Dissertations / Theses on the topic 'Insecticides; Mosquitoes'

The rotation of the use of chemically unrelated insecticides has been advocated to delay the build up of resistance. To examine this concept in the laboratory, Anopheles albimanus and Culex guinquefasciatus were subjected to two kinds of rotational selection which may be referred to as short term pre-planned rotation and "opportunistic" rotation. No difference was observed between these two methods in term of the time for each resistance level to reach 5Q%. The effectiveness of selection for resistance depends on the protection conferred by resistance genes and this was tested by laboratory releases of the DDT resistant and susceptible homozygotes and heterozygotes of An. gambiae into DDT sprayed and unsprayed miniature huts. All the genotypes were killed with freshly applied DDT but survivors were observed from month 2, 3 and 5 onwards for RR, RS and SS genotypes, respectively. Persistence of DDT on the sprayed wall and roof of the mud hut was studied by bioassays and biochemical assays on scrapings from the wall and roof. Linkage between resistance genes could effect the rotational use of insecticides. No linkage was found between propoxur and dieldrin resistance genes by combining bioassay and biochemical methods. As a preparation for a field studies in Malaysian field collected Aedes aegypti and Culex guinguefasciatus larvae were selected with temephos and Bti. Cx guinguefasciatus responded to temephos selection but Ae. aegypti did not respond to temephos and neither species responded to Bti selection. ~ Caged adults were exposed to thermal fogging in the field. The partially resistant Cx guinguefasciatus strain hardly survived any better than susceptible strains. Resistant and susceptible larvae were exposed to water samples from containers which had been treated with temephos sand granules. 100% mortality was obtained for all the strains up to week 6. Resistant Culex started to survive at week 7 but susceptibles did not do so until week ten. The prospects for the various proposed strategies for resistance management are discussed.

Bacillus sphaericus 2362 is a mosquito pathogenic bacterium. Its greatest industrial potential may be in developing countries where mosquitos are often vectors for diseases. This strain is typical of the species in that it is unable to grow using carbohydrates as a sole source of carbon. The goal of this research was to determine the metabolic deficiency(s) responsible for the inability of this organism to grow on carbohydrates. Compounds that supported light growth of this organism on an agar-solidified, defined medium included acetate, glycerol, and gluconate. Growth in a defined liquid medium with acetate as the source of carbon was much slower than growth in a complex, protein-based broth. B. sphaericus grew poorly in a defined, liquid medium with glycerol or gluconate as the carbon source. Activity of enzymes responsible for the initiation of metabolism of some substrates was not detected in cell extracts. These enzymes were: glucokinase, hexokinase, beta-galactosidase, and amylase. Growth of this bacterium on glycerol as a sole source of carbon implies the presence of the enzymes from the lower half of the Embden-Meyerhof-Parnas (EMP) pathway. Two enzymes of the upper half of the EMF pathway, phosphofructokinase and fructose diphosphate aldolase, were undetected in cell extracts. In addition, glucose dehydrogenase activity was not detected. The inability to form glucose-6-phosphate from glucose prevents the catabolism of this and related substrates via the Entner-Doudoroff (ED), hexose monophosphate (HMP), and EMF pathways. Oxygen uptake studies indicated that B. sphaericus oxidized gluconate slightly but only when the cells were grown in a complex, protein-based medium supplemented with gluconate. Although gluconokinase activity was detected in cell extracts, no activity was detected for the key enzymes of the ED (phosphogluconate dehydratase/KDPG aldolase), or HMP (6-phosphogluconate dehydrogenase) pathway. It is unclear how B. sphaericus grows on a defined medium with gluconate as the sole source of carbon. In addition to enzymatic deficiencies, whole cells were unable to accumulate [¹⁴C]glucose or [¹⁴C]sucrose.
Master of Science

Acetylcholinesterase (AChE) is an essential enzyme with an evolutionary conserved function: to terminate nerve signaling by rapid hydrolysis of the neurotransmitter acetylcholine. AChE is an important target for insecticides. Vector control by the use of insecticide-based interventions is today the main strategy for controlling mosquito-borne diseases that affect millions of people each year. However, the efficiency of many insecticides is challenged by resistant mosquito populations, lack of selectivity and off-target toxicity of currently used compounds. New selective and resistance-breaking insecticides are needed for an efficient vector control also in the future. In the work presented in this thesis, we have combined structural biology, biochemistry and medicinal chemistry to characterize mosquito AChEs and to develop selective and resistance-breaking inhibitors of this essential enzyme from two disease-transmitting mosquitoes.We have identified small but important structural and functional differences between AChE from mosquitoes and AChE from vertebrates. The significance of these differences was emphasized by a high throughput screening campaign, which made it evident that the evolutionary distant AChEs display significant differences in their molecular recognition. These findings were exploited in the design of new inhibitors. Rationally designed and developed thiourea- and phenoxyacetamide-based non-covalent inhibitors displayed high potency on both wild type and insecticide insensitive AChE from mosquitoes. The best inhibitors showed over 100-fold stronger inhibition of mosquito than human AChE, and proved insecticide potential as they killed both adult and larvae mosquitoes.We show that mosquito and human AChE have different molecular recognition and that non-covalent selective inhibition of AChE from mosquitoes is possible. We also demonstrate that inhibitors can combine selectivity with sub-micromolar potency for insecticide resistant AChE.

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.

La résistance des moustiques aux insecticides pyréthrinoïdes (PYRs) menace les programmes de lutte anti-vectorielle à l'échelle mondiale. Chez le moustique Aedes aegypti, vecteur de la dengue et du Chikungunya, les principaux mécanismes causant cette résistance ont été identifiés. La résistance métabolique joue alors un rôle important et consiste en une biodégradation accrue de l'insecticide par des enzymes de détoxication. Néanmoins, les bases moléculaires de ce mécanisme restent méconnues. La plupart des gènes impliqués dans la résistance métabolique aux PYRs ont été identifiés par des approches transcriptomiques, mais les modifications génomiques à l'origine de leur sur-expression dans les populations résistantes ainsi que les modifications structurales des enzymes en lien avec la résistance restent méconnues. Cette thèse vise alors à utiliser les nouvelles approches de séquençage à haut débit (NGS) pour caractériser les mécanismes moléculaires de la résistance aux PYRs chez le moustique Ae. Aegypti. La première partie de la thèse présente une étude pilote RNA-seq menée sur des populations de laboratoire sélectionnées avec des insecticides. Cette étude a pour objectif d'évaluer les avantages des NGS pour l'étude des mécanismes de résistance chez les moustiques. Le rôle des enzymes de détoxication dans la résistance a ainsi été clairement confirmé. Plusieurs gènes codant pour ces enzymes apparaissent sur-exprimés dans les populations résistantes et un important regroupement de P450 montre une forte empreinte de sélection en lien avec la résistance aux PYRs. La seconde partie de la thèse présente une étude sur des populations naturelles échantillonnées sur divers continents. Cette étude combine les technologies d'enrichissement génomique et de DNA-seq afin d'étudier les variations génomiques liées à la résistance au PYR Deltaméthrine. La comparaison de la couverture de séquençage entre populations résistantes et sensibles a permis d'identifier des variations de nombre de copies (CNVs) de certains gènes de détoxication associées à la résistance à la Deltaméthrine. Des mutations non-synonymes fortement liées au phénotype de résistance ont également été mises en évidence. La comparaison de ces marqueurs de la résistance entre les différentes populations a révélé que les gènes/mutations associés à la résistance à la Deltaméthrine sont peu conservés entre continents, probablement à cause des différences de fond génétique des populations, de leur histoire démographique et des pressions de sélections. La troisième partie de la thèse décrit une étude par RNA-seq portant sur les mêmes populations naturelles, visant à croiser des données de transcriptomique (expression des gènes et polymorphisme des transcrits) avec les données génomiques générées par l'étude précédente. Plusieurs enzymes de détoxications ont été retrouvées sur-exprimées chez les populations résistantes en lien avec les CNVs précédemment identifiées. Des centaines de variations de polymorphisme ont été identifiées par DNA-seq dans les zones cis-promotrices des différents gènes étudiés. Parmi ces variations, plusieurs apparaissent associées à la sur-régulation d'enzymes de détoxication. Enfin, la comparaison des données de polymorphismes obtenues par DNA-seq et RNA-seq a permis d'étudier les phénomènes d'expression d'allèles spécifiques en lien avec la résistance. Cette étude confirme l'intérêt de croiser des données de transcriptomique et de génomique pour caractériser les bases moléculaires de la résistance aux insecticides. D'un point de vue général, cette thèse permet de mieux appréhender les mécanismes de résistance du moustique Ae. aegypti aux PYRs mais aussi d'identifier de nouveaux marqueurs de la résistance potentiellement utilisables pour développer de nouveaux outils moléculaires diagnostiques de la résistance sur le terrain. Ce travail met également en avant les apports des NGS pour l'étude fine des bases moléculaires de l'adaptation d'organismes modèles
Mosquito control programs worldwide are increasingly threatened by resistance to pyrethroid insecticides (PYRs). In the dengue and chikungunya vector Aedes aegypti, the key resistance mechanisms include modifications in the protein targeted by insecticides (target-site mutations) and metabolic resistance, consisting in an increased insecticide biodegradation by so called detoxification enzymes. However, as opposed to target-site mutations, the molecular basis of metabolic resistance remains poorly understood. Most metabolic resistance genes have been detected by transcriptomic approaches based on their over-expressed in resistant populations, but genomic changes leading to these expression changes as well as structural changes in enzymes potentially involved in resistance remain unknown. In this context, this thesis aims at using next-generation sequencing approaches for characterizing PYR resistance mechanisms in the mosquito Ae. aegypti.The first chapter of this thesis describes a pilot study on laboratory insecticide-selected populations of Ae. aegypti. This study aims at investigating the benefits of next-generation sequencing for studying resistance mechanisms in mosquitoes. This study confirmed that detoxification enzymes play a key role in resistance, with several of them being over-expressed in resistant populations and a large cluster of cytochrome P450 genes showing a selection imprint associated with resistance to PYRs.The second chapter of this thesis describes a study conducted on natural mosquito populations from various continents. Combining genomic target enrichment (targeting about 800 genes potentially involved in resistance) and DNA-seq allowed unravelling genomic changes associated with resistance to the PYR deltamethrin. Comparing normalized sequencing coverage between resistant and susceptible populations identified significant copy number variations (CNVs) in several detoxification genes strongly associated to deltamethrin resistance. Non-synonymous mutations affecting detoxification enzymes associated to the resistance phenotype were also detected. Comparing resistance markers between populations from various continents revealed that genes/mutations associated with deltamethrin resistance are poorly conserved across continents, probably due to differences in the genetic background of populations but also differences in terms of demographic history and selection pressures.The third chapter describes an RNA-seq study performed on the same natural mosquito populations in order to cross-link transcriptomic data (gene expression and transcript polymorphism) with genomic data obtained from the previous study. Multiple detoxification enzymes were found over-transcribed in resistant populations linked with previously identified CNVs. Hundreds polymorphism variations were identified by targeted DNA-seq in cis-promoter regions of detoxification genes. Among them, several were associated with the upper-regulation of detoxification enzymes in resistant populations. Finally, cross-comparing polymorphism data obtain from DNA-seq and RNA-seq allowed investigating allele specific expression (ASE) events related to PYR resistance. Overall, this study confirmed the benefits of combining transcriptomic and genomic NGS approaches for studying the molecular basis of insecticide resistance.As a whole, this thesis not only contributed to better understand PYR resistance mechanisms in the dengue vector Ae. aegypti but also identified novel genomic markers of resistance opening the way for developing new molecular diagnostic to early detect and monitor resistance mechanisms in the field. This work also highlights the benefits of using NGS technologies for unravelling the molecular bases of adaptation in model organisms

The emergence and spread of insecticide resistance compromises the control of mosquito borne diseases that are responsible for millions of deaths every year in tropical and subtropical areas. Mathematical modelling is a valuable tool that can be used to explore different aspects of the development and management of insecticide resistance. We have used standard population genetics theory and ecological modelling techniques for developing models to evaluate the spread of resistance in the field. We started by developing a methodology to quantify the strength of selection for resistance occurring in nature. We used data from Mexico on the mosquito Aedes aegypti and a maximum likelihood methodology to estimate the selection and dominance coefficients driving the evolution of resistance in the field. We additionally explored the impact of poor data collection, data that combine information from different locations, and the consequences of selection and dominance coefficients varying over the sampling time period. This analysis highlighted factors highly relevant to field work such as the need for frequent surveillance in discrete sentinel sites. The use of insecticidal bed nets represents the primary tool for the prevention of malaria worldwide. It is of extreme importance to maintain their efficacy against mosquitoes, which has been undermined by the development of insecticide resistance. We assed the contribution of a novel design of bed nets in delaying insecticide resistance while at the same time determining the important parameters in driving resistance in an heterogeneous environment. We showed that this new bed net can indeed contribute to the delay of the spread of resistance, but surprisingly could have the reverse effect in specific circumstances. Finally we developed a model for the vector of malaria, that considers the stage-structured nature of the mosquito life cycle and, most importantly, explicitly incorporates insecticide resistance. It can be used to understand the population dynamics of mosquitoes throughout their entire lifecycle while analysing the impact of vector control interventions, alone and in combination, and the spread of insecticide resistance that those interventions induce. We showed that targeting the larval stages has the greatest effect on the adult population followed by targeting non host-seeking female adults. According to our results, low levels of resistance can induce failure of interventions, and the rate of spread of resistance is faster when insecticides target the larval stages.

Culex mosquitoes, as well as being vectors of filariasis and Japanese encephalitis, are a world wide biting nuisance. Organophosphorus insecticides (OPs) have been widely used to control Culex populations. Resistance to OPs has occurred and is typically mediated by the increase in non-specific esterase activity. The two esterases involved are classified as 'A' and 'B' esterases with respect to their preference for the substrates α- or β- naphthyl acetate. The commonest phenotype involves two elevated esterases, A2 and B2, which occur in complete linkage disequilibrium. The over expression of esterase B1 is due to gene amplification. Initially, in order to further study the molecular biology of OP resistance, full length cDNAs coding for both A2 and B2 esterases were isolated and sequenced from an OP resistant Sri Lankan strain of Culex quinquefasciatus, PelRR. The B2 esterase cDNA was isolated with PCR using primers sharing homology with the B1 esterase cDNA and has 97.4% homology with esterase B1 at the amino acid level. This confirmed that the B esterases belong to an allelic series. Partial genomic sequences of B2 esterase from PelRR and four other OP resistant Culex strains were identical. This suggests that the initial B2 esterase amplification has occurred only once. However, the cDNA sequence of a B1 esterase cDNA isolated from an OP resistant Cuban strain of Culex quinquefasciatus, MRES, was different to that of the previously published B1 esterase gene sequence. At the genomic level, the haplotype of the Cuban B1 esterase gene, based on EcoRI endonuclease analysis, was also different, suggesting that the initial B1 esterase gene amplification event has occurred at least twice. AB esterase cDNA from an OP susceptible strain, PelSS, has also been partially sequenced. PelSS was derived from the same origins as PelRR but its B esterase cDNA sequence and haplotype of the gene are different. Thus, the B2 esterase gene conferring OP resistance, as well as being amplified, is only found in the resistant strain, PelRR. The A2 esterase cDNA was isolated by screening a PelRR cDNA expression library with an anti-A2 antiserum. The cDNA coded for a protein of 540 amino acids (the same as B2 esterase) and shared 47% amino acid homology with B2 esterase. This strongly suggests that the two genes arose from a duplication of an ancestral counterpart. Furthermore, screening of a PelRR genomic library with A2 and B2 esterase gene probes suggests that the two esterase genes, A2 and B2 are situated in tandem within the genome. PCR was used to amplify the coding region of the PelRR A2 esterase cDNA and this was co-transfected into the baculovirus expression system. The recombinant virus expressed an active A esterase.

The development of pyrethroid resistance in mosquitoes threatens the goal of malaria elimination in Africa. Alternative insecticides, e.g. organophosphates, can be considered to control pyrethroid resistant mosquitoes. The problem associated with the deployment of organophosphate-based insecticides is their high volatility. Conventional application forms have a fairly short residual efficacy. This study aimed at extending the residual efficacy of an organophosphate insecticide by using a polymer matrix as a slow release device. A multilayer film blower was used to produce a trilayer film. The middle layer comprised poly(ethylene-co-vinyl acetate), i.e. EVA polymer, impregnated with malathion. This was sandwiched by two low density polyethylene (LDPE) outer layers. These acted as semi-permeable membrane-like barriers that slowed down the release of the contact insecticide to the surfaces of the film. In theory, such a film could be deployed as a long-lasting insecticide-treated wall lining in pyrethroid resistant settings. Scanning electron microscopy (SEM) confirmed the trilayer film structure of the blown film. The malathion release from the film was tracked with Fourier transform infrared spectroscopy (FTIR). The malathion absorption band in the FTIR spectra disappeared gradually over time. Confocal Raman analysis showed a malathion concentration gradient across the thickness of the polyethylene layers. These results suggested diffusion-controlled transport through the LDPE membranes. Bioassays indicated that the residual efficacy of the malathion, against mosquitoes, was increased to about six months. This means that trilayer films, impregnated with an organophosphate, may have potential as alternative mosquito control interventions in pyrethroid resistant settings.
Dissertation (MEng)--University of Pretoria, 2017.
Chemical Engineering
MEng
Unrestricted

One of the most effective ways of managing adult mosquitoes that vector human and animal pathogens is the use of ultra-low-volume (ULV) insecticides. Due to a lack of studies examining the environmental fate of ULV insecticides and because previous risk assessments have shown that environmental concentrations of insecticides contributed the largest amount of variance to the estimated total exposure, I measured deposition onto surfaces and air concentrations of permethrin and naled. I also conducted risk assessments for human and other non-target organisms using the values I measured. Deposition concentrations of permethrin and naled generally decreased as distance from the spray source increased. Overall, approximately 3.2% of the permethrin and 15% of the naled applied deposited on the ground within 75 m from the spray source 1 h after application. Concentrations of permethrin and naled 12 h after application were not significantly different than concentrations 1 h after application. The results of my probabilistic human-health risk assessment using actual environmental concentrations showed that previous risk assessments overestimated risks. Thus they were conservative in protecting human health. The non-target risk assessment and field bioassay using the house cricket, Acheta domesticus (L.), as a surrogate for medium- to large-bodied ground dwelling insects showed that ULV applications most likely would not result in impacts on populations. I also measured actual environmental concentrations of pyrethrins and piperonyl butoxide (PBO) after aerial ULV applications. Pyrethrins were not detected in the water or on deposition pads. However PBO was detected in the water and on deposition samples, but concentrations rapidly decreased to background levels by 36 h after application. The estimated risks of pyrethrins and PBO to aquatic surrogates were lower than those estimated by previous ecological risk assessments. My study is the first to relate actual environmental concentrations of ULV insecticides to estimates of human-health risks. Results of my environmental fate studies, human-health and non-target risk assessments, and the current weight of scientific evidence, demonstrate that the risks to humans and the environment after ULV applications of insecticides most likely are below regulatory levels of concern.

Culex mosquitoes are known for carrying several harmful viruses in the United States. Culex tarsalis is found in rural as well as some residential areas in the Western United States, so they are under insecticide pressure from both agricultural spraying and vector control. In response to insecticide pressure, mosquitoes can evolve two primary resistance mechanisms: target site insensitivity, as a result of DNA mutation, and elevated levels of detoxifying enzymes (GST, alpha and beta esterases, and P450 oxidases). The two types of target site insensitivity studied here in Cx. tarsalis are kdr, which is a mutation in the para-type voltage gated sodium channel and ace-1, which is a mutation in acetylcholinesterase gene. This study focused on a population of Cx. tarsalis in Sutter County, where insecticide use shifted from sumithrin to Naled over the course of the summer. The goal of this study was to determine if there was resistance to insecticides and characterize the mechanisms of resistance. Mosquitoes were separated into resistance levels based on CDC bottle bioassay results using Naled, sumithrin, and permethrin insecticides. Mosquitoes were used to test for elevated levels of detoxifying enzymes and genetic qPCR testing for either kdr and ace-1 mutations. Bottle bioassay results suggest Cx. tarsalis populations from Sutter County are mostly resistant to pyrethroids while not being resistant to organophosphates. Enzymatic assays suggest high concentrations or activities of detoxifying enzymes are commonly seen in resistant individuals, occasionally elevated levels of multiple enzymes within an individual. The ace-1 mutation was seen in a single susceptible individual (0.036%). Either one or two kdr alleles were present in every single semi-resistant or resistant mosquito tested.

Malaria is a vector-borne disease that presents the most persistent and serious public health burden in Liberia. Numerous studies have examined the relationship between ITN use and malaria prevalence. However, little research has explored the effectiveness of ITNs in controlling malaria among children in postwar Liberia. The aim of this study was to examine the association between ITN ownership, parental economic status, ITN installation support, and malaria prevalence among children. This was a quantitative cross-sectional study guided by the health belief model. The study used secondary data from the 2011 Liberia Malaria Indicator Survey. Chi-square for association and Logistic regression were used to analyze the data. The results revealed a significant association between parental education and malaria prevalence. There was also a significant association between parental economic status and malaria prevalence. However, there was no significant association between ITN ownership and malaria prevalence after controlling for parental education and ownership of structure. These findings may foster social change by helping public health authorities in Liberia integrate ITN use with other strategies like mosquito larvae elimination and indoor/outdoor insecticide spraying as part of a comprehensive approach to malaria control. Additionally, massive awareness and economic capacity building should be undertaken to empower malaria endemic communities with the understanding that malaria can be rapidly reduced with other robust strategies in combination with ITN use. These strategies, if implemented, may effectively control malaria prevalence among children and the emotional and financial burdens endure by their families.

L'évolution de la résistance aux insecticides chez les moustiques responsables de la transmission de maladies infectieuses compromet notre capacité à contrôler ces populations de vecteurs et pose de graves problèmes de santé publique. Mais les nombreuses modifications physiologiques associées au phénomène de résistance aux insecticides pourraient altérer l'épidémiologie de ces maladies de manière plus indirecte en modifiant la capacité vectorielle des moustiques. Afin d'étudier cette question nous avons mis en place un nouveau système expérimental composé du parasite aviaire Plasmodium relictum SGS1 et de son vecteur naturel le moustique Culex pipiens. Nous avons étudié l'effet de différents allèles de résistance aux insecticides (représentant deux mécanismes principaux i.e. la résistance métabolique ou la modification de la cible) sur une série de traits d'histoire de vie du parasite et du moustique. L'impact de ces différents allèles a été étudié d'une part, dans les conditions contrôlées de leur expression dans un même fond génétique (en utilisant plusieurs souches de moustiques isogéniques), et d'autre part, dans les conditions plus réalistes de leur expression dans un fond génétique hétérogène (utilisation de moustiques échantillonnés sur le terrain). Nous montrons que la résistance aux insecticides a des effets pleïotropes sur l'immunocompétence et les traits d'histoire de vie des moustiques. Son effet sur le développement de Plasmodium semble en revanche limité. Nous discutons d'une part, de la nécessité de poursuivre une approche multifactorielle (impliquant la physiologie, l'immunité et le comportement des moustiques) afin de mieux comprendre l'impact de la résistance aux insecticides sur la transmission de Plasmodium, et d'autre part des perspectives intéressantes qu'offrent ce nouveau système expérimental pour l'étude de l'écologie évolutive des maladies à vecteurs
The evolution of insecticide resistance in mosquitoes threatens our ability to control many-vector-transmitted diseases, thereby raising serious public health issues. Insecticide resistance entails numerous physiological changes in mosquitoes. This thesis investigates whether these physiological changes alter the quality of mosquitoes as vectors of malaria. To address this issue, we developed a new experimental system consisting in the avian malaria parasite Plasmodium relictum SGS1 and its natural vector, the mosquito Culex pipiens. We investigated the impact of two insecticide resistance mechanisms (target site resistance and metabolic resistance) on several mosquito and parasite life history traits relevant for malaria transmission. The effect of different insecticide resistant genes was investigated using both isogenic laboratory mosquito strains (i.e. against a homogeneous genetic background) and sympatric field caught mosquitoes (i.e. under the more realistic, albeit noisier, conditions of a heterogeneous genetic background). We show that insecticide resistance has a pleiotropic effect on several mosquito traits (immunocompetence, longevity, fecundity), whereas it has only a limited effect on Plasmodium development. We discuss, on the one hand, the need to pursue such a multi-factorial approach (combining the mosquito physiology, immunity and behavior) to better understand the impact of insecticide resistance on malaria transmission and, on the other hand, the promising perspectives offered by this new experimental system for studying the evolutionary-ecology of infectious diseases

Malaria is vectored by the mosquito Anopheles gambiae (Ag) in Sub-Saharan Africa and infects approximately 500 million people annually. The increasing prevalence of pyrethroid-resistant mosquitoes has amplified the need for development of new, selective mosquitocides for use on insecticide-treated nets.

We have developed several phenyl-substituted N-methylcarbamates producing a high degree of selectivity for Anopheles gambiae acetylcholinesterase (AgAChE) over human AChE. Molecular models suggest alternate conformations (flexibility) of W84 and W431 (Ag numbering) at the hydrophobic subpocket of the AgAChE active site and poor flexibility within human AChE, allowing for the high selectivity of our novel carbamates. Initial selectivity data was obtained through screening of these insecticides while using ethanol as a solvent. Re-screening of these carbamates in the presence of 0.1% DMSO (v/v) resulted in antagonism of inhibition for AgAChE, thus reducing the AgAChE-selectivity by at least 10-fold. However, the presence of 0.1% DMSO did not antagonize the inhibition of human, Drosophila melanogaster, or Musca domestica AChE. Non-selective carbamates also displayed no solvent-dependent antagonism of inhibition in any species studied, including AgAChE.

Molecular models provide an explanation for antagonism of inhibition when DMSO is present. I, and collaborators, propose that W84 and W431 in AgAChE comprise an allosteric pocket that is stabilized by DMSO and is responsible for the solvent-dependent antagonism of inhibition observed with AgAChE.
Master of Science in Life Sciences

Insecticide resistance in disease-transmitting arthropods has become a serious hindrance for successful vector control. Mosquitoes, in particular, are notorious vectors of potentially deadly diseases like malaria, dengue fever, and West Nile virus. Anopheles gambiae and Culex quinquefasciatus are just two examples of mosquito vectors that possess genetic mutations (denoted kdr and ace-1 ) and/or enhanced detoxifying enzymes (oxidases, esterases, and glutathione-s-transferases) that confer insecticide resistance. Culex tarsalis, a primary vector for West Nile virus among other arboviruses in Northern California, is a target for insecticide application and is under constant insecticide pressure, making it likely to adapt resistance mechanisms like kdr or ace-1 or increased detoxifying enzymes. Culex tarsalis adult females were collected from Yolo and Sutter counties. A bottle bioassay was completed to determine prevalence of resistance to Sumithrin (a pyrethroid; N=217) and Naled (an organophosphate; N=154). A susceptible lab-reared colony was used for comparison. Microplate assays were completed to investigate elevated levels of detoxification enzymes present as well as AChE. PCR was used to amplify the VGSC and ace-1 genes. Amplicons were sequenced and aligned to determine if mutations were present. No evidence of the ace-1 mutation was found in any mosquitoes, but the kdr mutation was seen in all semi-resistant and resistant individuals exposed to Sumithrin. Microplate data revealed significant differences between certain detoxifying enzymes within mosquitoes collected from Sutter and Yolo Counties exposed to both Sumithrin and Naled. The data obtained from this study suggests that resistance to Sumithrin in both populations is carried out by both metabolic and target site insensitivity, while resistance to Naled is caused by metabolic resistance only.

The escalation of Long-Lasting Insecticidal Nets (LLINs) mass distribution marked the beginning of a period of malaria decline in sub-Saharan Africa. However, the emergence and spread of insecticide resistance in malaria mosquitoes is a threat to the effectiveness and sustainability of this vector control method. In this context, it is necessary to design and evaluate new compounds and methods that attenuate or even reverse the insecticide resistance trend. Olyset Duo is a novel LLIN that combines the insecticide properties of permethrin and the chemosterilising effect of pyriproxyfen (PPF). The rationale is that resistant mosquitoes that survive the contact with the net would not be able to transfer the resistance genes to the offspring, eventually influencing the resistant phenotype of the mosquito population. In the first part of this study the sterilising and sub-lethal effect of PPF and Olyset Duo was evaluated by a range of bioassays with laboratory and wild mosquito populations. PPF significantly affected the longevity, oogenesis, oviposition and hatching rate of susceptible and resistant mosquitoes, although the effect was partially diminished on the resistant colonies. The possible reasons and implications of PPF performance under controlled conditions are discussed. The second part of this thesis was done within the context of an Olyset Duo Randomised Controlled Trial (RCT) set in Banfora District, Burkina Faso. The RCT had a stepped-wedge design which ensured that Olyset Duo nets gradually replaced Olyset nets in randomly allocated cluster of villages until the Olyset Duo coverage was total. Wild mosquitoes collected in sentinel sites with Olyset Duo showed evident signs of reproductive impairment even after 1 year of deployment. Insecticide resistance strength was monitored during the RCT in several sentinel sites, and time-response data showed an overall reduction of permethrin resistance strength after the distribution of Olyset Duo. This is the most detailed study on the effect of Olyset Duo on key entomological factors of wild mosquito populations. The standardised protocols as well as the dataset obtained are valuable information for ongoing evaluation of Olyset Duo and PPF as a tool for controlling malaria mosquitoes and as a potential alternative for insecticide resistance management.

The aim of this study is to investigate the role of physico-chemical environmental factors present in mosquito breeding ecologies as sources of selection pressure for the emergence and development of insecticides resistance in Anopheles gambiae from Northern Nigeria. Prospecting for, and sampling of An. gambiae larvae was carried out from many breeding sites in towns and villages spread out across Kano and Jigawa states of Northern Nigeria. The breeding sites visited and sampled were grouped into three study zones according to the type of human related activities taking place around the mosquito breeding environments. Larval densities (per litre of breeding water) and water chemistry analysis were carried out from all the breeding sites visited. Detoxification enzymes; Cytochrome P4S0, Glutathione S-transferases and Esterases as well as glutathione assays were carried out on the three life stages of the sampled An. gambiae using the relevant assay protocols. The results showed that the levels of the physical environmental factors; pH, temperature, conductivity, transparency, dissolved oxygen and biological oxygen demand did not vary significantly (p<0.05) across the three studied zones. The levels of the chemical environmental factors; total dissolved solids, sulphate, phosphate, nitrite and nitrate ions were significantly (p0.05) higher in study zone A compare to the other two zones, while carbon content and oil and grease were significantly (p<0.05) distributed in study C than the other two zones. Likewise, the activity of P4S0s was higher in study zone C while those of GST and CEs were significantly (p