild rodents (MURINAE).

BACKGROUND: In vertebrates, it has been repeatedly demonstrated that genes encoding proteins involved in pathogen-recognition by adaptive immunity (e.g. MHC) are subject to intensive diversifying selection. On the other hand, the role and the type of selection processes shaping the evolution of innate-immunity genes are currently far less clear. In this study we analysed the natural variation and the evolutionary processes acting on two genes involved in the innate-immunity recognition of Microbe-Associated Molecular Patterns (MAMPs). RESULTS: We sequenced genes encoding Toll-like receptor 4 (Tlr4) and 7 (Tlr7), two of the key bacterial- and viral-sensing receptors of innate immunity, across 23 species within the subfamily Murinae. Although we have shown that the phylogeny of both Tlr genes is largely congruent with the phylogeny of rodents based on a comparably sized non-immune sequence dataset, we also identified several potentially important discrepancies. The sequence analyses revealed that major parts of both Tlrs are evolving under strong purifying selection, likely due to functional constraints. Yet, also several signatures of positive selection have been found in both genes, with more intense signal in the bacterial-sensing Tlr4 than in the viral-sensing Tlr7. 92% and 100% of sites evolving under positive selection in Tlr4 and Tlr7, respectively, were located in the extracellular domain. Directly in the Ligand-Binding Region (LBR) of TLR4 we identified two rapidly evolving amino acid residues and one site under positive selection, all three likely involved in species-specific recognition of lipopolysaccharide of gram-negative bacteria. In contrast, all putative sites of LBRTLR7 involved in the detection of viral nucleic acids were highly conserved across rodents. Interspecific differences in the predicted 3D-structure of the LBR of both Tlrs were not related to phylogenetic history, while analyses of protein charges clearly discriminated Rattini and Murini clades. CONCLUSIONS: In consequence of the constraints given by the receptor protein function purifying selection has been a dominant force in evolution of Tlrs. Nevertheless, our results show that episodic diversifying parasite-mediated selection has shaped the present species-specific variability in rodent Tlrs. The intensity of diversifying selection was higher in Tlr4 than in Tlr7, presumably due to structural properties of their ligands.

Invasion genetics of the introduced black rat (Rattus rattus) in Senegal, West Africa.

An understanding of the evolutionary history and dynamics of invasive species is required for the construction of predictive models of future spread and the design of biological management measures. The black rat (Rattus rattus) is a major vertebrate invader with a worldwide distribution. Despite the severe ecological, economic and health impacts of this species, its evolutionary history has been little studied. We carried out extensive specimen sampling in Senegal, West Africa, and used microsatellite markers to describe the pattern and processes of invasion in this large continental area. The genetic data obtained were combined with historical knowledge concerning the presence of this species in Senegal. Data were analysed by a combination of Bayesian clustering and approximate Bayesian computation methods. The invasion pathways closely paralleled the history of human trade routes in Senegal. In several places, we detected the occurrence of multiple introductions from genetically different sources. Long-distance migration between towns and villages was also observed. Our findings suggest that genetic bottlenecks and admixture have played a major role in shaping the genetics of invasive black rats. These two processes may generate genetic novelty and favour rapid evolution along the invasion pathways.

Cytonuclear discordance among Southeast Asian black rats (Rattus rattus complex).

Black rats are major invasive vertebrate pests with severe ecological, economic and health impacts. Remarkably, their evolutionary history has received little attention, and there is no firm agreement on how many species should be recognized within the black rat complex. This species complex is native to India and Southeast Asia. According to current taxonomic classification, there are three taxa living in sympatry in several parts of Thailand, Cambodia and Lao People's Democratic Republic, where this study was conducted: two accepted species (Rattus tanezumi, Rattus sakeratensis) and an additional mitochondrial lineage of unclear taxonomic status referred to here as 'Rattus R3'. We used extensive sampling, morphological data and diverse genetic markers differing in rates of evolution and parental inheritance (two mitochondrial DNA genes, one nuclear gene and eight microsatellite loci) to assess the reproductive isolation of these three taxa. Two close Asian relatives, Rattus argentiventer and Rattus exulans, were also included in the genetic analyses. Genetic analyses revealed discordance between the mitochondrial and nuclear data. Mitochondrial phylogeny studies identified three reciprocally monophyletic clades in the black rat complex. However, studies of the phylogeny of the nuclear exon interphotoreceptor retinoid-binding protein gene and clustering and assignation analyses with eight microsatellites failed to separate R. tanezumi and R3. Morphometric analyses were consistent with nuclear data. The incongruence between mitochondrial and nuclear (and morphological) data rendered R. tanezumi/R3 paraphyletic for mitochondrial lineages with respect to R. sakeratensis. Various evolutionary processes, such as shared ancestral polymorphism and incomplete lineage sorting or hybridization with massive mitochondrial introgression between species, may account for this unusual genetic pattern in mammals.

Candidatus Neoehrlichia mikurensis in bank voles, France.

To further assess the geographic occurrence, possible vectors, and prevalence of Candidatus Neoehrlichia mikurensis, we analyzed spleen tissues from 276 voles trapped close to human settlements in France; 5 were infected with the organism. Sequencing showed the isolates carried the same genotype as the bacteria that caused disease in humans and animals elsewhere in Europe.

Meteorological and climatic variables predict the phenology of Ixodes ricinus nymph activity in France, accounting for habitat heterogeneity.

Ixodes ricinus ticks (Acari: Ixodidae) are the most important vector for Lyme borreliosis in Europe. As climate change might affect their distributions and activities, this study aimed to determine the effects of environmental factors, i.e., meteorological, bioclimatic, and habitat characteristics on host-seeking (questing) activity of I. ricinus nymphs, an important stage in disease transmissions, across diverse climatic types in France over 8 years. Questing activity was observed using a repeated removal sampling with a cloth-dragging technique in 11 sampling sites from 7 tick observatories from 2014 to 2021 at approximately 1-month intervals, involving 631 sampling campaigns. Three phenological patterns were observed, potentially following a climatic gradient. The mixed-effects negative binomial regression revealed that observed nymph counts were driven by different interval-average meteorological variables, including 1-month moving average temperature, previous 3-to-6-month moving average temperature, and 6-month moving average minimum relative humidity. The interaction effects indicated that the phenology in colder climates peaked differently from that of warmer climates. Also, land cover characteristics that support the highest baseline abundance were moderate forest fragmentation with transition borders with agricultural areas. Finally, our model could potentially be used to predict seasonal human-tick exposure risks in France that could contribute to mitigating Lyme borreliosis risk.

Concomitant influence of helminth infection and landscape on the distribution of Puumala hantavirus in its reservoir, Myodes glareolus.

BACKGROUND: Puumala virus, the agent of nephropathia epidemica (NE), is the most prevalent hantavirus in Europe. The risk for human infection seems to be strongly correlated with the prevalence of Puumala virus (PUUV) in populations of its reservoir host species, the bank vole Myodes glareolus. In humans, the infection risks of major viral diseases are affected by the presence of helminth infections. We therefore proposed to analyse the influence of both helminth community and landscape on the prevalence of PUUV among bank vole populations in the Ardennes, a PUUV endemic area in France. RESULTS: Among the 313 voles analysed, 37 had anti-PUUV antibodies. Twelve gastro-intestinal helminth species were recorded among all voles sampled. We showed that PUUV seroprevalence strongly increased with age or sexual maturity, especially in the northern forests (massif des Ardennes). The helminth community structure significantly differed between this part and the woods or hedgerows of the southern cretes pre-ardennaises. Using PUUV RNA quantification, we identified significant coinfections between PUUV and gastro-intestinal helminths in the northern forests only. More specifically, PUUV infection was positively associated with the presence of Heligmosomum mixtum, and in a lesser extent, Aonchotheca muris-sylvatici. The viral load of PUUV infected individuals tended to be higher in voles coinfected with H. mixtum. It was significantly lower in voles coinfected with A. muris-sylvatici, reflecting the influence of age on these latter infections. CONCLUSIONS: This is the first study to emphasize hantavirus--helminth coinfections in natural populations. It also highlights the importance to consider landscape when searching for such associations. We have shown that landscape characteristics strongly influence helminth community structure as well as PUUV distribution. False associations might therefore be evidenced if geographic patterns of helminths or PUUV repartition are not previously identified. Moreover, our work revealed that interactions between helminths and landscape enhance/deplete the occurrence of coinfections between PUUV and H. mixtum or A. muris-sylvatici. Further experimental analyses and long-term individual surveys are now required to confirm these correlative results, and to ascertain the causal links between helminth and PUUV infection risks.

Are water vole resistant to anticoagulant rodenticides following field treatments?

The anti-vitamin Ks (AVKs) are widely used to control rodent populations. They inhibit Vitamin K regeneration by the Vitamin K Epoxide Reductase (VKOR) and cause a fatal hemorrhagic syndrome. Because of repeated use, some populations of commensal rodents have expressed resistance to these compounds. In Franche-Comté (France), the water vole exhibits cyclic population outbreaks. A second generation AVK, bromadiolone, has been used for the last 20 years to control vole populations. The aim of this study is to determine whether these repeated treatments could have led to the development of resistance to AVKs in water vole populations. We conducted enzymatic and genetic studies on water voles trapped in treated and non treated plot. The results indicate that voles from the most heavily treated area exhibit enzymatic changes in VKOR activity hence arguing for resistance to AVKs and that an intronic haplotype on the vkorc1 gene seems to be associated with these enzymatic changes.

Impact of Landscape on Host-Parasite Genetic Diversity and Distribution Using the Puumala orthohantavirus-Bank Vole System.

In nature, host specificity has a strong impact on the parasite's distribution, prevalence, and genetic diversity. The host's population dynamics is expected to shape the distribution of host-specific parasites. In turn, the parasite's genetic structure is predicted to mirror that of the host. Here, we study the tandem Puumala orthohantavirus (PUUV)-bank vole system. The genetic diversity of 310 bank voles and 33 PUUV isolates from 10 characterized localities of Northeast France was assessed. Our findings show that the genetic diversity of both PUUV and voles, was positively correlated with forest coverage and contiguity of habitats. While the genetic diversity of voles was weakly structured in space, that of PUUV was found to be strongly structured, suggesting that the dispersion of voles was not sufficient to ensure a broad PUUV dissemination. Genetic diversity of PUUV was mainly shaped by purifying selection. Genetic drift and extinction events were better reflected than local adaptation of PUUV. These contrasting patterns of microevolution have important consequences for the understanding of PUUV distribution and epidemiology.

Are Orienteers Protected Enough against Tick Bites? Estimating Human Exposure to Tick Bites through a Participative Science Survey during an Orienteering Competition.

Mass-participation events in temperate forests are now well-established features of outdoor activities and represent high-risk activities regarding human exposition to tick bites. In this study we used a citizen science approach to quantify the space-time frequency of tick bites and undetected tick bites among orienteers that participated in a 6-day orienteering competition that took place in July 2018 in the forests of Eastern France, and we looked at the use and efficacy of different preventive behaviors. Our study confirms that orienteers are a high-risk population for tick bites, with 62.4% of orienteers bitten at least once during the competition, and 2.4 to 12.1 orienteers per 100 orienteers were bitten by ticks when walking 1 km. In addition, 16.7% of orienteers bitten by ticks had engorged ticks, meaning that they did not detect and remove their ticks immediately after the run. Further, only 8.5% of orienteers systematically used a repellent, and the use of repellent only partially reduced the probability of being bitten by ticks. These results represent the first attempt to quantify the risk of not immediately detecting a tick bite and provide rare quantitative data on the frequency of tick bites for orienteers according to walking distance and time spent in the forest. The results also provide information on the use of repellent, which will be very helpful for modeling risk assessment. The study also shows that prevention should be increased for orienteers in France.

Revisiting the taxonomy of the Rattini tribe: a phylogeny-based delimitation of species boundaries.

BACKGROUND: Rodents are recognized as hosts for at least 60 zoonotic diseases and may represent a serious threat for human health. In the context of global environmental changes and increasing mobility of humans and animals, contacts between pathogens and potential animal hosts and vectors are modified, amplifying the risk of disease emergence. An accurate identification of each rodent at a specific level is needed in order to understand their implications in the transmission of diseases. Among the Muridae, the Rattini tribe encompasses 167 species inhabiting South East Asia, a hotspot of both biodiversity and emerging and re-emerging diseases. The region faces growing economical development that affects habitats, biodiversity and health. Rat species have been demonstrated as significant hosts of pathogens but are still difficult to recognize at a specific level using morphological criteria. DNA-barcoding methods appear as accurate tools for rat species identification but their use is hampered by the need of reliable identification of reference specimens. In this study, we explore and highlight the limits of the current taxonomy of the Rattini tribe. RESULTS: We used the DNA sequence information itself as the primary information source to establish group membership and estimate putative species boundaries. We sequenced two mitochondrial and one nuclear genes from 122 rat samples to perform phylogenetic reconstructions. The method of Pons and colleagues (2006) that determines, with no prior expectations, the locations of ancestral nodes defining putative species was then applied to our dataset. To give an appropriate name to each cluster recognized as a putative species, we reviewed information from the literature and obtained sequences from a museum holotype specimen following the ancient DNA criteria. CONCLUSIONS: Using a recently developed methodology, this study succeeds in refining the taxonomy of one of the most difficult groups of mammals. Most of the species expected within the area were retrieved but new putative species limits were also indicated, in particular within Berylmys and Rattus genera, where future taxonomic studies should be directed. Our study lays the foundations to better investigate rodent-born diseases in South East Asia and illustrates the relevance of evolutionary studies for health and medical sciences.

A 454 multiplex sequencing method for rapid and reliable genotyping of highly polymorphic genes in large-scale studies.

BACKGROUND: High-throughput sequencing technologies offer new perspectives for biomedical, agronomical and evolutionary research. Promising progresses now concern the application of these technologies to large-scale studies of genetic variation. Such studies require the genotyping of high numbers of samples. This is theoretically possible using 454 pyrosequencing, which generates billions of base pairs of sequence data. However several challenges arise: first in the attribution of each read produced to its original sample, and second, in bioinformatic analyses to distinguish true from artifactual sequence variation. This pilot study proposes a new application for the 454 GS FLX platform, allowing the individual genotyping of thousands of samples in one run. A probabilistic model has been developed to demonstrate the reliability of this method. RESULTS: DNA amplicons from 1,710 rodent samples were individually barcoded using a combination of tags located in forward and reverse primers. Amplicons consisted in 222 bp fragments corresponding to DRB exon 2, a highly polymorphic gene in mammals. A total of 221,789 reads were obtained, of which 153,349 were finally assigned to original samples. Rules based on a probabilistic model and a four-step procedure, were developed to validate sequences and provide a confidence level for each genotype. The method gave promising results, with the genotyping of DRB exon 2 sequences for 1,407 samples from 24 different rodent species and the sequencing of 392 variants in one half of a 454 run. Using replicates, we estimated that the reproducibility of genotyping reached 95%. CONCLUSIONS: This new approach is a promising alternative to classical methods involving electrophoresis-based techniques for variant separation and cloning-sequencing for sequence determination. The 454 system is less costly and time consuming and may enhance the reliability of genotypes obtained when high numbers of samples are studied. It opens up new perspectives for the study of evolutionary and functional genetics of highly polymorphic genes like major histocompatibility complex genes in vertebrates or loci regulating self-compatibility in plants. Important applications in biomedical research will include the detection of individual variation in disease susceptibility. Similarly, agronomy will benefit from this approach, through the study of genes implicated in productivity or disease susceptibility traits.

Associations between MHC genes and Puumala virus infection in Myodes glareolus are detected in wild populations, but not from experimental infection data.

We analysed the influence of MHC class II Dqa and Drb genes on Puumala virus (PUUV) infection in bank voles (Myodes glareolus). We considered voles sampled in five European localities or derived from a previous experiment that showed variable infection success of PUUV. The genetic variation observed in the Dqa and Drb genes was assessed by using single-strand conformation polymorphism and pyrosequencing methods, respectively. Patterns were compared with those obtained from 13 microsatellites. We revealed significant genetic differentiation between PUUV-seronegative and -seropositive bank voles sampled in wild populations, at the Drb gene only. The absence of genetic differentiation observed at neutral microsatellites confirmed the important role of selective pressures in shaping these Drb patterns. Also, we found no significant associations between infection success and MHC alleles among laboratory-colonized bank voles, which is explained by a loss of genetic variability that occurred during the captivity of these voles.