Stéphane Reynaud

Cross-induction of detoxification genes by environmental xenobiotics and insecticides in the mosquito Aedes aegypti: impact on larval tolerance to chemical insecticides.

The effect of exposure of Aedes aegypti larvae to sub-lethal doses of the pyrethroid insecticide permethrin, the organophosphate temephos, the herbicide atrazine, the polycyclic aromatic hydrocarbon fluoranthene and the heavy metal copper on their subsequent tolerance to insecticides, detoxification enzyme activities and expression of detoxification genes was investigated. Bioassays revealed a moderate increase in larval tolerance to permethrin following exposure to fluoranthene and copper while larval tolerance to temephos increased moderately after exposure to atrazine, copper and permethrin. Cytochrome P450 monooxygenases activities were induced in larvae exposed to permethrin, fluoranthene and copper while glutathione S-transferase activities were induced after exposure to fluoranthene and repressed after exposure to copper. Microarray screening of the expression patterns of all detoxification genes following exposure to each xenobiotic with the Aedes Detox Chip identified multiple genes induced by xenobiotics and insecticides. Further expression studies using real-time quantitative PCR confirmed the induction of multiple CYP genes and one carboxylesterase gene by insecticides and xenobiotics. Overall, this study reveals the potential of xenobiotics found in polluted mosquito breeding sites to affect their tolerance to insecticides, possibly through the cross-induction of particular detoxification genes. Molecular mechanisms involved and impact on mosquito control strategies are discussed.

Insecticide Resistance in the Dengue Vector Aedes aegypti from Martinique: Distribution, Mechanisms and Relations with Environmental Factors

Dengue is an important mosquito borne viral disease in Martinique Island (French West Indies). The viruses responsible for dengue are transmitted by Aedes aegypti, an indoor day-biting mosquito. The most effective proven method for disease prevention has been by vector control by various chemical or biological means. Unfortunately insecticide resistance has already been observed on the Island and recently showed to significantly reduce the efficacy of vector control interventions. In this study, we investigated the distribution of resistance and the underlying mechanisms in nine Ae. aegypti populations. Statistical multifactorial approach was used to investigate the correlations between insecticide resistance levels, associated mechanisms and environmental factors characterizing the mosquito populations. Bioassays revealed high levels of resistance to temephos and deltamethrin and susceptibility to Bti in the 9 populations tested. Biochemical assays showed elevated detoxification enzyme activities of monooxygenases, carboxylesterases and glutathione S-tranferases in most of the populations. Molecular screening for common insecticide target-site mutations, revealed the presence of the “knock-down resistance” V1016I Kdr mutation at high frequency (>87%). Real time quantitative RT-PCR showed the potential involvement of several candidate detoxification genes in insecticide resistance. Principal Component Analysis (PCA) performed with variables characterizing Ae. aegypti from Martinique permitted to underline potential links existing between resistance distribution and other variables such as agriculture practices, vector control interventions and urbanization. Insecticide resistance is widespread but not homogeneously distributed across Martinique. The influence of environmental and operational factors on the evolution of the resistance and mechanisms are discussed.

Impact of glyphosate and benzo[a]pyrene on the tolerance of mosquito larvae to chemical insecticides. Role of detoxification genes in response to xenobiotics☆

The effect of exposure of Aedes aegypti larvae for 72h to sub-lethal concentrations of the herbicide glyphosate and the polycyclic aromatic hydrocarbon benzo[a]pyrene on their subsequent tolerance to the chemical insecticides imidacloprid, permethrin and propoxur, detoxification enzyme activities and transcription of detoxification genes was investigated. Bioassays revealed a significant increase in larval tolerance to imidacloprid and permethrin following exposure to benzo[a]pyrene and glyphosate. Larval tolerance to propoxur increased moderately after exposure to benzo[a]pyrene while a minor increased tolerance was observed after exposure to glyphosate. Cytochrome P450 monooxygenases activities were strongly induced in larvae exposed to benzo[a]pyrene and moderately induced in larvae exposed to imidacloprid and glyphosate. Larval glutathione S-transferases activities were strongly induced after exposure to propoxur and moderately induced after exposure to benzo[a]pyrene and glyphosate. Larval esterase activities were considerably induced after exposure to propoxur but only slightly induced by other xenobiotics. Microarray screening of 290 detoxification genes following exposure to each xenobiotic with the DNA microarray Aedes Detox Chip identified multiple detoxification and red/ox genes induced by xenobiotics and insecticides. Further transcription studies using real-time quantitative RT-PCR confirmed the induction of multiple P450 genes, 1 carboxy/cholinelesterase gene and 2 red/ox genes by insecticides and xenobiotics. Overall, this study reveals the potential of benzo[a]pyrene and glyphosate to affect the tolerance of mosquito larvae to chemical insecticides, possibly through the cross-induction of particular genes encoding detoxification enzymes.

Transcription profiling of eleven cytochrome P450s potentially involved in xenobiotic metabolism in the mosquito Aedes aegypti

Transcription profiles of 11 Aedes aegypti P450 genes from CYP6 and CYP9 subfamilies potentially involved in xenobiotic metabolism were investigated. Many genes were preferentially transcribed in tissues classically involved in xenobiotic metabolism including midgut and Malpighian tubules. Life‐stage transcription profiling revealed important variations amongst larvae, pupae, and adult males and females. Exposure of mosquito larvae to sub‐lethal doses of three xenobiotics induced the transcription of several genes with an induction peak after 48 to 72 h exposure. Several CYP genes were also induced by oxidative stress and one gene strongly responded to 20‐hydroxyecdysone. Overall, this study revealed that these P450s show different transcription profiles according to xenobiotic exposures, life stages or sex. Their putative chemoprotective functions are discussed.

The central role of mosquito cytochrome P450 CYP6Zs in insecticide detoxification revealed by functional expression and structural modelling

The resistance of mosquitoes to chemical insecticides is threatening vector control programmes worldwide. Cytochrome P450 monooxygenases (CYPs) are known to play a major role in insecticide resistance, allowing resistant insects to metabolize insecticides at a higher rate. Among them, members of the mosquito CYP6Z subfamily, like Aedes aegypti CYP6Z8 and its Anopheles gambiae orthologue CYP6Z2, have been frequently associated with pyrethroid resistance. However, their role in the pyrethroid degradation pathway remains unclear. In the present study, we created a genetically modified yeast strain overexpressing Ae. aegypti cytochrome P450 reductase and CYP6Z8, thereby producing the first mosquito P450–CPR (NADPH-cytochrome P450-reductase) complex in a yeast recombinant system. The results of the present study show that: (i) CYP6Z8 metabolizes PBAlc (3-phenoxybenzoic alcohol) and PBAld (3-phenoxybenzaldehyde), common pyrethroid metabolites produced by carboxylesterases, producing PBA (3-phenoxybenzoic acid); (ii) CYP6Z8 transcription is induced by PBAlc, PBAld and PBA; (iii) An. gambiae CYP6Z2 metabolizes PBAlc and PBAld in the same way; (iv) PBA is the major metabolite produced in vivo and is excreted without further modification; and (v) in silico modelling of substrate–enzyme interactions supports a similar role of other mosquito CYP6Zs in pyrethroid degradation. By playing a pivotal role in the degradation of pyrethroid insecticides, mosquito CYP6Zs thus represent good targets for mosquito-resistance management strategies.

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.

Interactions between immune and biotransformation systems in fish: A review

In animals biotransformation and immune system are not totally independent, there are numerous functional interrelationships between these two systems. They are both implicated in the capacity of organisms to resist to a wide variety of environmental components such as viruses, bacteria and xenobiotics. It is known for a long time that the immune system functions as a physiologic system and interacts with all the other components of the organism including nervous or endocrine ones. In the same manner, the biotransformation system (especially the cytochrome P450 monooxygenases) is involved in the regulation of numerous hormone productions. In this way, many studies in mammals have revealed the possible interaction between immune and biotransformation systems. Among these interactions, the capacity of the activation of host defense mechanisms to down-regulate microsomal cytochrome P450 and the role of biotransformation system in the xenobiotic-mediated immunotoxicity have been underlined. Advances in the basic knowledge of fish immune and biotransformation systems should lead to a better understanding of the possible interactions between both systems and should improve fish health monitoring which is a crucial ecotoxicological goal.

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 showing > 50% 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.

Molecular mechanisms associated with increased tolerance to the neonicotinoid insecticide imidacloprid in the dengue vector Aedes aegypti

Mosquitoes are vectors of several major human diseases and their control is mainly based on the use of chemical insecticides. Resistance of mosquitoes to organochlorines, organophosphates, carbamates and pyrethroids led to a regain of interest for the use of neonicotinoid insecticides in vector control. The present study investigated the molecular basis of neonicotinoid resistance in the mosquito Aedes aegypti. A strain susceptible to insecticides was selected at the larval stage with imidacloprid. After eight generations of selection, larvae of the selected strain (Imida-R) showed a 5.4-fold increased tolerance to imidacloprid while adult tolerance level remained low. Imida-R larvae showed significant cross-tolerance to other neonicotinoids but not to pyrethroids, organophosphates and carbamates. Transcriptome profiling identified 344 and 108 genes differentially transcribed in larvae and adults of the Imida-R strain compared to the parental strain. Most of these genes encode detoxification enzymes, cuticle proteins, hexamerins as well as other proteins involved in cell metabolism. Among detoxification enzymes, cytochrome P450 monooxygenases (CYPs) and glucosyl/glucuronosyl transferases (UDPGTs) were over-represented. Bioassays with enzyme inhibitors and biochemical assays confirmed the contribution of P450s with an increased capacity of the Imida-R microsomes to metabolize imidacloprid in presence of NADPH. Comparison of substrate recognition sites and imidacloprid docking models of six CYP6s over-transcribed in the Imida-R strain together with Bemisia tabaci CYP6CM1vQ and Drosophila melanogaster CYP6G1, both able to metabolize imidacloprid, suggested that CYP6BB2 and CYP6N12 are good candidates for imidacloprid metabolism in Ae. aegypti. The present study revealed that imidacloprid tolerance in mosquitoes can arise after few generations of selection at the larval stage but does not lead to a significant tolerance of adults. As in other insects, P450-mediated insecticide metabolism appears to play a major role in imidacloprid tolerance in mosquitoes.

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.