Methodological optimization and standardization of the metabarcoding of insects gut microbiome

Metabarcoding analysis of microbiota could help understand how Orthopteran species cope with challenges associated with environmental changes. Since microbial symbionts have a mutually beneficial relationship with its host and play important roles in the immune and physiological systems, they likely impact its ecology and evolution (i.e. plant range, life history, behaviour). In addition, the analysis of the complex pathogenic communities associated with locusts could be useful to discover unexplored pathogens and develop future research on biological control innovation. Yet, current knowledge of Orthopteran-associated microbial communities is limited. This is partly because recognizing cryptic, diverse, and numerous microorganisms hosted by insects is a difficult task. Despite the design of standard genes for their identification and the latest advances in high throughput sequencing, difficulties persist when we look at the microbiota of insects, including Orthopterans. (1) DNA purification is an essential step in all cultivationindependent approaches to characterize microbial diversity. Indeed, the microbial composition is mainly biased by the efficiency of cell lysis. (2) Another critical step for unbiased representation analysis and high taxonomic resolution is the choice of amplicon and primers. In particular, we showed that Enterobacteriacea, common in insects, were poorly resolved with some of currently used amplicons. (3) Moreover, in the case of phytophagous insects, it is necessary to avoid the amplification of plant remains contained in the digestive tract. In this study, we use (1) three mock community standards that contained equal and logarithmic numbers of eight species (ZymoBIOMICS), and equal numbers of twenty other species (ATCC), and (2) six samplesrepresenting the six main orders of insects (Orthoptera, Diptera, Hemiptera, Coleoptera, Hymenoptera and Lepidoptera). On these dedicated samples, we first statistically evaluate the most commonly used DNA purification kit (Qiagen DNeasy Blood and Tissue), two microorganisms-specific DNA purification kits (ZymoBIOMICS-96 bashing beads and DNeasy UltraClean 96 Microbial Kit) and two homemade procedures (bashing beads and enzymatic cocktails added to Qiagen DNeasy Blood and Tissue). These methods are compared on the basis of DNA yield, DNA shearing, reproducibility, and most importantly representation of microbial diversityin 16S rRNA gene sequences. . Secondly, we are currently evaluating the taxonomic representativity and resolution of different 16S gene primers to avoid plant chloroplast genes amplifications. Second, we evaluate, using in silico analyses, (1) the PCR efficiency (representativity), (2) the taxonomic resolution and (3) the risk to amplify plant chloroplasts of already published primers on various variable regions of the 16S gene (V3, V4, V6, V9) and of the rpoB gene. We then test and validate in vitro the best primer candidates on the dedicated samples

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 trait

SESAME (SEquence Sorter & AMplicon Explorer): genotyping based on high-throughput multiplex amplicon sequencing

Summary: Characterizing genetic diversity through genotyping short amplicons is central to evolutionary biology. Next-generation sequencing (NGS) technologies changed the scale at which these type of data are acquired. SESAME is a web application package that assists genotyping of multiplexed individuals for several markers based on NGS amplicon sequencing. It automatically assigns reads to loci and individuals, corrects reads if standard samples are available and provides an intuitive graphical user interface (GUI) for allele validation based on the sequences and associated decision-making tools. The aim of SESAME is to help allele identification among a large number of sequences

Urban market gardening and rodent-borne pathogenic Leptospira in arid zones: a case study in Niamey, Niger

Leptospirosis essentially affects human following contact with rodent urine-contaminated water. As such, it was mainly found associated with rice culture, recreational activities and flooding. This is also the reason why it has mainly been investigated in temperate as well as warm and humid regions, while arid zones have been only very occasionally monitored for this disease. In particular, data for West African countries are extremely scarce. Here, we took advantage of an extensive survey of urban rodents in Niamey, Niger, in order to look for rodent-borne pathogenic[i] Leptospira[/i] species presence and distribution across the city. To do so, we used high throughput bacterial 16S-based metabarcoding, [i]lipL32[/i] gene-targeting RT-PCR, rrs gene sequencing and VNTR typing as well as GIS-based multivariate spatial analysis. Our results show that leptospires seem absent from the core city where usual [i]Leptospira[/i] reservoir rodent species (namely [i]R. rattus[/i] and [i]M. natalensis[/i]) are yet abundant. On the contrary, [i]L. kirschneri[/i] was detected in [i]Arvicanthis niloticus[/i] and [i]Cricetomys gambianus[/i], two rodent species that are restricted to irrigated cultures within the city. Moreover, the VNTR profiles showed that rodent-borne leptospires in Niamey belong to previously undescribed serovars. Altogether, our study points towards the importance of market gardening in maintain and circulation of leptospirosis within Sahelian cities. In Africa, irrigated urban agriculture constitutes a pivotal source of food supply, especially in the context of the ongoing extensive urbanization of the continent. With this in mind, we speculate that leptospirosis may represent a zoonotic disease of concern also in arid regions that would deserve to be more rigorously surveyed, especially in urban agricultural settings

No Difference between the Sexes in Fine-Scale Spatial Genetic Structure of Roe Deer

Background: Data on spatial genetic patterns may provide information about the ecological and behavioural mechanisms underlying population structure. Indeed, social organization and dispersal patterns of species may be reflected by the pattern of genetic structure within a population. [br/] Methodology/Principal Findings: We investigated the fine-scale spatial genetic structure of a roe deer (Capreolus capreolus) population in Trois-Fontaines (France) using 12 microsatellite loci. The roe deer is weakly polygynous and highly sedentary, and can form matrilineal clans. We show that relatedness among individuals was negatively correlated with geographic distance, indicating that spatially proximate individuals are also genetically close. More unusually for a large mammalian herbivore, the link between relatedness and distance did not differ between the sexes, which is consistent with the lack of sex-biased dispersal and the weakly polygynous mating system of roe deer. [br/] Conclusions/Significance: Our results contrast with previous reports on highly polygynous species with male-biased dispersal, such as red deer, where local genetic structure was detected in females only. This divergence between species highlights the importance of socio-spatial organization in determining local genetic structure of vertebrate populations

Comparison between Transcriptome Sequencing and 16S Metagenomics for Detection of Bacterial Pathogens in Wildlife

Background Rodents are major reservoirs of pathogens responsible for numerous zoonotic diseases in humans and livestock. Assessing their microbial diversity at both the individual and population level is crucial for monitoring endemic infections and revealing microbial association patterns within reservoirs. Recently, NGS approaches have been employed to characterize microbial communities of different ecosystems. Yet, their relative efficacy has not been assessed. Here, we compared two NGS approaches, RNA-Sequencing (RNA-Seq) and 16S-metagenomics, assessing their ability to survey neglected zoonotic bacteria in rodent populations.Methodology/Principal Findings : We first extracted nucleic acids from the spleens of 190 voles collected in France. RNA extracts were pooled, randomly retro-transcribed, then RNA-Seq was performed using HiSeq. Assembled bacterial sequences were assigned to the closest taxon registered in GenBank. DNA extracts were analyzed via a 16S-metagenomics approach using two sequencers: the 454 GS-FLX and the MiSeq. The V4 region of the gene coding for 16S rRNA was amplified for each sample using barcoded universal primers. Amplicons were multiplexed and processed on the distinct sequencers. The resulting datasets were de-multiplexed, and each read was processed through a pipeline to be taxonomically classified using the Ribosomal Database Project. Altogether, 45 pathogenic bacterial genera were detected. The bacteria identified by RNA-Seq were comparable to those detected by 16S-metagenomics approach processed with MiSeq (16S-MiSeq). In contrast, 21 of these pathogens went unnoticed when the 16S-metagenomics approach was processed via 454-pyrosequencing (16S-454). In addition, the 16S-metagenomics approaches revealed a high level of coinfection in bank voles. Conclusions/Significance :We concluded that RNA-Seq and 16S-MiSeq are equally sensitive in detecting bacteria. Although only the 16S-MiSeq method enabled identification of bacteria in each individual reservoir, with subsequent derivation of bacterial prevalence in host populations, and generation of intra-reservoir patterns of bacterial interactions. Lastly, the number of bacterial reads obtained with the 16S-MiSeq could be a good proxy for bacterial prevalence

Microevolution of bank voles (Myodes glareolus) at neutral and immune-related genes during multiannual dynamic cycles : Consequences for Puumala hantavirus epidemiology

Understanding howhost dynamics, including variations of population size and dispersal, may affect the epidemiology of infectious diseases through ecological and evolutionary processes is an active research area. Here we focus on a bank vole (Myodes glareolus) metapopulation surveyed in Finland between 2005 and 2009. Bank vole is the reservoir of Puumala hantavirus (PUUV), the agent of nephropathia epidemica (NE, a mild form of hemorrhagic fever with renal symptom) in humans. M. glareolus populations experience multiannual density fluctuations that may influence the level of genetic diversity maintained in bank voles, PUUV prevalence and NE occurrence. We examine bank vole metapopulation genetics at presumably neutral markers and immunerelated genes involved in susceptibility to PUUV (Tnf-promoter, Tlr4, Tlr7 and Mx2 gene) to investigate the links between population dynamics, microevolutionary processes and PUUV epidemiology. We show that genetic drift slightly and transiently affects neutral and adaptive genetic variability within the metapopulation. Gene flow seems to counterbalance its effects during the multiannual density fluctuations. The low abundance phase may therefore be too short to impact genetic variation in the host, and consequently viral genetic diversity. Environmental heterogeneity does not seem to affect vole gene flow, which might explain the absence of spatial structure previously detected in PUUV in this area. Besides, our results suggest the role of vole dispersal on PUUV circulation through sex-specific and density-dependent movements. We find little evidence of selection acting on immune-related genes within this metapopulation. Footprint of positive selection is detected at Tlr-4 gene in 2008 only. We observe marginally significant associations between Mx2 genotype and PUUV genogroups. These results show that neutral processes seem to be the main factors affecting the evolution of these immune-related genes at a contemporary scale, although the relative effects of neutral and adaptive forces could vary temporally with density fluctuations. Immune related gene polymorphism may in turn partly influence PUUV epidemiology in this metapopulation. (C) 2016 Published by Elsevier B.V.Peer reviewe

Detection of Orientia sp. DNA in rodents from Asia, West Africa and Europe

Article Open AccessInternational audienceOrientia bacterium is the agent of the scrub typhus, a seriously neglected life-threatening disease in Asia. Here, we report the detection of DNA of Orientia in rodents from Europe and Africa. These findings have important implications for public health. Surveillance outside Asia, where the disease is not expected by sanitary services, needs to be improved

Insights into Myodes glareolus / Puumala hantavirus interactions from rodent immunogenetics

Nephropathia epidemica (NE) is a mild form of hemorrhagic fever with renal syndrome (HFRS) caused by the hantavirus Puumala (PUUV). In Europe, its distribution is fragmented, whereas the bank vole Myodes glareolus, which is the reservoir of PUUV, is common all over the continent. Determining the causes underlying this heterogeneity is of main importance to better understand and prevent the risks of NE emergence. Besides climatic and ecological hypotheses, we have proposed that the geographic variability of bank vole immune responses to PUUV infection could shape differences in PUUV prevalence, and consequently NE incidence. We have tested this hypothesis by studying polymorphisms and / or expression levels of six candidate genes involved in the immune response to PUUV (DRB-MHC, TNF-alpha promoter, TLR4, TLR7, Mx2, Integrin bêta3) on ten populations of bank voles sampled in the French Ardennes, along a North-South transect including PUUV endemic and non-endemic areas. Signatures of selection have been evidenced for TNF-alpha and Mx2 genes using population genetics (FST scan) and genotype - phenotype association approaches. These genes have antiviral properties but also induce immunological damages, what make them central for driving a balance of resistance / tolerance to PUUV. Bank voles vary in their basal ability to tolerate/resist to PUUV. In high PUUV prevalence areas, TNF-alpha and Mx2 expression seemed down-regulated what suggest selection or phenotypic plasticity for higher tolerance to PUUV, at the benefit of lower immunopathological costs. Some of these results have been confirmed at the European scale.Le campagnol roussâtre Myodes glareolus est le réservoir de l’hantavirus Puumala (PUUV), responsable chez l’Homme d’une forme atténuée de Fièvre Hémorragique à Syndrome Rénal (FHSR), la Néphropathie Épidémique (NE). En Europe, l’incidence de la NE présente, malgré la distribution continue du réservoir, une forte variabilité géographique dont les causes ne sont à ce jour pas identifiées. Aux hypothèses climatiques et paysagères, nous proposons que des facteurs intrinsèques aux campagnols puissent également être impliqués. Une plus forte tolérance à l’infection par le virus PUUV, chez certains campagnols roussâtres, favoriserait la persistance et la transmission de ce virus, ce qui devrait accroître le risque de NE chez l’Homme. Nous avons testé cette hypothèse en étudiant les polymorphismes et/ou les niveaux d'expression de six gènes candidats impliqués dans la réponse immunitaire à PUUV (DRB-CMH, promoteur du TNF-alpha, TLR4, TLR7, Mx2, intégrine bêta3) chez dix populations de campagnols échantillonnées le long d’un axe nord/sud dans les Ardennes françaises, couvrant des zones endémiques et non endémiques à PUUV. Des signatures de sélection ont été détectées pour TNF-alpha et Mx2 grâce à des approches de génétique des populations (scan FST) et d’associations génotypes / phénotypes. Ces gènes codent des protéines dont les propriétés antivirales sont connues, mais qui induisent des coûts immunopathologiques importants. Ils pourraient donc jouer un rôle central dans une balance de tolérance / résistance à PUUV. De plus, dans les zones d’endémie, les gènes TNF-alpha et Mx2 sont sous-exprimés, ce qui suggère l’évolution d’une plus forte tolérance à PUUV, potentiellement au bénéfice d’un moindre coût immunopathologique. Certains de ces résultats ont été confirmés à l’échelle européenn