Thierry Gaude

Identifying genomic changes associated with insecticide resistance in the dengue mosquito Aedes aegypti by deep targeted sequencing

The capacity of mosquitoes to resist insecticides threatens the control of diseases such as dengue and malaria. Until alternative control tools are implemented, characterizing resistance mechanisms is crucial for managing resistance in natural populations. Insecticide biodegradation by detoxification enzymes is a common resistance mechanism; however, the genomic changes underlying this mechanism have rarely been identified, precluding individual resistance genotyping. In particular, the role of copy number variations (CNVs) and polymorphisms of detoxification enzymes have never been investigated at the genome level, although they can represent robust markers of metabolic resistance. In this context, we combined target enrichment with high-throughput sequencing for conducting the first comprehensive screening of gene amplifications and polymorphisms associated with insecticide resistance in mosquitoes. More than 760 candidate genes were captured and deep sequenced in several populations of the dengue mosquito Ae. aegypti displaying distinct genetic backgrounds and contrasted resistance levels to the insecticide deltamethrin. CNV analysis identified 41 gene amplifications associated with resistance, most affecting cytochrome P450s overtranscribed in resistant populations. Polymorphism analysis detected more than 30,000 variants and strong selection footprints in specific genomic regions. Combining Bayesian and allele frequency filtering approaches identified 55 nonsynonymous variants strongly associated with resistance. Both CNVs and polymorphisms were conserved within regions but differed across continents, confirming that genomic changes underlying metabolic resistance to insecticides are not universal. By identifying novel DNA markers of insecticide resistance, this study opens the way for tracking down metabolic changes developed by mosquitoes to resist insecticides within and among populations.

Genetic transformation of Arabidopsis lyrata: specific expression of the green fluorescent protein (GFP) in pistil tissues

Arabidopsis thaliana has become widely used as a model system for plant biology. Recent phylogenetic studies led to a severe revision of the systematic relationships across species of the Brassicaceae family. This provided an opportunity to examine close relatives of A. thaliana and to study the function and molecular evolution of genes that play roles in ecology and speciation. In this context, developing tools to genetically transform “non-model plants” appears as a major issue to ascertain gene function. Here, we report a method to transform A. lyrata, one of the closest relatives of A. thaliana.

Response of phase II detoxification enzymes in Phragmites australis plants exposed to organochlorines

Mixed pollution is a characteristic of many industrial sites and constructed wetlands. Plants possessing an enzymatic detoxifying system that is able to handle xenobiotics seems to be a viable option for the removal of mixed persistent contaminants such organochlorines (OCs: monochlorobenzene (MCB), 1,4-dichlorobenzene (DCB), 1,2,4-trichlorobenzene (TCB), γ-hexachlorocyclohexane (HCH)). In this study, Phragmites australis plants were exposed to sub-lethal concentrations of OCs (7xa0days), in single-exposure (0.8 to 10xa0mgu2009l−1) and in mixture of OCs (0.2xa0mgu2009l−1 MCBu2009+u20090.2xa0mgu2009l−1 DCBu2009+u20092.5xa0mgu2009l−1 TCBu2009+u20090.175xa0mgu2009l−1 HCH). Studies were conducted on the detoxification phase II enzymes; glutathione S-transferases (GST), and glucosyltransferases (UGT). Measurements of GST and UGT activities revealed that OCs may be buffered by glutathione and glucose conjugation. There appeared to be a correlation between the effects on phase II enzymes and the degree of chlorination of the benzene ring with, for example, the greatest effects being obtained for HCH exposure. In the case of mixed pollution, the induction of some GST isoenzymes (CDNB, 35xa0% non-significant) and UGT (118xa0%) in leaves and the inhibition of phase II enzymes in the other organs were measured. UGTs appear to be key enzymes in the detoxification of OCs.

Ubiquitous Water-Soluble Molecules in Aquatic Plant Exudates Determine Specific Insect Attraction

Plants produce semio-chemicals that directly influence insect attraction and/or repulsion. Generally, this attraction is closely associated with herbivory and has been studied mainly under atmospheric conditions. On the other hand, the relationship between aquatic plants and insects has been little studied. To determine whether the roots of aquatic macrophytes release attractive chemical mixtures into the water, we studied the behaviour of mosquito larvae using olfactory experiments with root exudates. After testing the attraction on Culex and Aedes mosquito larvae, we chose to work with Coquillettidia species, which have a complex behaviour in nature and need to be attached to plant roots in order to obtain oxygen. This relationship is non-destructive and can be described as commensal behaviour. Commonly found compounds seemed to be involved in insect attraction since root exudates from different plants were all attractive. Moreover, chemical analysis allowed us to identify a certain number of commonly found, highly water-soluble, low-molecular-weight compounds, several of which (glycerol, uracil, thymine, uridine, thymidine) were able to induce attraction when tested individually but at concentrations substantially higher than those found in nature. However, our principal findings demonstrated that these compounds appeared to act synergistically, since a mixture of these five compounds attracted larvae at natural concentrations (0.7 nM glycerol, <0.5 nM uracil, 0.6 nM thymine, 2.8 nM uridine, 86 nM thymidine), much lower than those found for each compound tested individually. These results provide strong evidence that a mixture of polyols (glycerol), pyrimidines (uracil, thymine), and nucleosides (uridine, thymidine) functions as an efficient attractive signal in nature for Coquillettidia larvae. We therefore show for the first time, that such commonly found compounds may play an important role in plant-insect relationships in aquatic eco-systems.

In the hunt for genomic markers of metabolic resistance to pyrethroids in the mosquito Aedes aegypti: An integrated next-generation sequencing approach

Background The capacity of Aedes mosquitoes to resist chemical insecticides threatens the control of major arbovirus diseases worldwide. Until alternative control tools are widely deployed, monitoring insecticide resistance levels and identifying resistance mechanisms in field mosquito populations is crucial for implementing appropriate management strategies. Metabolic resistance to pyrethroids is common in Aedes aegypti but the monitoring of the dynamics of resistant alleles is impeded by the lack of robust genomic markers. Methodology/Principal findings In an attempt to identify the genomic bases of metabolic resistance to deltamethrin, multiple resistant and susceptible populations originating from various continents were compared using both RNA-seq and a targeted DNA-seq approach focused on the upstream regions of detoxification genes. Multiple detoxification enzymes were over transcribed in resistant populations, frequently associated with an increase in their gene copy number. Targeted sequencing identified potential promoter variations associated with their over transcription. Non-synonymous variations affecting detoxification enzymes were also identified in resistant populations. Conclusion /Significance This study not only confirmed the role of gene copy number variations as a frequent cause of the over expression of detoxification enzymes associated with insecticide resistance in Aedes aegypti but also identified novel genomic resistance markers potentially associated with their cis-regulation and modifications of their protein structure conformation. As for gene transcription data, polymorphism patterns were frequently conserved within regions but differed among continents confirming the selection of different resistance factors worldwide. Overall, this study paves the way of the identification of a comprehensive set of genomic markers for monitoring the spatio-temporal dynamics of the variety of insecticide resistance mechanisms in Aedes aegypti.

Levels of insecticide resistance to deltamethrin, malathion, and temephos, and associated mechanisms in Aedes aegypti mosquitoes from the Guadeloupe and Saint Martin islands (French West Indies)

BackgroundIn the Guadeloupe and Saint Martin islands, Aedes aegypti mosquitoes are the only recognized vectors of dengue, chikungunya, and Zika viruses. For around 40xa0years, malathion was used as a mosquito adulticide and temephos as a larvicide. Since the European Union banned the use of these two insecticide molecules in the first decade of the 21st century, deltamethrin and Bacillus thuringiensis var. israelensis are the remaining adulticide and larvicide, respectively, used in Guadeloupe. In order to improve the management of vector control activities in Guadeloupe and Saint Martin, we investigated Ae. aegypti resistance to and mechanisms associated with deltamethrin, malathion, and temephos.MethodsAe. aegypti mosquitoes were collected from six different localities of Guadeloupe and Saint Martin. Larvae were used for malathion and temephos bioassays, and adult mosquitoes for deltamethrin bioassays, following World Health Organization recommendations. Knockdown resistance (Kdr) genotyping for V1016I and F1534C mutations, and expression levels of eight enzymes involved in detoxification mechanisms were examined in comparison with the susceptible reference Bora Bora strain.ResultsResistance ratios (RR50) calculated for Ae. aegypti larvae showed high resistance levels to temephos (from 8.9 to 33.1-fold) and low resistance levels to malathion (from 1.7 to 4.4-fold). Adult females displayed moderate resistance levels to deltamethrin regarding the time necessary to affect 50% of individuals, varying from 8.0 to 28.1-fold. Molecular investigations on adult mosquitoes showed high resistant allele frequencies for V1016I and F1534C (from 85 to 96% and from 90 to 98%, respectively), as well as an overexpression of the glutathione S-transferase gene, GSTe2, the carboxylesterase CCEae3a, and the cytochrome genes 014614, CYP6BB2, CYP6M11, and CYP9J23.ConclusionsAe. aegypti populations from Guadeloupe and Saint Martin exhibit multiple resistance to organophosphates (temephos and malathion), and pyrethroids (deltamethrin). The mechanisms associated with these resistance patterns show strong frequencies of F1534C and V1016I Kdr mutations, and an over-expression of CCEae3a, GSTe2, and four cytochrome P450 genes (014614, CYP9J23, CYP6M11, CYP6BB2). These results will form the baseline for a deeper understanding of the insecticide resistance levels and associated mechanisms of Ae. aegypti populations and will be used to improve vector control strategies in Guadeloupe and Saint Martin.

Pre-selecting resistance against individual Bti Cry toxins facilitates the development of resistance to the Bti toxins cocktail

The bioinsecticide Bacillus thuringiensis subsp. israelensis is a larvicide used worldwide for mosquito control, which contains three Cry toxins and one Cyt toxin. We investigated for the first time in Aedes aegypti (1) the evolution of resistance and cross-resistance of strains selected with each Cry toxin, and (2) the effect of pre-selection with Cry toxin on the evolution of resistance to a mix of Bti toxins. Cross resistance was higher between Cry4Ba and Cry11Aa than between Cry4Aa and either Cry4Ba or Cry11Aa, suggesting both common and specific mechanisms of resistance. Pre-selecting resistance to each Cry toxins facilitated the development of resistance to the full Bti toxins cocktail.

Self-incompatibility in flowering plants : The Brassica model

Self-incompatibility (SI) is a widespread mechanism in flowering plants that prevents self-fertilization. Self-pollen recognition relies on the products of genes located at the S (self-incompatibility) locus. Significant progress towards understanding molecular interactions allowing stigmatic cells to recognize and reject self-pollen in Brassica has been made during the past two years. Thus, the male and female determinants responsible of the self-incompatibility (SI) response have been identified. The structural features of these molecules strongly suggest that SI response is triggered by a ligand-receptor interaction.

The cell biology of pollen development in Brassica

Several aspects of cell and molecular biology of Brassica have been largely developed because this genus plays a central role in crop improvement (see list of cultivars of Brassica oleracea in Table 1). In addition to these applied aspects, Brassica oleracea and to a lesser degree, B. campestris, are among the best models to carry molecular investigations on sporophytic self-incompatibility (review in Nasrallah et al. 1991).

The molecular signatures of compatible and incompatible pollination

Fertilization in flowering plants depends on the early contact and acceptance of pollen grains by the receptive papilla cells of the stigma. To identify the associated molecular pathways, we developed a transcriptomic analysis based on single nucleotide polymorphisms (SNPs) present in two Arabidopsis thaliana accessions, one used as female and the other as male. We succeeded in distinguishing 80 % of transcripts according to their parental origins and drew up a catalog of genes whose expression is modified after pollen-stigma interaction. Global analysis of our data reveals pattern-triggered immunity (PTI). From this analysis, we predicted the activation of the Mitogen-activated Protein Kinase 3 on the female side after compatible pollination, which we confirmed through expression and mutant analysis. Altogether this works provides the molecular signatures of compatible and incompatible pollination, highlights the active status of incompatible stigmas, and unravels a new MPK3-dependent cell wall feature associated with stigma-pollen interaction.