The incidence of mosquito-borne human diseases is increasing worldwide, with effective chemical control limited due to widespread insecticide resistance in the insect. Recent evidence also suggests that bacterial symbionts of mosquitoes, known to be essential in nutritional homeostasis and pathogen defense, may play a significant role in facilitating mosquitocide resistance. Here, I examined the metabolic detoxification and toxicity of two mosquitocides, propoxur and naled, and the capacity of bacterial symbionts to modify the detoxification of the mosquitocides and, thus, alter their toxic action in the yellow fever mosquito, Aedes aegypti. The insecticide synergists piperonyl butoxide (PBO), triphenyl phosphate (TPP), and S,S,S-tributyl phosphorotrithioate (DEF) were used to examine the metabolic detoxification and toxic action of the two mosquitocides in mosquito larvae. A significant increase in the toxicity of propoxur was observed when applied in combination with PBO; however, there was no corresponding decrease in AChE activity. Naled applied in combination with PBO resulted in a decrease in anticholinesterase activity (higher residual AChE activity) and a subsequent decrease in toxicity of the insecticide. This suggests that esterases play a major role in the metabolic detoxification of both insecticides in mosquito larvae. The acute toxicities of naled and propoxur to Ae. aegypti larvae were also studied following a reduction of bacterial symbionts with the broad-spectrum antibiotics gentamycin, penicillin, and streptomycin. Antibiotic-treated mosquito larvae showed increased susceptibility and a reduction in cytochrome P450 monooxygenase and general esterase activities when treated with naled and propoxur. A reduction of bacteria in mosquito larvae treated with broad-spectrum antibiotics, therefore, appears to affect the metabolic detoxification of standard-use mosquitocides, such as propoxur and naled. The results also suggest that the bacteria themselves may contain metabolic detoxification enzymes that are functionally similar to those in the mosquito larvae. Additional experiments, however, are needed to fully elucidate the contribution of bacterial symbionts in Ae. aegypti larvae in the metabolic detoxification of mosquitocides. / Master of Science in Life Sciences

Les moustiques génèrent une nuisance importante et sont notamment contrôlés grâce à des traitements insecticides. Aujourd'hui, les gîtes où se développent leurs larves sont souvent pollués par des xénobiotiques environnementaux (hydrocarbures, herbicides, pesticides, toxines naturelles…). Jusqu'à présent, l'impact de ces xénobiotiques sur la capacité des larves de moustiques à résister aux insecticides chimiques reste méconnu. Cette thèse vise à étudier la réponse des larves de d'Aedes aegypti aux xénobiotiques environnementaux et leur impact sur leur tolérance et résistance aux insecticides chimiques. Une première étude, sur le court terme, montre que des larves exposées pendant 24h à divers xénobiotiques deviennent plus tolérantes à vis à vis de différents insecticides chimiques (Poupardin et al. 2008). Des études biochimiques et transcriptomiques suggèrent que l'induction de certaines familles d'enzymes (e.g. P450s et GSTs) par ces xénobiotiques peut être liée à l'augmentation de tolérance des larves vis-à-vis de l'insecticide. Dans le but de mieux caractériser le profil transcriptionnel des précédents gènes candidats, des expérimentations complémentaires ont été faites à différents niveaux (Poupardin et al., 2010). Cette étude a montré que de nombreux gènes étaient préférentiellement transcrits dans des tissus fortement impliqués dans la détoxication de composés exogènes, essentiellement des CYP6. Elle révèle aussi que la transcription de ces P450s varie beaucoup au cours des différents stades de développement et qu'ils étaient induits à des faibles de doses de polluants avec un pic d'induction après 48 et 72 heures d'exposition. Ces études mettent en évidence le rôle potentiel des gènes de détoxication dans la réponse à l'exposition à des xénobiotiques et dans l'augmentation de tolérance aux insecticides chimiques. Concernant l'étude sur le long terme de l'impact des polluants sur la résistance des moustiques aux insecticides, la question est de savoir si les polluants trouvés dans l'environnement influencent la sélection de la résistance aux insecticides et si oui, favorisent-ils la sélection de gènes en particulier? Pour répondre à ces questions, trois souches d'Aedes aegypti ont été sélectionnées à la perméthrine. Ces souches sont exposées ou non à différents polluants avant sélection. Après 10 générations de sélection, des bioessais montrent une résistance de ces 3 souches vis-à-vis de la perméthrine. Aucune différence significative de niveau de résistance n'est observée entre les trois souches sélectionnées pour le moment. Pour identifier les gènes différentiellement transcrits dans ces souches, la puce "Agilent Aedes chip" développée par l'école de médecine tropicale de Liverpool (LSTM) et contenant 14200 transcrits a été utilisée. Les microarrays ont révélé que la présence de polluants ou insecticides résiduels pouvait affecter la sélection des mécanismes de résistance aux insecticides chimiques, notamment par la sélection de gènes particuliers codant pour des enzymes de détoxication (Poupardin et al, en préparation). D'une manière globale, cette thèse permettra de mieux comprendre l'impact de l'environnement chimique sur la résistance des moustiques aux insecticides et fournira de nouvelles pistes afin d'optimiser les traitements insecticides utilisés en démoustication. / Mosquitoes have a major impact on public health due to their capacity to transmit human diseases such as viruses (dengue, yellow-fever, west-Nile, chikungunya…) and parasites (malaria, filariasis…). To control them, insecticides have been heavily used since the 1950's leading to the emergence of insecticide resistance. Today, wetlands where mosquito larvae develop are frequently contaminated by environmental xenobiotics (e.g. residual insecticides, agrochemicals, pollutants and plant allelochemicals) and little is known about the impact of these molecules on the capacity of mosquitoes to resist insecticides. The aim of my thesis is to study the response of mosquito larvae to xenobiotic exposures and the impact of these molecules on the tolerance (single generation) and resistance (multiple generations) of mosquitoes to chemical insecticides. A first ‘short term' study revealed that mosquito larvae exposed for few hours to sub-lethal doses of various xenobiotics become more tolerant to several chemical insecticides (Poupardin et al., 2008, Riaz et al., 2009) and that this increased tolerance is linked with an increase of detoxification enzyme activities. Thanks to the “Aedes detox chip” developed in LSTM, we showed that several detoxification genes, especially P450s, were induced by various xenobiotics which could explain the increased tolerance of mosquito larvae to insecticides. In order to better characterize these genes, their transcription profiles were studied at different life stages and in various organs (Poupardin et al., 2010). We demonstrated that several of these P450s are preferentially transcribed in gastric caeca, midgut and malpighian tubules, known to play an important role in xenobiotic metabolism. Moreover, we found that the transcription levels of these genes vary according to life stages. Finally, several genes were induced by environmental doses of xenobiotics with a maximum induction peak at 48-72h after exposure. Overall, these studies evidenced of the potential role of mosquito detoxification genes to respond to xenobiotic exposure and to affect their tolerance to chemical insecticides. The other aim of my thesis was to understand the ‘long term' (across several generations) impact of xenobiotics on the selection of insecticide resistance mechanisms in mosquitoes. In other words, ‘Do pollutants affect the selection of insecticides resistance mechanism by insecticides treatments' and if yes, ‘are particular genes favoured?' To answer these questions, three strains of the mosquito Aedes aegypti were selected with the pyrethroid insecticide permethrin. Before the selection process, larvae were exposed or not to sub-lethal dose of various pollutants. After 11 generations of selection, the three strains showed elevated resistance to permethrin compared to the susceptible strain. To identify the genes differentially transcribed in these resistant strains, we used the new ‘Agilent Aedes chip' representing more than 14,200 transcripts developed by the LSTM. Microarray results showed that the presence pollutants or residual insecticide can affect the selection of insecticide resistance mechanisms by favouring the selection of particular genes such as those encoding for detoxification enzymes (Poupardin et al., in prep). Globally, this research work will provide a better understanding of the impact of environmental factors on insecticide resistances in mosquitoes and will provide new ways to optimize the control of vectors with insecticides.

Résumé trop long / Mosquitoes transmit several human and animal diseases and their control represents a public health challenge worldwide. In most tropical countries, efficient control of mosquitoes relies on the use of chemical insecticides targeting adults or larvae. However, resistance to the four main classes of chemical insecticides has been reported worldwide and threatens vector control programs. In this context, there is an urgent need to find alternatives to conventional insecticides used in vector control. In this thesis, I explored the potential use of the neonicotinoid insecticide imidacloprid for mosquito control, focusing on the identification of metabolic resistance mechanisms, cross-resistance with other insecticides and the impact of environmental pollutants on imidacloprid tolerance. The mosquito Aedes aegypti was used as a model species for this research work. Basal tolerance of Ae. aegypti to imidacloprid was first evaluated at the larval and adult stages. Effects of a larval exposure across a single generation to a sub-lethal dose of imidacloprid were then investigated at the toxicological and molecular levels using transcriptome profiling. Short sub-lethal exposures were also used to identify potential cross-responses between imidacloprid, other chemical insecticides and anthropogenic pollutants. Long-term adaptive response of Ae. aegypti to imidacloprid was then investigated across several generations by selecting an insecticide-susceptible strain (Bora-Bora strain) with imidacloprid at the larval stage for 14 generations in the laboratory. Such artificial selection allowed obtaining the Imida-R strain. This strain showed an increased resistance to imidacloprid in larvae while no significant resistance was measured in adults. Resistance mechanisms were then investigated using various approaches including the use of detoxification enzyme inhibitors, biochemical assays and transcriptome profiling with DNA microarray and massive mRNA sequencing. Several protein families potentially involved in resistance were identified including detoxifications enzymes and cuticle proteins. Among the formers, 8 cytochrome P450s and 1 glutathione S-transferase appears as good candidates for a role in imidacloprid metabolism. The role of P450s in the elevated resistance of the Imida-R strain was confirmed by comparative P450-dependent in vitro metabolism assays conducted on microsomal fractions of the susceptible and Imida-R strains. At the gene level, substrate binding modeling allowed restricting the panel of P450 candidates. Meantime, heterologous expression of one P450 was performed and its ability to metabolize imidacloprid confirmed. Bioassay with other insecticides revealed potential cross-resistance of the Imida-R at the larval stage to other neonicotinoids but also to an insect growth inhibitor and in a lesser extent to DDT, confirming the probable role of detoxification enzymes. Relaxing the selection pressure of the Imida-R strain for few generations led to a rapid decrease of resistance, suggesting a cost of resistance mechanisms. Comparing the inducibility of candidate detoxification genes by imidacloprid in susceptible and resistant strains revealed a higher induction of these genes in the resistant strain, suggesting the selection of both a higher constitutive expression but also a greater phenotypic plasticity of these enzymes in the Imida-R strain. Finally, the potential role of cuticle protein in resistance was preliminary investigated by exposing larvae to a chitin synthesis inhibitor before bioassays. Overall, although this research work requires additional functional validation experiments, these data provide a better understanding of imidacloprid resistance mechanisms in mosquitoes and its potential use as an alternative to conventional insecticides in vector control.

L’afide del pesco Myzus persicae rappresenta uno degli insetti più dannosi in agricoltura. Estremamente polifago e cosmopolita, viene combattuto principalmente con trattamenti insetticidi. Nel corso degli ultimi anni neonicotinoidi e piretroidi hanno rappresentato i componenti principali delle strategie di difesa contro questa specie, ma recenti programmi di monitoraggio condotti in Sud Europa hanno rivelato la presenza di popolazioni resistenti, mettendo in dubbio l’efficacia a lungo termine di queste classi di prodotti. Il presente lavoro prende in esame la diffusione dei principali meccanismi di resistenza agli insetticidi nelle popolazioni di M. persicae presenti sul territorio italiano. La prima parte si concentra sulle resistenze target-site e considera la distribuzione delle principali mutazioni che sono state associate alla resistenza a neonicotinoidi e piretroidi. La seconda parte riguarda le resistenze metaboliche e analizza le principali classi di enzimi associate ad attività di sequestro o detossificazione delle molecole di insetticida. Infine, il progetto si focalizza sulla caratterizzazione delle possibili interazioni tra tali enzimi detossificanti e molecole sinergizzanti quali il ben noto piperonil butossido (PBO). I risultati ottenuti consentiranno di migliorare le strategie di difesa per evitare trattamenti inefficaci e mantenere il più a lungo possibile l’efficacia dei prodotti oggi disponibili per il controllo di M. persicae. / The green peach aphid Myzus persicae is a globally significant crop pest, controlled mainly by chemical treatments. In recent years neonicotinoids and pyrethroids have been the main components of pest management strategies used by growers. However, recent monitoring programmes in Southern Europe have shown the widespread presence of resistant populations, posing a serious threat to the long-term efficacy of these insecticide classes. The present work aims to characterise the main biochemical and molecular mechanisms responsible for insecticide resistance in Italian populations of M. Persicae. The first part is focused on target-site resistance and consider the frequency and distribution of the main target-site mutations associated with neonicotinoid and pyrethroid resistance. The second part is related to metabolic resistance and analyses the involvement of detoxifying enzymes able to sequester or metabolise the insecticide molecules. Furthermore, the project aims to characterise possible interactions between those enzymes and synergistic compounds like the well-known piperonyl butoxide (PBO). Results obtained by this investigation will help to improve insecticide resistance management strategies, in order to avoid ineffective applications and maintain the long-term sustainability of chemical control against M. persicae.