Aurélie Bonin

obitools: a unix-inspired software package for DNA metabarcoding

DNA metabarcoding offers new perspectives in biodiversity research. This recently developed approach to ecosystem study relies heavily on the use of next‐generation sequencing (NGS) and thus calls upon the ability to deal with huge sequence data sets. The obitools package satisfies this requirement thanks to a set of programs specifically designed for analysing NGS data in a DNA metabarcoding context. Their capacity to filter and edit sequences while taking into account taxonomic annotation helps to set up tailor‐made analysis pipelines for a broad range of DNA metabarcoding applications, including biodiversity surveys or diet analyses. The obitools package is distributed as an open source software available on the following website: http://metabarcoding.org/obitools. A Galaxy wrapper is available on the GenOuest core facility toolshed: http://toolshed.genouest.org.

Comparative analysis of response to selection with three insecticides in the dengue mosquito Aedes aegypti using mRNA sequencing

BackgroundMosquito control programmes using chemical insecticides are increasingly threatened by the development of resistance. Such resistance can be the consequence of changes in proteins targeted by insecticides (target site mediated resistance), increased insecticide biodegradation (metabolic resistance), altered transport, sequestration or other mechanisms. As opposed to target site resistance, other mechanisms are far from being fully understood. Indeed, insecticide selection often affects a large number of genes and various biological processes can hypothetically confer resistance. In this context, the aim of the present study was to use RNA sequencing (RNA-seq) for comparing transcription level and polymorphism variations associated with adaptation to chemical insecticides in the mosquito Aedes aegypti. Biological materials consisted of a parental susceptible strain together with three child strains selected across multiple generations with three insecticides from different classes: the pyrethroid permethrin, the neonicotinoid imidacloprid and the carbamate propoxur.ResultsAfter ten generations, insecticide-selected strains showed elevated resistance levels to the insecticides used for selection. RNA-seq data allowed detecting over 13,000 transcripts, of which 413 were differentially transcribed in insecticide-selected strains as compared to the susceptible strain. Among them, a significant enrichment of transcripts encoding cuticle proteins, transporters and enzymes was observed. Polymorphism analysis revealed over 2500 SNPs showingu2009>u200950% allele frequency variations in insecticide-selected strains as compared to the susceptible strain, affecting over 1000 transcripts. Comparing gene transcription and polymorphism patterns revealed marked differences among strains. While imidacloprid selection was linked to the over transcription of many genes, permethrin selection was rather linked to polymorphism variations. Focusing on detoxification enzymes revealed that permethrin selection strongly affected the polymorphism of several transcripts encoding cytochrome P450 monooxygenases likely involved in insecticide biodegradation.ConclusionsThe present study confirmed the power of RNA-seq for identifying concomitantly quantitative and qualitative transcriptome changes associated with insecticide resistance in mosquitoes. Our results suggest that transcriptome modifications can be selected rapidly by insecticides and affect multiple biological functions. Previously neglected by molecular screenings, polymorphism variations of detoxification enzymes may play an important role in the adaptive response of mosquitoes to insecticides.

Effect of DNA extraction and sample preservation method on rumen bacterial population

The comparison of the bacterial profile of intracellular (iDNA) and extracellular DNA (eDNA) isolated from cow rumen content stored under different conditions was conducted. The influence of rumen fluid treatment (cheesecloth squeezed, centrifuged, filtered), storage temperature (RT,xa0-80xa0°C) and cryoprotectants (PBS-glycerol, ethanol) on quality and quantity parameters of extracted DNA was evaluated by bacterial DGGE analysis, real-time PCR quantification and metabarcoding approach using high-throughput sequencing. Samples clustered according to the type of extracted DNA due to considerable differences between iDNA and eDNA bacterial profiles, while storage temperature and cryoprotectants additives had little effect on sample clustering. The numbers of Firmicutes and Bacteroidetes were lower (Pxa0<xa00.01) in eDNA samples. The qPCR indicated significantly higher amount of Firmicutes in iDNA sample frozen with glycerol (Pxa0<xa00.01). Deep sequencing analysis of iDNA samples revealed the prevalence of Bacteroidetes and similarity of samples frozen with and without cryoprotectants, which differed from sample stored with ethanol at room temperature. Centrifugation and consequent filtration of rumen fluid subjected to the eDNA isolation procedure considerably changed the ratio of molecular operational taxonomic units (MOTUs) of Bacteroidetes and Firmicutes. Intracellular DNA extraction using bead-beating method from cheesecloth sieved rumen content mixed with PBS-glycerol and stored atxa0-80xa0°C was found as the optimal method to study ruminal bacterial profile.

Oral Samples as Non-Invasive Proxies for Assessing the Composition of the Rumen Microbial Community

Microbial community analysis was carried out on ruminal digesta obtained directly via rumen fistula and buccal fluid, regurgitated digesta (bolus) and faeces of dairy cattle to assess if non-invasive samples could be used as proxies for ruminal digesta. Samples were collected from five cows receiving grass silage based diets containing no additional lipid or four different lipid supplements in a 5 x 5 Latin square design. Extracted DNA was analysed by qPCR and by sequencing 16S and 18S rRNA genes or the fungal ITS1 amplicons. Faeces contained few protozoa, and bacterial, fungal and archaeal communities were substantially different to ruminal digesta. Buccal and bolus samples gave much more similar profiles to ruminal digesta, although fewer archaea were detected in buccal and bolus samples. Bolus samples overall were most similar to ruminal samples. The differences between both buccal and bolus samples and ruminal digesta were consistent across all treatments. It can be concluded that either proxy sample type could be used as a predictor of the rumen microbial community, thereby enabling more convenient large-scale animal sampling for phenotyping and possible use in future animal breeding programs aimed at selecting cattle with a lower environmental footprint.

Gene expression patterns and sequence polymorphisms associated with mosquito resistance to Bacillus thuringiensis israelensis toxins

BackgroundDespite the intensive use of Bacillus thuringiensis israelensis (Bti) toxins for mosquito control, little is known about the long term effect of exposure to this cocktail of toxins on target mosquito populations. In contrast to the many cases of resistance to Bacillus thuringiensis Cry toxins observed in other insects, there is no evidence so far for Bti resistance evolution in field mosquito populations. High fitness costs measured in a Bti selected mosquito laboratory strain suggest that evolving resistance to Bti is costly. The aim of the present study was to identify transcription level and polymorphism variations associated with resistance to Bti toxins in the dengue vector Aedes aegypti. We used RNA sequencing (RNA-seq) for comparing a laboratory-selected strain showing elevated resistance to Bti toxins and its parental non-selected susceptible strain. As the resistant strain displayed two marked larval development phenotypes (slow and normal), each phenotype was analyzed separately in order to evidence potential links between resistance mechanisms and mosquito life-history traits.ResultsA total of 12,458 genes were detected of which 844 were differentially transcribed between the resistant and susceptible strains. Polymorphism analysis revealed a total of 68,541 SNPs of which 12,571 SNPs exhibited more than 40% frequency difference between the resistant and susceptible strains, affecting 2,953 genes. Bti resistance is associated with changes in the transcription level of enzymes involved in detoxification and chitin metabolism. Among previously described Bti-toxin receptors, four alkaline phosphatases (ALPs) were differentially transcribed between resistant and susceptible larvae, and non-synonymous changes affected the protein sequence of one cadherin, six aminopeptidases (APNs) and four α-amylases. Other putative Cry receptors located in lipid rafts, such as flotillin and glycoside hydrolases, were under-transcribed and/or contained non-synonymous substitutions. Finally, immunity-related genes showed contrasted transcription and polymorphisms patterns between the two developmental resistant phenotypes, suggesting the existence of trade-offs between Bti-resistance, life-history traits and immunity.ConclusionsThe present study is the first to analyze the whole transcriptome of Bti-resistant mosquitoes by RNA-seq, shedding light on the importance of studying both transcription levels and sequence polymorphism variations to get a comprehensive view of insecticide resistance.

Impaired liver function in Xenopus tropicalis exposed to benzo[a]pyrene: transcriptomic and metabolic evidence

BackgroundDespite numerous studies suggesting that amphibians are highly sensitive to cumulative anthropogenic stresses, the role pollutants play in the decline of amphibian populations remains unclear. Amongst the most common aquatic contaminants, polycyclic aromatic hydrocarbons (PAHs) have been shown to induce several adverse effects on amphibian species in the larval stages. Conversely, adults exposed to high concentrations of the ubiquitous PAH, benzo[a]pyrene (BaP), tolerate the compound thanks to their highly efficient hepatic detoxification mechanisms. Due to this apparent lack of toxic effect on adults, no studies have examined in depth the potential toxicological impact of PAH on the physiology of adult amphibian livers. This study sheds light on the hepatic responses of Xenopus tropicalis when exposed to high environmentally relevant concentrations of BaP, by combining a high throughput transcriptomic approach (mRNA deep sequencing) and a characterization of cellular and physiological modifications to the amphibian liver.ResultsTranscriptomic changes observed in BaP-exposed Xenopus were further characterized using a time-dependent enrichment analysis, which revealed the pollutant-dependent gene regulation of important biochemical pathways, such as cholesterol biosynthesis, insulin signaling, adipocytokines signaling, glycolysis/gluconeogenesis and MAPK signaling. These results were substantiated at the physiological level with the detection of a pronounced metabolic disorder resulting in a possible insulin resistance-like syndrome phenotype. Hepatotoxicity induced by lipid and cholesterol metabolism impairments was clearly identified in BaP-exposed individuals.ConclusionsOur data suggested that BaP may disrupt overall liver physiology, and carbohydrate and cholesterol metabolism in particular, even after short-term exposure. These results are further discussed in terms of how this deregulation of liver physiology can lead to general metabolic impairment in amphibians chronically exposed to contaminants, thereby illustrating the role xenobiotics might play in the global decline in amphibian populations.

Contrasting nuclear and cytoplasmic exchanges between phylogenetically distant oak species (Quercus suber L. and Q. ilex L.) in Southern France: inferring crosses and dynamics.

Gene flow is particularly frequent in the genus Quercus (oaks), especially between closely related species. We focus here on Quercus ilex and the cork-producing Quercus suber, which occasionally hybridize although they are phylogenetically markedly separated. Morphological observations were combined with both allozymic and chloroplastic diagnostic markers to characterize hybridization and introgression and to infer their dynamics in two French regions (French Catalonia and Provence), which are separated by several hundred kilometres. Some hybrids were found in both regions, indicating recent hybridization events. As expected from previous studies, most hybrids resulted from female symbol Q. ilex x male symbol Q. suber crosses, but our data showed that the reciprocal cross is also possible. Partial independence between nuclear and chloroplastic introgression was observed in the two species. Nuclear introgression was limited in both species and both regions, with no preferred direction. In Provence, chloroplastic introgression was very rare in both species. Conversely, all Q. suber individuals from French Catalonia were introgressed by Q. ilex chlorotypes. This might be explained by introgression in the Iberian Peninsula antedating the first occurrence of the two species in French Catalonia. We also observed a new chlorotype that was created locally, and was exchanged between the two species. However, the two species still remain genetically differentiated. The dynamics and complexity of exchanges and the factors determining them (including human management of Q. suber) are discussed.

Soil community assembly varies across body sizes in a tropical forest

The relative influence of deterministic niche-based (i.e. abiotic conditions, biotic interactions) and stochastic-distance dependent neutral processes (i.e. demography, dispersal) in shaping communities has been extensively studied for various organisms, but is far less explored jointly across the tree of life, in particular in soil environments. Here, using a thorough DNA-based census of the whole soil biota in a large tropical forest plot, we show that soil aluminium, topography, and plant species identity are all important drivers of soil richness and community composition. Body size emerges as an important feature of the comparative ecology of the different taxa at the studied spatial scale, with microorganisms being more importantly controlled by environmental factors, while soil mesofauna rather display random spatial distribution. We infer that niche-based processes contribute differently to community assembly across trophic levels due to spatial scaling. Body size could hence help better quantifying important properties of multitrophic assemblages.

Double-digest RAD-sequencing: do wet and dry protocol parameters impact biological results?

Next-generation sequencing technologies have opened a new era of research in genomics. Among these, restriction enzyme-based techniques such as restriction-site associated DNA sequencing (RADseq) or double-digest RAD-sequencing (ddRADseq) are now widely used in many population genomics fields. From DNA sampling to SNP calling, both wet and dry protocols have been discussed in the literature to identify key parameters for an optimal loci reconstruction. The impact of these parameters on downstream analyses and biological results drawn from RADseq or ddRADseq data has however not been fully explored yet. In this study, we tackled this issue by investigating the effects of ddRADseq laboratory (i.e. wet protocol) and bioinformatics (i.e. dry protocol) settings on loci reconstruction and inferred biological signal at two evolutionary scale using two systems: a complex of butterfly species (Coenonympha sp.) and populations of Common beech (Fagus sylvatica). Results suggest an impact of wet protocol parameters (DNA quantity, number of PCR cycles during library preparation) on the number of recovered reads and SNPs, the number of unique alleles and individual heterozygosity. We also found that bioinformatic settings (i.e. clustering and minimum coverage thresholds) impact loci reconstruction (e.g. number of loci, mean coverage) and SNP calling (e.g. number of SNPs, heterozygosity). We however do not detect an impact of parameter settings on three types of analysis performed with ddRADseq data: measure of genetic differentiation, estimation of individual admixture, and demographic inferences. In addition, our work demonstrates the high reproducibility and low rate of genotyping inconsistencies of the ddRADseq protocol. Thus, our study highlights the impact of wet parameters on ddRADseq protocol with strong consequences on experimental success and biological conclusions. Dry parameters affects loci reconstruction and descriptive statistics but not biological conclusion for the two studied systems. Overall, this study illustrates, with others, the relevance of ddRADseq for population and evolutionary genomics at the inter- or intraspecific scales.

Diet shifts by adult flightless dung beetles Circellium bacchus, revealed using DNA metabarcoding, reflect complex life histories

Life history changes may change resource use. Such shifts are not well understood in the dung beetles, despite recognised differences in larval and adult feeding ability. We use the flightless dung beetle Circellium bacchus to explore such shifts, identifying dung sources of adults using DNA metabarcoding, and comparing these with published accounts of larval dung sources. C. bacchus is traditionally considered to specialise on the dung of large herbivores for both larval and adult feeding. We successfully extracted mammal DNA from 151 adult C. bacchus fecal samples, representing 16 mammal species (ranging from elephants to small rodents), many of which are hitherto undescribed in the diet. Adult C. bacchus showed clear dung source preferences, especially for large herbivores inhabiting dense-cover vegetation. Our approach also confirmed the presence of cryptic taxa in the study area, and we propose that this may be used for biodiversity survey and monitoring purposes. Murid rodent feces were the most commonly fed-upon dung source (77.5%) for adult C. bacchus, differing markedly from the large and megaherbivore dung sources used for larval rearing. These findings support the hypothesis of life history-specific shifts in resource use in dung beetles, and reveal a hitherto unsuspected, but ecologically important, role of these dung beetles in consuming rodent feces. The differences in feeding abilities of the larval and adult life history stages have profound consequences for their resource use and foraging strategies, and hence the ecological role of dung beetles. This principle and its ecological consequences should be explored in other scarabaeids.