Exploring the potential effects of forest urbanization on the interplay between small mammal communities and their gut microbiota.

Urbanization significantly impacts wild populations, favoring urban dweller species over those that are unable to adapt to rapid changes. These differential adaptative abilities could be mediated by the microbiome, which may modulate the host phenotype rapidly through a high degree of flexibility. Conversely, under anthropic perturbations, the microbiota of some species could be disrupted, resulting in dysbiosis and negative impacts on host fitness. The links between the impact of urbanization on host communities and their gut microbiota (GM) have only been scarcely explored. In this study, we tested the hypothesis that the bacterial composition of the GM could play a role in host adaptation to urban environments. We described the GM of several species of small terrestrial mammals sampled in forested areas along a gradient of urbanization, using a 16S metabarcoding approach. We tested whether urbanization led to changes in small mammal communities and in their GM, considering the presence and abundance of bacterial taxa and their putative functions. This enabled to decipher the processes underlying these changes. We found potential impacts of urbanization on small mammal communities and their GM. The urban dweller species had a lower bacterial taxonomic diversity but a higher functional diversity and a different composition compared to urban adapter species. Their GM assembly was mostly governed by stochastic effects, potentially indicating dysbiosis. Selection processes and an overabundance of functions were detected that could be associated with adaptation to urban environments despite dysbiosis. In urban adapter species, the GM functional diversity and composition remained relatively stable along the urbanization gradient. This observation can be explained by functional redundancy, where certain taxa express the same function. This could favor the adaptation of urban adapter species in various environments, including urban settings. We can therefore assume that there are feedbacks between the gut microbiota and host species within communities, enabling rapid adaptation.

Evaluation of 96-well high-throughput DNA extraction methods for 16S rRNA gene metabarcoding.

Gaining meaningful insights into bacterial communities associated with animal hosts requires the provision of high-quality nucleic acids. Although many studies have compared DNA extraction methods for samples with low bacterial biomass (e.g. water) or specific PCR inhibitors (e.g. plants), DNA extraction bias in samples without inherent technical constraint (e.g. animal samples) has received little attention. Furthermore, there is an urgent need to identify a DNA extraction methods in a high-throughput format that decreases the cost and time for processing large numbers of samples. We here evaluated five DNA extraction protocols, using silica membrane-based spin columns and a 96-well microplate format and based on either mechanical or enzymatic lysis or a combination of both, using three bacterial mock communities and Illumina sequencing of the V4 region of the 16SrRNA gene. Our results showed that none of the DNA extraction methods fully eliminated bias associated with unequal lysis efficiencies. However, we identified a DNA extraction method with a lower bias for each mock community standard. Of these methods, those including an enzymatic lysis showed biases specific to some bacteria. Altogether, these results again demonstrate the importance of DNA extraction standardization to be able to compare the microbiome results of different samples. In this attempt, we advise for the use of the 96-well DNeasy Blood and Tissue kit (Qiagen) with a zirconia bead-beating procedure, which optimizes altogether the cost, handling time and bacteria-specific effects associated with enzymatic lysis.

Small terrestrial mammals (Rodentia and Soricomorpha) along a gradient of forest anthropisation (reserves, managed forests, urban parks) in France.

Background: Understanding the relationships between wildlife biodiversity and zoonotic infectious diseases in a changing climate is a challenging issue that scientists must address to support further policy actions. We aim at tackling this challenge by focusing on small mammal-borne diseases in temperate forests and large urban green spaces. Small mammals are important reservoirs of zoonotic agents, with a high transmission potential for humans and domestic animals. Forests and large urban green spaces are ecosystems where efforts are undertaken to preserve biodiversity. They are put forward for their contribution to human well-being in addition to other ecosystem services (e.g. provisioning and regulating services). Moreover, forests and large urban green spaces are environments where small mammals are abundant and human/domestic-wildlife interactions are plausible to occur. These environments are, therefore, focal points for conservation management and public health issues. New information: The European Biodiversa BioRodDis project (https://www6.inrae.fr/biodiversa-bioroddis) aims at better understanding the relationships between small terrestrial mammal biodiversity and health in the context of global change and, in particular, of forest anthropisation and urbanisation. Here, we present the data gathered in France. The dataset will enable us to describe the diversity of small terrestrial mammal communities in forested areas corresponding to different levels of anthropisation and to evaluate the variability of this diversity over time, between seasons and years.The dataset contains occurrences of small terrestrial mammals (Rodentia and Soricomorpha) trapped in forested areas in eastern France (administrative Departments: Rhône, Ain, Jura). The sampling sites correspond to different degrees of anthropisation. Forests included in biological reserves are the least anthropised sites. Then, public forests and urban parks experience increasing levels of anthropisation. Data were collected during spring and autumn 2020 (three to four sampling sites), 2021 (six sampling sites) and 2022 (four sampling sites). These variations in the number of sites between years were due to lockdown restrictions in 2020 or to the legal authorisation to trap around biological reserves granted in 2021 only. The capture of animals was carried out in various types of forests (pine, deciduous, mixed) and in different habitats within urban parks (wooded areas, buildings, hay storage yards, riverside vegetation, restaurants, playground for kids, botanical garden, landfills). Animals were captured using live traps that were set on the ground for one to 11 nights. During this study period, 1593 small mammals were trapped and identified. They belong to 15 species, amongst which were nine species of rodents (Muridae, Cricetidae, Gliridae) and six species of shrews (Soricidae). They were weighted (gram) and measured (cm): head-body length, tail length and hind-foot length. Sexual characteristics were also recorded.

Increasing helminth infection burden depauperates the diversity of the gut microbiota and alters its composition in mice.

The gut microbiota constitutes a diverse community of organisms with pervasive effects on host homeostasis. The diversity and composition of the gut microbiota depend on both intrinsic (host genetics) and extrinsic (environmental) factors. Here, we investigated the reaction norms of fecal microbiota diversity and composition in three strains of mice infected with increasing doses of the gastrointestinal nematode Heligmosomoides polygyrus. We found that α-diversity (bacterial taxonomic unit richness) declined along the gradient of infective doses, and ß-diversity (dissimilarity between the composition of the microbiota of uninfected and infected mice) increased as the infective dose increased. We did not find evidence for genotype by environment (host strain by infective dose) interactions, except when focusing on the relative abundance of the commonest bacterial families. A simulation approach also showed that significant genotype by environment interactions would have been hardly found even with much larger sample size. These results show that increasing parasite burden progressively depauperates microbiota diversity and contributes to rapidly change its composition, independently from the host genetic background.

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.

Isolation and Genetic Characterization of Puumala Orthohantavirus Strains from France.

Puumala orthohantavirus (PUUV) causes a mild form of haemorrhagic fever with renal syndrome (HFRS) called nephropathia epidemica (NE), regularly diagnosed in Europe. France represents the western frontier of the expansion of NE in Europe with two distinct areas: an endemic area (north-eastern France) where PUUV circulates in rodent populations, with the detection of many human NE cases, and a non-endemic area (south-western France) where the virus is not detected, with only a few human cases being reported. In this study, we describe the different stages of the isolation of two PUUV strains from two distinct French geographical areas: Ardennes (endemic area) and Loiret (non-endemic area). To isolate PUUV efficiently, we selected wild bank voles (Myodes glareolus, the specific reservoir of PUUV) captured in these areas and that were seronegative for anti-PUUV IgG (ELISA) but showed a non-negligible viral RNA load in their lung tissue (qRT-PCR). With this study design, we were able to cultivate and maintain these two strains in Vero E6 cells and also propagate both strains in immunologically neutral bank voles efficiently and rapidly. High-throughput and Sanger sequencing results provided a better assessment of the impact of isolation methods on viral diversity.

Differential immune gene expression associated with contemporary range expansion in two invasive rodents in Senegal.

Biological invasions are major anthropogenic changes associated with threats to biodiversity and health. However, what determines the successful establishment and spread of introduced populations remains unclear. Here, we explore several hypotheses linking invasion success and immune phenotype traits, including those based on the evolution of increased competitive ability concept. We compared gene expression profiles between anciently and recently established populations of two major invading species, the house mouse Mus musculus domesticus and the black rat Rattus rattus, in Senegal (West Africa). Transcriptome analyses identified differential expression between anciently and recently established populations for 364 mouse genes and 83 rat genes. All immune-related genes displaying differential expression along the mouse invasion route were overexpressed at three of the four recently invaded sites studied. Complement activation pathway genes were overrepresented among these genes. By contrast, no particular immunological process was found to be overrepresented among the differentially expressed genes of black rat. Changes in transcriptome profiles were thus observed along invasion routes, but with different specific patterns between the two invasive species. These changes may be driven by increases in infection risks at sites recently invaded by the house mouse, and by stochastic events associated with colonization history for the black rat. These results constitute a first step toward the identification of immune eco-evolutionary processes potentially involved in the invasion success of these two rodent species.

How Bank Vole-PUUV Interactions Influence the Eco-Evolutionary Processes Driving Nephropathia Epidemica Epidemiology-An Experimental and Genomic Approach.

In Europe, Puumala virus (PUUV) is responsible for nephropathia epidemica (NE), a mild form of hemorrhagic fever with renal syndrome (HFRS). Despite the presence of its reservoir, the bank vole, on most of French territory, the geographic distribution of NE cases is heterogeneous and NE endemic and non-endemic areas have been reported. In this study we analyzed whether bank vole-PUUV interactions could partly shape these epidemiological differences. We performed crossed-experimental infections using wild bank voles from French endemic (Ardennes) and non-endemic (Loiret) areas and two French PUUV strains isolated from these areas. The serological response and dynamics of PUUV infection were compared between the four cross-infection combinations. Due to logistical constraints, this study was based on a small number of animals. Based on this experimental design, we saw a stronger serological response and presence of PUUV in excretory organs (bladder) in bank voles infected with the PUUV endemic strain. Moreover, the within-host viral diversity in excretory organs seemed to be higher than in other non-excretory organs for the NE endemic cross-infection but not for the NE non-endemic cross-infection. Despite the small number of rodents included, our results showed that genetically different PUUV strains and in a lesser extent their interaction with sympatric bank voles, could affect virus replication and diversity. This could impact PUUV excretion/transmission between rodents and to humans and in turn at least partly shape NE epidemiology in France.

In silico and empirical evaluation of twelve metabarcoding primer sets for insectivorous diet analyses.

During the most recent decade, environmental DNA metabarcoding approaches have been both developed and improved to minimize the biological and technical biases in these protocols. However, challenges remain, notably those relating to primer design. In the current study, we comprehensively assessed the performance of ten COI and two 16S primer pairs for eDNA metabarcoding, including novel and previously published primers. We used a combined approach of in silico, in vivo-mock community (33 arthropod taxa from 16 orders), and guano-based analyses to identify primer sets that would maximize arthropod detection and taxonomic identification, successfully identify the predator (bat) species, and minimize the time and financial costs of the experiment. We focused on two insectivorous bat species that live together in mixed colonies: the greater horseshoe bat (Rhinolophus ferrumequinum) and Geoffroy's bat (Myotis emarginatus). We found that primer degeneracy is the main factor that influences arthropod detection in silico and mock community analyses, while amplicon length is critical for the detection of arthropods from degraded DNA samples. Our guano-based results highlight the importance of detecting and identifying both predator and prey, as guano samples can be contaminated by other insectivorous species. Moreover, we demonstrate that amplifying bat DNA does not reduce the primers' capacity to detect arthropods. We therefore recommend the simultaneous identification of predator and prey. Finally, our results suggest that up to one-third of prey occurrences may be unreliable and are probably not of primary interest in diet studies, which may decrease the relevance of combining several primer sets instead of using a single efficient one. In conclusion, this study provides a pragmatic framework for eDNA primer selection with respect to scientific and methodological constraints.

Spatio-temporal trends in richness and persistence of bacterial communities in decline-phase water vole populations.

Understanding the driving forces that control vole population dynamics requires identifying bacterial parasites hosted by the voles and describing their dynamics at the community level. To this end, we used high-throughput DNA sequencing to identify bacterial parasites in cyclic populations of montane water voles that exhibited a population outbreak and decline in 2014-2018. An unexpectedly large number of 155 Operational Taxonomic Units (OTUs) representing at least 13 genera in 11 families was detected. Individual bacterial richness was higher during declines, and vole body condition was lower. Richness as estimated by Chao2 at the local population scale did not exhibit clear seasonal or cycle phase-related patterns, but at the vole meta-population scale, exhibited seasonal and phase-related patterns. Moreover, bacterial OTUs that were detected in the low density phase were geographically widespread and detected earlier in the outbreak; some were associated with each other. Our results demonstrate the complexity of bacterial community patterns with regard to host density variations, and indicate that investigations about how parasites interact with host populations must be conducted at several temporal and spatial scales: multiple times per year over multiple years, and at both local and long-distance dispersal scales for the host(s) under consideration.

DNA Metabarcoding as a Tool for Disentangling Food Webs in Agroecosystems.

Better knowledge of food webs and related ecological processes is fundamental to understanding the functional role of biodiversity in ecosystems. This is particularly true for pest regulation by natural enemies in agroecosystems. However, it is generally difficult to decipher the impact of predators, as they often leave no direct evidence of their activity. Metabarcoding via high-throughput sequencing (HTS) offers new opportunities for unraveling trophic linkages between generalist predators and their prey, and ultimately identifying key ecological drivers of natural pest regulation. Here, this approach proved effective in deciphering the diet composition of key predatory arthropods (nine species.; 27 prey taxa), insectivorous birds (one species, 13 prey taxa) and bats (one species; 103 prey taxa) sampled in a millet-based agroecosystem in Senegal. Such information makes it possible to identify the diet breadth and preferences of predators (e.g., mainly moths for bats), to design a qualitative trophic network, and to identify patterns of intraguild predation across arthropod predators, insectivorous vertebrates and parasitoids. Appropriateness and limitations of the proposed molecular-based approach for assessing the diet of crop pest predators and trophic linkages are discussed.

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.

Disentangling the effect of host genetics and gut microbiota on resistance to an intestinal parasite.

Resistance to infection is a multifactorial trait, and recent work has suggested that the gut microbiota can also contribute to resistance. Here, we performed a fecal microbiota transplant to disentangle the contribution of the gut microbiota and host genetics as drivers of resistance to the intestinal nematode Heligmosomoides polygyrus. We transplanted the microbiota of a strain of mice (SJL), resistant to H. polygyrus, into a susceptible strain (CBA) and vice-versa. We predicted that if the microbiota shapes resistance to H. polygyrus, the FMT should reverse the pattern of resistance between the two host strains. The two host strains had different microbiota diversities and compositions before the start of the experiment, and the FMT altered the microbiota of recipient mice. One mouse strain (SJL) was more resistant to colonization by the heterologous microbiota, and it maintained its resistance profile to H. polygyrus (lower parasite burden) independently of the FMT. On the contrary, CBA mice harbored parasites with lower fecundity during the early stage of the infection, and had an up-regulated expression of the cytokine IL-4 (a marker of H. polygyrus resistance) after receiving the heterologous microbiota. Therefore, while host genetics remains the main factor shaping the pattern of resistance to H. polygyrus, the composition of the gut microbiota also seems to play a strain-specific role.

rpoB, a promising marker for analyzing the diversity of bacterial communities by amplicon sequencing.

BACKGROUND: Microbiome composition is frequently studied by the amplification and high-throughput sequencing of specific molecular markers (metabarcoding). Various hypervariable regions of the 16S rRNA gene are classically used to estimate bacterial diversity, but other universal bacterial markers with a finer taxonomic resolution could be employed. We compared specificity and sensitivity between a portion of the rpoB gene and the V3 V4 hypervariable region of the 16S rRNA gene. RESULTS: We first designed universal primers for rpoB suitable for use with Illumina sequencing-based technology and constructed a reference rpoB database of 45,000 sequences. The rpoB and V3 V4 markers were amplified and sequenced from (i) a mock community of 19 bacterial strains from both Gram-negative and Gram-positive lineages; (ii) bacterial assemblages associated with entomopathogenic nematodes. In metabarcoding analyses of mock communities with two analytical pipelines (FROGS and DADA2), the estimated diversity captured with the rpoB marker resembled the expected composition of these mock communities more closely than that captured with V3 V4. The rpoB marker had a higher level of taxonomic affiliation, a higher sensitivity (detection of all the species present in the mock communities), and a higher specificity (low rates of spurious OTU detection) than V3 V4. We compared the performance of the rpoB and V3 V4 markers in an animal ecosystem model, the infective juveniles of the entomopathogenic nematode Steinernema glaseri carrying the symbiotic bacteria Xenorhabdus poinarii. Both markers showed the bacterial community associated with this nematode to be of low diversity (< 50 OTUs), but only rpoB reliably detected the symbiotic bacterium X. poinarii. CONCLUSIONS: Our results confirm that different microbiota composition data may be obtained with different markers. We found that rpoB was a highly appropriate marker for assessing the taxonomic structure of mock communities and the nematode microbiota. Further studies on other ecosystems should be considered to evaluate the universal usefulness of the rpoB marker. Our data highlight two crucial elements that should be taken into account to ensure more reliable and accurate descriptions of microbial diversity in high-throughput amplicon sequencing analyses: i) the need to include mock communities as controls; ii) the advantages of using a multigenic approach including at least one housekeeping gene (rpoB is a good candidate) and one variable region of the 16S rRNA gene. This study will be useful to the growing scientific community describing bacterial communities by metabarcoding in diverse ecosystems.

Deciphering host-parasitoid interactions and parasitism rates of crop pests using DNA metabarcoding.

An accurate estimation of parasitism rates and diversity of parasitoids of crop insect pests is a prerequisite for exploring processes leading to efficient natural biocontrol. Traditional methods such as rearing have been often limited by taxonomic identification, insect mortality and intensive work, but the advent of high-throughput sequencing (HTS) techniques, such as DNA metabarcoding, is increasingly seen as a reliable and powerful alternative approach. Little has been done to explore the benefits of such an approach for estimating parasitism rates and parasitoid diversity in an agricultural context. In this study, we compared the composition of parasitoid species and parasitism rates between rearing and DNA metabarcoding of host eggs and larvae of the millet head miner, Heliocheilus albipunctella De Joannis (Lepidoptera, Noctuidae), collected from millet fields in Senegal. We first assessed the detection threshold for the main ten endoparasitoids, by sequencing PCR products obtained from artificial dilution gradients of the parasitoid DNAs in the host moth. We then assessed the potential of DNA metabarcoding for diagnosing parasitism rates in samples collected from the field. Under controlled conditions, our results showed that relatively small quantities of parasitoid DNA (0.07 ng) were successfully detected within an eight-fold larger quantity of host DNA. Parasitoid diversity and parasitism rate estimates were always higher for DNA metabarcoding than for host rearing. Furthermore, metabarcoding detected multi-parasitism, cryptic parasitoid species and differences in parasitism rates between two different sampling sites. Metabarcoding shows promise for gaining a clearer understanding of the importance and complexity of host-parasitoid interactions in agro-ecosystems, with a view to improving pest biocontrol strategies.

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.

Preliminary insights into the genetics of bank vole tolerance to Puumala hantavirus in Sweden.

Natural reservoirs of zoonotic pathogens generally seem to be capable of tolerating infections. Tolerance and its underlying mechanisms remain difficult to assess using experiments or wildlife surveys. High-throughput sequencing technologies give the opportunity to investigate the genetic bases of tolerance, and the variability of its mechanisms in natural populations. In particular, population genomics may provide preliminary insights into the genes shaping tolerance and potentially influencing epidemiological dynamics. Here, we addressed these questions in the bank vole Myodes glareolus, the specific asymptomatic reservoir host of Puumala hantavirus (PUUV), which causes nephropathia epidemica (NE) in humans. Despite the continuous spatial distribution of M. glareolus in Sweden, NE is endemic to the northern part of the country. Northern bank vole populations in Sweden might exhibit tolerance strategies as a result of coadaptation with PUUV. This may favor the circulation and maintenance of PUUV and lead to high spatial risk of NE in northern Sweden. We performed a genome-scan study to detect signatures of selection potentially correlated with spatial variations in tolerance to PUUV. We analyzed six bank vole populations from Sweden, sampled from northern NE-endemic to southern NE-free areas. We combined candidate gene analyses (Tlr4, Tlr7, and Mx2 genes) and high-throughput sequencing of restriction site-associated DNA (RAD) markers. Outlier loci showed high levels of genetic differentiation and significant associations with environmental data including variations in the regional number of NE human cases. Among the 108 outliers that matched to mouse protein-coding genes, 14 corresponded to immune-related genes. The main biological pathways found to be significantly enriched corresponded to immune processes and responses to hantavirus, including the regulation of cytokine productions, TLR cascades, and IL-7, VEGF, and JAK-STAT signaling. In the future, genome-scan replicates and functional experimentations should enable to assess the role of these biological pathways in M. glareolus tolerance to PUUV.

Evaluating metabarcoding to analyse diet composition of species foraging in anthropogenic landscapes using Ion Torrent and Illumina sequencing.

DNA metabarcoding of faecal samples is being successfully used to study the foraging niche of species. We assessed the ability of two benchtop high-throughput sequencing (HTS) platforms, to identify a large taxonomic array of food items from domestic cats Felis silvestris catus, including prey and human-related food taxa (pet food and leftovers leaving undetectable solid remains in faeces). Scats from a captive feeding trial (n = 41) and from free-ranging individuals (n = 326) were collected and analysed using a cytb mini-barcode in independent PCR replicates on the Ion PGM and the MiSeq platforms. Outputs from MiSeq were more sensitive and reproducible than those from Ion PGM due to a higher sequencing depth and sequence quality on MiSeq. DNA from intact prey taxa was detected more often (82% of the expected occurrences) than DNA from pet food (54%) and raw fish and meat (31%). We assumed that this variability was linked to different degree of DNA degradation: The Ion PGM detected significantly less human-linked food, birds, field voles, murids and shrews in the field-collected samples than the MiSeq platform. Pooling the replicates from both platforms and filtering the data allowed identification of at least one food item in 87.4% of the field-collected samples. Our DNA metabarcoding approach identified 29 prey taxa, of which 25 to species level (90% of items) including 9 rodents, 3 insectivores, 12 birds and 1 reptile and 33 human-related food taxa of which 23 were identified to genus level (75% of items). Our results demonstrate that using HTS platforms such as MiSeq, which provide reads of sufficiently high quantity and quality, with sufficient numbers of technical replicates, is a robust and non-invasive approach for further dietary studies on animals foraging on a wide range of food items in anthropogenic landscapes.

Early life infection and host senescence.

Advanced age is often associated with a chronic inflammatory status and inflammatory diseases. It has been suggested that exposure to infectious agents that stimulate the inflammatory response at early ages might have carry over effects in terms of accelerated senescence and increased mortality at late ages. However, not all pathogens and parasites have pro-inflammatory effects. In particular, parasitic nematodes have been shown to dampen the inflammatory response and to prevent or alleviate the symptoms of inflammatory diseases. We, therefore, tentatively predicted that early infection with a parasite that has anti-inflammatory properties might postpone aging. We tested this idea using the association between the nematode Heligmosomoides polygyrus and its rodent host. In addition to the infection with H. polygyrus, we also activated the systemic inflammatory response with an Escherichia coli LPS injection, to explore the effect of H. polygyrus under control and inflammatory conditions. In addition to lifespan, we also assessed several biomarkers of aging, once the infection had been cleared. We found that both treatments (H. polygyrus infection and LPS challenge) reduced longevity. Most of the biomarkers of aging were affected by the previous infection status, suggesting that mice exposed to the nematode had an accentuated senescent phenotype. These results show that infection with immunomodulatory parasites per se does not prolong host lifespan and rather support the view that infection in early life accelerates the rate of aging.