Margot Paris

Persistence of Bacillus thuringiensis israelensis (Bti) in the environment induces resistance to multiple Bti toxins in mosquitoes.

BACKGROUND The simultaneous production of six different toxins by Bacillus thuringiensis israelensis (Bti) is thought to delay the evolution of resistance in treated mosquito populations. Recent studies have shown that Bti can persist and proliferate in the environment, thereby imposing continuous selective pressure on mosquito populations, raising concerns about the long-term effectiveness of this bioinsecticide. In order to evaluate the effect of Bti persistence on the evolution of resistance, the authors selected a laboratory Aedes aegypti L. strain with field-collected leaf litter containing Bti toxins. RESULTS It is shown that resistance to each individual Bti toxin (up to 30-fold) can be obtained after only a few generations of selection. However, the resistance to commercial Bti and to environmental Bti remains low (twofold and 3.4-fold respectively) in the selected strain. Furthermore, some selected individuals exhibited resistance to Cry4B but not to Cry4A, suggesting that two distinct resistance mechanisms are involved in the resistance to these two toxins. CONCLUSION Considering that resistance to Cry toxins might act as a first step to resistance to a complete Bti toxin mixture, the present results highlight the importance of testing each toxin individually in order accurately to monitor Bti toxin resistance evolution in field populations.

Genome scan in the mosquito Aedes rusticus: population structure and detection of positive selection after insecticide treatment

Identification of genes involved in local adaptation is particularly challenging for species functioning as a network of interconnected populations undergoing frequent extinctions–recolonizations, because populations are submitted to contrasted evolutionary pressures. Using amplified fragment length polymorphism markers, population genetic structure of the mosquito Aedes rusticus was analysed in five geographical areas of the French Rhône‐Alpes region. We included a number of sites that were treated with the bio‐insecticide Bacillus thuringiensis israelensis (Bti) for more than 15 years. Analysis of molecular variance revealed that most of the genetic variability was found within populations (96%), with no significant variation among geographical areas, although variation among populations within areas (4%) was significant. The global genetic differentiation index FST was low (0.0366 ± 0.167). However, pairwise FST values were significant and no isolation‐by‐distance at the regional level was observed, suggesting a metapopulation structure in this species. Bti‐treatment had no effect on genetic structure and on within‐population genetic diversity. Potential signatures of positive selection associated with Bti‐treatment were detected for five loci, even though toxicological bioassays performed on field‐collected larvae showed no significant difference in mortality between Bti‐treated and nontreated sites. The difficulty of detecting moderate resistance in field‐collected larvae together with possible differential persistence of toxins in the environment may explain our inability to detect a toxicological response to Bti in treated sites. The evidence for positive selection occurring at several genomic regions suggests a first step towards Bti resistance in natural mosquito populations treated with this bio‐insecticide. Furthermore, this signal was detectable using genomic tools before any toxicological evidence for resistance could be identified.

Long Lasting Persistence of Bacillus thuringiensis Subsp. israelensis ( Bti ) in Mosquito Natural Habitats

Background The detrimental effects of chemical insecticides on the environment and human health have lead to the call for biological alternatives. Today, one of the most promising solutions is the use of spray formulations based on Bacillus thuringiensis subsp. israelensis (Bti) in insect control programs. As a result, the amounts of Bti spread in the environment are expected to increase worldwide, whilst the common belief that commercial Bti is easily cleared from the ecosystem has not yet been clearly established. Methodology/Main Findings In this study, we aimed to determine the nature and origin of the high toxicity toward mosquito larvae found in decaying leaf litter collected in several natural mosquito breeding sites in the Rhône-Alpes region. From the toxic fraction of the leaf litter, we isolated B. cereus-like bacteria that were further characterized as B. thuringiensis subsp. israelensis using PCR amplification of specific toxin genes. Immunological analysis of these Bti strains showed that they belong to the H14 group. We finally used amplified length polymorphism (AFLP) markers to show that the strains isolated from the leaf litter were closely related to those present in the commercial insecticide used for field application, and differed from natural worldwide genotypes. Conclusions/Significance Our results raise the issue of the persistence, potential proliferation and environmental accumulation of human-spread Bti in natural mosquito habitats. Such Bti environmental persistence may lengthen the exposure time of insects to this bio-insecticide, thereby increasing the risk of resistance acquisition in target insects, and of a negative impact on non-target insects.

Larval midgut modifications associated with Bti resistance in the yellow fever mosquito using proteomic and transcriptomic approaches

BackgroundBacillus thuringiensis var. israelensis (Bti) is a natural larval mosquito pathogen producing pore-forming toxins targeting the midgut of Diptera larvae. It is used worldwide for mosquito control. Resistance mechanisms of an Aedes aegypti laboratory strain selected for 30 generations with field-collected leaf litter containing Bti toxins were investigated in larval midguts at two levels: 1. gene transcription using DNA microarray and RT-qPCR and 2. differential expression of brush border membrane proteins using DIGE (Differential In Gel Electrophoresis).ResultsSeveral Bti Cry toxin receptors including alkaline phosphatases and N-aminopeptidases and toxin-binding V-ATPases exhibited altered expression levels in the resistant strain. The under-expression of putative Bti- receptors is consistent with Bt-resistance mechanisms previously described in Lepidoptera. Four soluble metalloproteinases were found under-transcribed together with a drastic decrease of metalloproteinases activity in the resistant strain, suggesting a role in resistance by decreasing the amount of activated Cry toxins in the larval midgut.ConclusionsBy combining transcriptomic and proteomic approaches, we detected expression changes at nearly each step of the ingestion-to-infection process, providing a short list of genes and proteins potentially involved in Bti-resistance whose implication needs to be validated. Collectively, these results open the way to further functional analyses to better characterize Bti- resistance mechanisms in mosquitoes.

Identifying insecticide resistance genes in mosquito by combining AFLP genome scans and 454 pyrosequencing

AFLP‐based genome scans are widely used to study the genetics of adaptation and to identify genomic regions potentially under selection. However, this approach usually fails to detect the actual genes or mutations targeted by selection owing to the difficulties of obtaining DNA sequences from AFLP fragments. Here, we combine classical AFLP outlier detection with 454 sequencing of AFLP fragments to obtain sequences from outlier loci. We applied this approach to the study of resistance to Bacillus thuringiensis israelensis (Bti) toxins in the dengue vector Aedes aegypti. A genome scan of Bti‐resistant and Bti‐susceptible A. aegypti laboratory strains was performed based on 432 AFLP markers. Fourteen outliers were detected using two different population genetic algorithms. Out of these, 11 were successfully sequenced. Three contained transposable elements (TEs) sequences, and the 10 outliers that could be mapped at a unique location in the reference genome were located on different supercontigs. One outlier was in the vicinity of a gene coding for an aminopeptidase potentially involved in Bti toxin‐binding. Patterns of sequence variability of this gene showed significant deviation from neutrality in the resistant strain but not in the susceptible strain, even after taking into account the known demographic history of the selected strain. This gene is a promising candidate for future functional analysis.

Candidate genes revealed by a genome scan for mosquito resistance to a bacterial insecticide: sequence and gene expression variations

BackgroundGenome scans are becoming an increasingly popular approach to study the genetic basis of adaptation and speciation, but on their own, they are often helpless at identifying the specific gene(s) or mutation(s) targeted by selection. This shortcoming is hopefully bound to disappear in the near future, thanks to the wealth of new genomic resources that are currently being developed for many species. In this article, we provide a foretaste of this exciting new era by conducting a genome scan in the mosquito Aedes aegypti with the aim to look for candidate genes involved in resistance to Bacillus thuringiensis subsp. israelensis (Bti) insecticidal toxins.ResultsThe genome of a Bti-resistant and a Bti-susceptible strains was surveyed using about 500 MITE-based molecular markers, and the loci showing the highest inter-strain genetic differentiation were sequenced and mapped on the Aedes aegypti genome sequence. Several good candidate genes for Bti-resistance were identified in the vicinity of these highly differentiated markers. Two of them, coding for a cadherin and a leucine aminopeptidase, were further examined at the sequence and gene expression levels. In the resistant strain, the cadherin gene displayed patterns of nucleotide polymorphisms consistent with the action of positive selection (e.g. an excess of high compared to intermediate frequency mutations), as well as a significant under-expression compared to the susceptible strain.ConclusionBoth sequence and gene expression analyses agree to suggest a role for positive selection in the evolution of this cadherin gene in the resistant strain. However, it is unlikely that resistance to Bti is conferred by this gene alone, and further investigation will be needed to characterize other genes significantly associated with Bti resistance in Ae. aegypti. Beyond these results, this article illustrates how genome scans can build on the body of new genomic information (here, full genome sequence and MITE characterization) to finally hold their promises and help pinpoint candidate genes for adaptation and speciation.

Transcription profiling of resistance to Bti toxins in the mosquito Aedes aegypti using next-generation sequencing.

The control of mosquitoes transmitting infectious diseases relies mainly on the use of chemical insecticides. However, resistance to most chemical insecticides threatens mosquito control programs. In this context, the spraying of toxins produced by the bacteria Bacillus thuringiensis subsp. israelensis (Bti) in larval habitats represents an alternative to chemical insecticides and is now widely used for mosquito control. Recent studies suggest that resistance of mosquitoes to Bti toxin may occur locally but mechanisms have not been characterized so far. In the present study, we investigated gene transcription level variations associated with Bti toxin resistance in the mosquito Aedes aegypti using a next-generation sequencing approach. More than 6 million short cDNA tags were sequenced from larvae of two strains sharing the same genetic background: a Bti toxins-resistant strain and a susceptible strain. These cDNA tags were mapped with a high coverage (308 reads per position in average) to more than 6000 genes of Ae. aegypti genome and used to quantify and compare the transcription level of these genes between the two mosquito strains. Among them, 86 genes were significantly differentially transcribed more than 4-fold in the Bti toxins resistant strain comparatively to the susceptible strain. These included gene families previously associated with Bti toxins resistance such as serine proteases, alkaline phosphatase and alpha-amylase. These results are discussed in regards of potential Bti toxins resistance mechanisms in mosquitoes.

Amplified fragment length homoplasy: in silico analysis for model and non-model species

BackgroundAFLP markers are widely used in evolutionary genetics and ecology. However the frequent occurrence of non-homologous co-migrating fragments (homoplasy) both at the intra- and inter-individual levels in AFLP data sets is known to skew key parameters in population genetics. Geneticists can take advantage of the growing number of full genome sequences available for model species to study AFLP homoplasy and to predict it in non-model species.ResultsIn this study we performed in silico AFLPs on the complete genome of three model species to predict intra-individual homoplasy in a prokaryote (Bacillus thuringiensis ser. konkukian), a plant (Arabidopsis thaliana) and an animal (Aedes aegypti). In addition, we compared in silico AFLPs to empirical data obtained from three related non-model species (Bacillus thuringiensis ser. israelensis, Arabis alpina and Aedes rusticus). Our results show that homoplasy rate sharply increases with the number of peaks per profile. However, for a given number of peaks per profile, genome size or taxonomical range had no effect on homoplasy. Furthermore, the number of co-migrating fragments in a single peak was dependent on the genome richness in repetitive sequences: we found up to 582 co-migrating fragments in Ae. aegypti. Finally, we show that in silico AFLPs can help to accurately predict AFLP profiles in related non-model species.ConclusionsThese predictions can be used to tackle current issues in the planning of AFLP studies by limiting homoplasy rate and population genetic estimation bias. ISIF (In SIlico Fingerprinting) program is freely available at http://www-leca.ujf-grenoble.fr/logiciels.htm.

Adaptive evolution and segregating load contribute to the genomic landscape of divergence in two tree species connected by episodic gene flow

Speciation often involves repeated episodes of genetic contact between divergent populations before reproductive isolation (RI) is complete. Whole‐genome sequencing (WGS) holds great promise for unravelling the genomic bases of speciation. We have studied two ecologically divergent, hybridizing species of the ‘model tree’ genus Populus (poplars, aspens, cottonwoods), Populus alba and P. tremula, using >8.6 million single nucleotide polymorphisms (SNPs) from WGS of population pools. We used the genomic data to (i) scan these species’ genomes for regions of elevated and reduced divergence, (ii) assess key aspects of their joint demographic history based on genomewide site frequency spectra (SFS) and (iii) infer the potential roles of adaptive and deleterious coding mutations in shaping the genomic landscape of divergence. We identified numerous small, unevenly distributed genome regions without fixed polymorphisms despite high overall genomic differentiation. The joint SFS was best explained by ancient and repeated gene flow and allowed pinpointing candidate interspecific migrant tracts. The direction of selection (DoS) differed between genes in putative migrant tracts and the remainder of the genome, thus indicating the potential roles of adaptive divergence and segregating deleterious mutations on the evolution and breakdown of RI. Genes affected by positive selection during divergence were enriched for several functionally interesting groups, including well‐known candidate ‘speciation genes’ involved in plant innate immunity. Our results suggest that adaptive divergence affects RI in these hybridizing species mainly through intrinsic and demographic processes. Integrating genomic with molecular data holds great promise for revealing the effects of particular genetic pathways on speciation.

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.