Evolution de la résistance au bactério-insecticide Bti chez les moustiques

Mosquito control represents a major public health concern, as mosquitoes transmit many severe human diseases. After the massive use of chemical insecticides since the fifties, the bacterio-insecticide Bacillus thuringiensis subsp. israelensis (Bti) represents a safe alternative and is now widely used to control mosquito populations. High toxicity due to commercial Bti proliferation and persistence was found in decaying leaf litter collected in mosquito breeding sites. In order to evaluate the evolution of resistance, a laboratory Aedes aegypti strain was selected with field-collected leaf litter containing Bti toxins. Resistance of Aedes aegypti to Bti Cry toxins evolved after only few generations of larval selection. Several approaches were used to study the genetic bases of Bti resistance in the resistant strain. First, the genetic comparison by two genome scans (one with markers linked to transposable elements and one with neutral AFLP markers) of the sensible and the resistant strains revealed genetic loci related to this resistance. Those loci were sequenced and located in the Ae. aegypti genome and allowed the detection of several genomic regions under selection. Then, a transcriptome scan allowed the comparison of the transcription of more than 6000 between the susceptible and the resistant strain. Several genes potentially involved in Bti resistance were detected by combining these results with a candidate gene approach. Among them, a cadherin gene displayed patterns of nucleotide polymorphisms consistent with the action of positive selection and appeared a good candidate for Bti resistance. Furthermore, signatures of selection linked to Bti treatment and high gene flow were detected by a population genomic approach in the Rhône-Alpine mosquito strain Aedes rusticus. Information about genetic factors linked to Bti resistance and biological factors linked to treated species are essential to use adapted resistance management strategies.La résistance aux insecticides chez les moustiques pose des problèmes de santé publique car ils sont vecteurs de nombreuses maladies. Une alternative aux insecticides chimiques est l'utilisation du bactério-insecticide Bacillus thuringiensis subsp israelensis (Bti) qui a l'avantage de produire un mélange de six toxines spécifiques des Diptères. Cependant le Bti commercial peut proliférer et s'accumuler, entrainant une forte toxicité dans les litières végétales de certains gîtes à moustiques. Afin d'étudier l'évolution de la résistance au Bti chez les moustiques, j'ai sélectionné en laboratoire une souche d'Aedes aegypti avec des litières végétales contenant des toxines de Bti. Une résistance multigénique aux toxines Cry du Bti est apparue en seulement quelques générations chez la souche sélectionnée. Plusieurs approches ont été utilisées pour rechercher les bases génétiques de la résistance au Bti chez la souche d'Ae. aegypti résistante. Deux "scans génomiques" ont permis de déterminer plusieurs régions du génome présentant des signatures de sélection. Ensuite, les niveaux de transcription de plus de 6000 gènes ont été étudiés par séquençage haut débit. La combinaison de ces résultats avec une approche "gènes candidats" a permis d'obtenir une liste de gènes potentiellement liés à la résistance au Bti. Parmi les gènes identifiés, un gène codant pour une cadhérine présente des signatures de sélection chez la souche résistante et semble donc impliqué dans la résistance au Bti. De plus, une étude de génomique des populations de l'espèce de terrain Aedes rusticus traitée depuis 20 ans au Bti a mis en évidence des signatures de sélection liées au traitement et des flux de gènes importants chez cette espèce dans la région Rhône-Alpes. La caractérisation de facteurs génétiques liés à la résistance et de facteurs biologiques liés aux espèces traitées peut aider à la mise en place de stratégies de gestion limitant l'évolution de la résistance au Bti dans ces populations

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

<p>Abstract</p> <p>Background</p> <p>Genome 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 <it>Aedes aegypti </it>with the aim to look for candidate genes involved in resistance to <it>Bacillus thuringiensis </it>subsp. <it>israelensis </it>(<it>Bti</it>) insecticidal toxins.</p> <p>Results</p> <p>The genome of a <it>Bti</it>-resistant and a <it>Bti</it>-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 <it>Aedes aegypti </it>genome sequence. Several good candidate genes for <it>Bti</it>-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.</p> <p>Conclusion</p> <p>Both 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 <it>Bti </it>is conferred by this gene alone, and further investigation will be needed to characterize other genes significantly associated with <it>Bti </it>resistance in <it>Ae. aegypti</it>. 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.</p

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

<p>Abstract</p> <p>Background</p> <p>AFLP 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.</p> <p>Results</p> <p>In this study we performed <it>in silico </it>AFLPs on the complete genome of three model species to predict intra-individual homoplasy in a prokaryote (<it>Bacillus thuringiensis </it>ser. <it>konkukian</it>), a plant (<it>Arabidopsis thaliana</it>) and an animal (<it>Aedes aegypti</it>). In addition, we compared <it>in silico </it>AFLPs to empirical data obtained from three related non-model species (<it>Bacillus thuringiensis </it>ser. <it>israelensis, Arabis alpina </it>and <it>Aedes rusticus</it>). 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 <it>Ae. aegypti</it>. Finally, we show that <it>in silico </it>AFLPs can help to accurately predict AFLP profiles in related non-model species.</p> <p>Conclusions</p> <p>These 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 <url>http://www-leca.ujf-grenoble.fr/logiciels.htm</url>.</p

Incidence and developmental timing of endosperm failure in post-zygotic isolation between wild tomato lineages

Background and AimsDefective hybrid seed development in angiosperms might mediate the rapid establishment of intrinsic post-zygotic isolation between closely related species. Extensive crosses within and among three lineages of wild tomatoes (Solanum section Lycopersicon) were performed to address the incidence, developmental timing and histological manifestations of hybrid seed failure. These lineages encompass different, yet fairly recent, divergence times and both allopatric and partially sympatric pairs.MethodsMature seeds were scored visually 2 months after hand pollinations, and viable-looking seeds were assessed for germination success. Using histological sections from early-developing seeds from a sub-set of crosses, the growth of three major seed compartments (endosperm, embryo and seed coat) was measured at critical developmental stages up to 21 d after pollination, with a focus on the timing and histological manifestations of endosperm misdevelopment in abortive hybrid seeds.Key ResultsFor two of three interspecific combinations including the most closely related pair that was also studied histologically, almost all mature seeds appeared ‘flat’ and proved inviable; histological analyses revealed impaired endosperm proliferation at early globular embryo stages, concomitant with embryo arrest and seed abortion in both cross directions. The third interspecific combination yielded a mixture of flat, inviable and plump, viable seeds; many of the latter germinated and exhibited near-normal juvenile phenotypes or, in some instances, hybrid necrosis and impaired growth.ConclusionsThe overall results suggest that near-complete hybrid seed failure can evolve fairly rapidly and without apparent divergence in reproductive phenology/biology. While the evidence accrued here is largely circumstantial, early-acting disruptions of normal endosperm development are most probably the common cause of seed failure regardless of the type of endosperm (nuclear or cellular)

A MITE-based genotyping method to reveal hundreds of DNA polymorphisms in an animal genome after a few generations of artificial selection

<p>Abstract</p> <p>Background</p> <p>For most organisms, developing hundreds of genetic markers spanning the whole genome still requires excessive if not unrealistic efforts. In this context, there is an obvious need for methodologies allowing the low-cost, fast and high-throughput genotyping of virtually any species, such as the Diversity Arrays Technology (DArT). One of the crucial steps of the DArT technique is the genome complexity reduction, which allows obtaining a genomic representation characteristic of the studied DNA sample and necessary for subsequent genotyping. In this article, using the mosquito <it>Aedes aegypti </it>as a study model, we describe a new genome complexity reduction method taking advantage of the abundance of miniature inverted repeat transposable elements (MITEs) in the genome of this species.</p> <p>Results</p> <p><it>Ae. aegypti </it>genomic representations were produced following a two-step procedure: (1) restriction digestion of the genomic DNA and simultaneous ligation of a specific adaptor to compatible ends, and (2) amplification of restriction fragments containing a particular MITE element called <it>Pony </it>using two primers, one annealing to the adaptor sequence and one annealing to a conserved sequence motif of the <it>Pony </it>element. Using this protocol, we constructed a library comprising more than 6,000 DArT clones, of which at least 5.70% were highly reliable polymorphic markers for two closely related mosquito strains separated by only a few generations of artificial selection. Within this dataset, linkage disequilibrium was low, and marker redundancy was evaluated at 2.86% only. Most of the detected genetic variability was observed between the two studied mosquito strains, but individuals of the same strain could still be clearly distinguished.</p> <p>Conclusion</p> <p>The new complexity reduction method was particularly efficient to reveal genetic polymorphisms in <it>Ae. egypti</it>. Overall, our results testify of the flexibility of the DArT genotyping technique and open new prospects as regards its application to a wider range of species, including animals which have been refractory to it so far. DArT has also a role to play in the current burst of whole-genome scans carried out in various organisms, which track signatures of selection in order to unravel the basis of genetic adaptation.</p

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 th

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

A dedicated target capture approach reveals variable genetic markers across micro- and macro-evolutionary time scales in palms

Understanding the genetics of biological diversification across micro‐ and macro‐ evolutionary time scales is a vibrant field of research for molecular ecologists as rapid advances in sequencing technologies promise to overcome former limitations. In palms, an emblematic, economically and ecologically important plant family with high diversity in the tropics, studies of diversification at the population and species levels are still hampered by a lack of genomic markers suitable for the genotyping of large numbers of recently diverged taxa. To fill this gap, we used a whole genome sequencing approach to develop target sequencing for molecular markers in 4,184 genome regions, including 4,051 genes and 133 non‐genic putatively neutral regions. These markers were chosen to cover a wide range of evolutionary rates allowing future studies at the family, genus, species and population levels. Special emphasis was given to the avoidance of copy number variation during marker selection. In addition, a set of 149 well‐known sequence regions previously used as phylogenetic markers by the palm biological research community were included in the target regions, to open the possibility to combine and jointly analyse already available data sets with genomic data to be produced with this new toolkit. The bait set was effective for species belonging to all three palm sub‐families tested (Arecoideae, Ceroxyloideae and Coryphoideae), with high mapping rates, specificity and efficiency. The number of high‐quality single nucleotide polymorphisms (SNPs) detected at both the sub‐family and population levels facilitates efficient analyses of genomic diversity across micro‐ and macro‐evolutionary time scales

Development of the Observation Schedule for Children with Autism-Anxiety, Behaviour and Parenting (OSCA-ABP): A New Measure of Child and Parenting Behavior for Use with Young Autistic Children.

Co-occurring emotional and behavioral problems (EBPs) frequently exist in young autistic children. There is evidence based on parental report that parenting interventions reduce child EBPs. More objective measures of child EBPs should supplement parent reported outcomes in trials. We describe the development of a new measure of child and parenting behavior, the Observation Schedule for Children with Autism-Anxiety, Behaviour and Parenting (OSCA-ABP). Participants were 83 parents/carers and their 4-8-year-old autistic children. The measure demonstrated good variance and potential sensitivity to change. Child and parenting behavior were reliably coded among verbal and minimally verbal children. Associations between reports from other informants and observed behavior showed the measure had sufficient convergent validity. The measure has promise to contribute to research and clinical practice in autism mental health beyond objective measurement in trials