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.

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.

In the Amazonian region occurs a variation in the malaria incidence, which is related to the pluviometric variation annual. The mathematical model proposed here considers this seasonality and different treatment intensities accessible to the infected people. The numerical evidence the seasonal fluctuation and the relationship between the environment temperature and treatment efficiency, showing that the temperature increase strongly affects the extrinsic latent period,reducing the healthy care efficiency. Because malaria treatment already exists it should be import. For another hand, even the investment in treatment is an efficient form to block the epidemy, it is not always sufficient, because the protozoan has been more resistent to the medicine; then scientists are creating transgenic mosquitoes refractory to malaria to couple with wild one, generating descending transgenic. To avaliate this situation, we consider here a mathematical model that describes the relatioship between these populations. Then, we formulate and solve an optimal control problem indicating how the transgenic mosquitoes should be introduced in the environment. The numerical simulations show the effectiveness of the control. / Na Amazônia ocorre uma variação na incidência de malária que está intimamente relacionada à variação pluviométrica ao longo do ano. O modelo matemático aqui proposto considera esta sazonalidade e diferentes intensidades de tratamento acessíveis às pessoas infectadas. Experimentos numéricos descrevem a flutuação sazonal e evidenciam uma relação inversa entre a temperatura e eficiência do tratamento, mostrando que um aumento na temperatura afeta fortemente o período latente extrínseco, reduzindo a eficiência do investimento em saúde. Como o tratamento para os infectados existe, é importante concentrar esforços nesse sentido para obter sucesso no controle da malária. Por outro lado, embora o investimento em tratamento seja uma forma eficaz de impedir a epidemia, isso nem sempre é suficiente, pois é fato que o protozoário tem se mostrado cada vez mais resistente aos medicamentos; por esse motivo, cientistas estão criando mosquitos transgênicos refratários à malária que devem acasalar com os mosquitos selvagens, gerando descendência transgência. Para avaliar esta situação, consideramos neste trabalho um modelo matemático que descreve de maneira simplificada a relação entre estas duas populações. A partir desse modelo, formulamos e resolvemos um problema de controle ótimo indicando uma forma adequada de introduzir esses mosquitos transgênicos. Experimentos numéricos mostram a eficácia do controle adotado.

Mosquitoes are found in extremely large numbers in the lower parts of the River Dalälven. In the year 2000 the mosquito nuisance was especially high, resulting in foundation of the Biological mosquito control project. Since 2001 mosquito larvae are controlled by using a biological pesticide BTI (Bacillus thuringiensis ssp israelensis). The mosquito fauna in the area is dominated by flood water mosquitoes, a group of mosquitoes that are very aggressive and form new generations of mosquitoes during every single flooding event during the summer. To be able to efficiently control the mosquitoes it is essential to know the extension and locality of the flooding. A flooding event is evaluated by how high the water level will reach and at which time the top of the hydrograph is accruing. There is a need for a tool for short time prediction of flooding events in the Nedre Dalälven region. In this study a hydraulic model in the software HEC-RAS has been used, here called HEC-RAS NEDA. The hydraulic model came up with good results when predicting water surface levels. The validation process made from figures of a period with large amplitude in water levels showed that the modelled water level was within ± 0,05 m from the observed water stands. It is a stable model that can handle 100-year flows with quick changes in the flow. The correspondence is good concerning the water levels and it functions well when studying the top of the hydrograph. HEC-RAS NEDA is an accurate and easy to manage tool for prediction of flooding in the lake of Bysjön, Österviken and Färnebofjärden. Modelled results have been compared with the model made by Swedish meteorological and hydrological institute on the request of Räddningsverket. The results from the two models shows large discrepancies, probably because of low accuracy data entered into the model from Räddningsverket. Räddningsverket has used the Geographical Sweden Data height data bank´and I used the height data from a laser scanned digital terrain model. Area studies have also been made according to how the area of the water surface changes with the water stand. These studies show that during the flood in year 2000, 55 km2 was flooded based on laserscanning in Färnebofjärden. / Mygg förekommer i extremt stora antal i området kring nedre delen av Dalälven. År 2000 var myggplågan särskilt stor vilket medförde att projekt Biologisk Myggkontroll bildades. Myggbekämpning har bedrivits i Nedre Dalälven sedan 2001 med ett biologiskt bekämpningsmedel, Bacillus thuringiensis ssp israelensis (BTI). Områdets myggfauna domineras av så kallade översvämningsmygg, en grupp stickmyggor som är mycket aggressiva och bildar nya generationer efter varje enskild översvämning under sommaren. För en effektiv bekämpning av mygglarverna krävs att man vet översvämningens omfattning och lokalisering. En översvämning bedöms med avseende på hur högt vattenståndet når samt vid vilken tidpunkt hydrografens toppvärde inträffar. Det finns ett behov av ett verktyg som kan utföra korttidsprognoser av översvämningar i Nedre Dalälven. I denna studie har en hydraulisk modell i programvaran HEC-RAS använts, här kallad för HEC-RAS NEDA. Den hydrauliska modellen fungerar bra till att användas för prediktering av vattenstånd i Nedre Dalälven. Valideringen av modellen mot värden under en testperiod med stor amplitud i vattenståndet visade att modellerade vattenstånd var inom ± 0,05 m från de uppmätta värdena. Modellen är stabil och klarar av att hantera 100-årsflöden med snabba flödesfluktuationer. HEC-RAS NEDA är ett noggrant och lätthanterligt predikteringsverktyg för översvämningar i Bysjön, Österviken och Färnebofjärden. Överensstämmelsen är god gällande nivån av vattenståndet och den fungerar bra för att studera hydrografens topp temporärt. Modellerade resultat har jämförts med karteringar som Sveriges meteorologiska och hydrologiska institut har gjort på Räddningsverkets begäran. Resultaten skiljer sig betydligt mellan HEC-RAS NEDA och Räddningsverkets modell, troligen på grund av att Räddningsverket har stora svagheter i indata till modellen. Räddningsverket har använt Geografiska Sverige Data-Höjddata och modellen HEC-RAS NEDA är framtagen med höjddata från en digital terrängmodell baserad på laserscanning. Areella studier i GIS över hur arean av vattenspegeln förändras med vattenståndet visar att Färnebofjärden under augusti år 2000 lade 55 km2 under vatten.

Submitted by Cláudia Bueno (claudiamoura18@gmail.com) on 2016-01-20T16:01:25Z No. of bitstreams: 2 Dissertação - Luaciana Silva Lobo - 2011.pdf: 1289874 bytes, checksum: 72f8fd364b738135e2d30a5d5322d572 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-01-21T06:45:14Z (GMT) No. of bitstreams: 2 Dissertação - Luaciana Silva Lobo - 2011.pdf: 1289874 bytes, checksum: 72f8fd364b738135e2d30a5d5322d572 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2016-01-21T06:45:14Z (GMT). No. of bitstreams: 2 Dissertação - Luaciana Silva Lobo - 2011.pdf: 1289874 bytes, checksum: 72f8fd364b738135e2d30a5d5322d572 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2011-11-16 / Aedes aegypti is the principal vector of dengue virus in the tropics. Metropolitan Goiânia had the highest incidence of this arbovirose in Central Brazil between 2010 and 2011. Improved integrated control strategies are necessary in order to face vector resistance to chemicals. The entomopathogenic fungus Metarhizium anisopliae is effective against A. aegypti eggs, larvae and adults under laboratory conditions. There is still little knowledge on specific formulation and application techniques to control this mosquito with this and other fungi. The present study reports on the impact of substrates, previously treated or not with vegetable oil, diatomaceous earth and/or M. anisopliae IP 46 conidia on oviposition and survival of A. aegypti under laboratory conditions. In addition, the study presents results on field tests carried out in the city of Goiânia with adapted ovitraps. Laboratory reared adult A. aegypti females and males were exposed in cages to oviposition devices equipped with water, mud or filter paper. Moistened or dry filter paper treated with additives (vegetable oil Graxol® or diatomaceous earth KeepDry®) and/or M. anisopliae s. l. IP 46 conidia were tested as mono or poly-optional assays. Mortality and quantitative oviposition were assessed up to 15 days of exposure and eclosion of larvae after submersion of eggs in water up to 10 days. Field tests were run with adapted ovitraps treated with conidia of IP 46 formulated with water or oil-water. Females never laid eggs on dry filter paper and few eggs on water. Quantitative oviposition decreased at higher oil concentrations and gravid females laid eggs on fungus-treated filter paper regardless of the conidial concentration (poly-optional tests). Adult mortality reached 42.1% at a 15 day incubation when testing filter paper treated with oil-in-water conidia and this formulation reduced larval eclosion (mono-opcional tests). Under field conditions, eggs were detected on filter paper treated or not with water or oil-in-water formulated conidia. Gravid females detected gradient of humidity and oil but they were not influenced by the presence of the conidia. Toxicity of vegetable oil and virulence of IP 46 increased with time of exposure. M. anisopliae applied in a simulated breeding device and formulated in oil showed to be promising against A. aegypti under laboratory and Aedes sp. under field conditions. / Aedes aegypti é o principal vetor do vírus da dengue nos trópicos. A cidade de Goiânia teve a maior incidência dessa arbovirose no Centro-Oeste do Brasil entre 2010 e 2011. Melhores estratégias de controle integrado são necessárias para enfrentar a propagação da resistência desse vetor a produtos químicos. O fungo entomopatogênico Metarhizium anisopliae é efetivo contra ovos, larvas e adultos de A. aegypti em condições de laboratório. Entretanto, há pouco conhecimento sobre formulações e técnicas de aplicação específicas para controlar esse mosquito com esse e outros fungos. O presente estudo reporta o impacto de substratos previamente tratados ou não com óleo vegetal, terra diatomácea e/ou conídios de M. anisopliae s. l. IP 46 sobre a oviposição e sobrevivência de A. aegypti em condições de laboratório e apresenta resultados sobre testes de campo realizados na cidade de Goiânia em 2010 e 2011 com ovitrampas adaptadas. A. aegypti adultos colocados em laboratório foram expostos em gaiolas e expostos a dispositivos para oviposição equipados com água, lama ou papel filtro. Papel filtro úmido ou seco tratado com aditivos (óleo vegetal Graxol® ou terra diatomácea KeepDry®) e/ou conídios de IP 46 foram testados em ensaios de escolha poli ou mono-opcional. A mortalidade e oviposição quantitativa foram avaliadas até 15 dias de exposição e eclosão de larvas após submersão de ovos em água, até 10 dias. Os testes de campo foram executados com ovitrampas tratadas com conídios formulados em água ou óleo-água. Fêmeas ovipuseram mais sobre lama e papel filtro úmido, pouco sobre água e não ovipuseram sobre papel filtro seco. A oviposição quantitativa decresceu em concentrações mais elevadas do óleo, e fêmeas grávidas ovipuserm sobre papéis filtro tratados independente da concentração de conídios (testes poli-opcionais). A mortalidade de adultos alcançou até 42,1% em 15 dias de incubação ao testar papel filtro tratado com IP 46 formulado em óleo-água, e esse formulado reduziu a eclosão larval (testes mono-opcionais). Em condições de campo, ovos foram encontrados sobre papel filtro tratado ou não com conídios formulados em água ou óleo-água. Fêmeas grávidas de A. aegypti detectaram um gradiente de umidade e de óleo, mas não foram influenciadas pela presença de conídios. A toxicidade do óleo vegetal e a virulência de IP 46 aumentaram com o tempo de exposição. M. anisopliae formulado em óleo e aplicado em criadouro simulado mostrou ser promissor contra A. aegypti em condições de laboratório e contra e Aedes sp. em condições de campo.

Made available in DSpace on 2015-04-20T12:31:08Z (GMT). No. of bitstreams: 1 eleilza.pdf: 788700 bytes, checksum: 7900cf61a2a7a4d6a517f73e27656735 (MD5) Previous issue date: 2006-04-03 / Larvicidal activity of B. sphaericus from several places in Amazonia, was estimated to Anopheles darlingi and Culex quinquefasciatus. Pobit analysis was used to determine the median lethal concentration (LC50) and potency relative to the 2362 standard strain. Findings pointed out strains IB15 (LC50 = 0.040 ppm), IB19 and S1116 (LC50 = 0.048 ppm), IB16 (LC50 = 0.052 ppm) and S265 (LC50 = 0.057 ppm) as the most effective ones, being IB15 with a potency of close to 50%, higher to strain 2362 in the assays carried out with A. darlingi. IB16 and S1116 were more potent in the experiments with C. quinquefasciatus, showing to be nearly 300-400% higher. Through molecular characterisation were diagnosed the binary toxin gene in all twenty studied stains and MTX1 toxin was found only on fourteen of them. Twenty-three polymorphic sites on the sequences were observed on the basis of the BinA sequences, being only four of them informative for the parsimony. As a whole, ten haplotypes were obtained amongst the Amazonian strains, being haplotype 1 (nine strains) similar to that of 2362 (Type 2) and the rest presenting distinct sequences. The 16 polymorphic sites observed on the aminoacid sequences resulted into 19 variant aminoacids and, on the basis of the genetic distance among the strains, it was not possible to establish a correlation between variations on BinA toxin sequences and toxicity level, as well as precedence of the strains. As B. sphaericus raises biotechnological interest in the production of biolarvicides, IB15 strain microbial growth in NYSM medium during 24h of fermentation, was studied. It was found that IB15 presented a growth profile similar to 2362 strain to prduction of cells, spores, biomass and larvicidal activity throughout fermentation, with no statistically significant differences. At the end, 1.61x109 spores.mL-1 was obtained with IB15 and 6.46x108 spores.mL-1 with 2362. LT50 values were similar, being in average 2.5h in the experiments with one culture of the bacillus at the end of 24 hours. Strain IB15 showed to be suitable for growth in NYSM medium, presenting desirable levels of production of spores and toxins throughout fermentation. Further studies are needed on the large-scale production with that strain for the development of biolarvicides. Therefore, with the findings obtained in this study, one verifies the great importance of mosquito biological control with the use of entomopathogenic bacteria, showing to be a viable complement or alternative on the control of vector mosquitoes in Amazonia. / A atividade larvicida de B. sphaericus, procedentes de diversas localidades da Amazônia, foi estimada por meio de bioensaios com Anopheles darlingi e Culex quinquefasciatus. Pela análise de Probit foi determinada a concentração letal mediana (CL50) e a potência em relação à estirpe padrão 2362. Os resultados apontaram as estirpes IB15 (CL50 = 0,040 ppm), IB19 e S1116 (CL50 = 0,048 ppm), IB16 (CL50 = 0,052 ppm) e S265 (CL50 = 0,057 ppm) como mais efetivas, sendo IB15 com potência cerca de 50% superior à estirpe 2362 nos bioensaios realizados contra A. darlingi e IB16 e S1116 mais potentes nos testes com C. quinquefasciatus, mostrando-se cerca de 300-400% superiores. Na caracterização molecular o gene da toxina binária foi diagnosticado em todas as estirpes estudadas e a toxina MTX1 foi observada em apenas quatorze estirpes. Com base nas sequências de BinA, foram observados 23 sítios polimórficos nas sequências, sendo apenas quatro informativos para parcimônia. Ao total obteve-se dez haplótipos entre as estirpes da Amazônia, sendo o haplótipo 1 (nove estirpes) similar a 2362 (Tipo 2) e o restante com sequências distintas. Os 16 sítios polimórficos observados nas sequências de aminoácidos resultaram em 19 aminoácidos variantes e, com base na distância genética, não foi possível estabelecer uma correlação entre variações nas sequências da toxina BinA e o nível de toxicidade, bem como a procedência das estirpes. Como B. sphaericus desperta um interesse biotecnológico na produção de biolarvicidas para controle de mosquitos vetores, foi estudado o crescimento microbiano da estirpe IB15 em meio NYSM, durante 24 horas de fermentação. IB15 apresentou um perfil de crescimento semelhante à estirpe 2362 quanto às produções de células, esporos, biomassa e em atividade larvicida ao longo da fermentação. Ao final, obteve-se 1,61x109 esporos.mL-1 com IB15 e 6,46x108 esporos.mL-1 com 2362. Os valores de TL50 foram semelhantes, sendo em média 2,5h nos testes com uma cultura do bacilo ao final de 24 horas. A estirpe IB15 demonstrou ser adequada para crescimento em meio NYSM, apresentando níveis desejados de produção de esporos e toxinas ao longo da fermentação. São necessários estudos complementares sobre produção em grande escala com essa estirpe para o desenvolvimento de biolarvicidas. Portanto, com os resultados obtidos nesse trabalho, verifica-se a grande importância do controle biológico de mosquitos com o emprego de bactérias entomopatogênicas, mostrando ser uma alternativa ou complemento viável no controle de mosquitos vetores na Amazônia.

Aedes albopictus et Aedes aegypti sont des moustiques vecteurs d’arbovirus tels que le virus de la dengue et le virus du chikungunya. En plus des virus transmis, les moustiques hébergent également des bactéries dont certaines affectent la biologie des hôtes. Par exemple, la bactérie Wolbachia infecte naturellement Aedes albopictus. Comme chez la plupart des insectes, cette bactérie est un parasite qui manipule la reproduction du moustique et peut également interagir avec certains pathogènes, modifiant ainsi la transmission du pathogène par le moustique hôte. Hors Wolbachia, peu d’études ont été consacrées à l’étude des populations bactériennes hébergées par les moustiques du genre Aedes et aux interactions entre ces populations bactériennes et les arbovirus transmis. Dans ce contexte, le travail de cette thèse a consisté à caractériser la diversité des communautés bactériennes des moustiques du genre Aedes et à explorer l’interférence possible entre le compartiment bactérien et le virus chikungunya. L’utilisation de techniques telles que les isolements bactériens, l’amplification par PCR, la DGGE et l’hybridation in situ ont permis de détecter et localiser certaines bactéries présentes chez des populations naturelles et de laboratoires d’Aedes. Ces populations appartiennent aux Alpha, Beta et Gammaprotéobactéries ainsi qu’aux Firmicutes. L’étude de la dynamique des communautés bactériennes symbiotiques et de l’infection par le virus chikungunya chez Aedes albopictus par PCR quantitative et puces taxonomiques a révélé l’existence d’interactions entre les différents partenaires de ce système vectoriel / Aedes albopictus and Aedes aegypti transmit a large number of arboviruses, including dengue and chikungunya. In addition to viruses, mosquitoes harbour other symbionts that are able to affect its biology. For instance, the bacterium Wolbachia infects naturally Aedes albopictus. As for many insects, this bacterium is an obligate parasite that manipulates the host reproduction and can also interact with pathogens, modifying the transmission of the pathogens by the mosquitoes. Except Wolbachia, little is known about the bacteria associated with Aedes mosquitoes. First, we detected and localized bacteria in field-caught and laboratory populations of Aedes, using culture and non-culture methods including PCR, DGGE and in situ hybridization. The bacterial populations belonged to Alpha, Beta and Gammaproteobacteria as well as to Firmicutes. Then, the effects of chikungunya infection on Wolbachia and total bacterial community were measured using quantitative PCR and taxonomic microarrays. Results showed interactions between the different partners in this vectorial system

La lutte anti-vectorielle est l’un des volets le plus important de l’entomologie médicale et nécessite une identification précise des vecteurs. Cette dernière décennie, la technique du MALDI-TOF MS a prouvé son potentiel comme outil rapide et efficace pour l'identification des arthropodes hématophages adultes. Dès lorsNous nous sommes intéressés à la mise au point d’un protocole d’identification des stades aquatiques de moustique par MALDI-TOF MS d’une part. D’autre part la détection d’un pathogène dans un arthropode n’implique pas forcement sa capacité à transmettre. L’incrimination d’un arthropode comme vecteur respecte certaines règles allant de la suspicion à la démonstration de sa compétence vectorielle au laboratoire. Afin de mieux comprendre l’épidémiologie de R. felis nous avons d’abord participé à une investigation conduite à l’Ile de la Réunion, en testant des puces, les seuls vecteurs biologiques connus jusqu’à présent. Ensuite Nous avons démontré le rôle potentiel des moustiques en particulier d’Anopheles gambiae à transmettre Rickettsia felis. Enfin nous avons utilisé le MALDI-TOF MS pour la détermination du statut infectieux d’Anopheles gambiae à R. felis. Nous proposons également un cycle probable de transmission de R. felis à l’homme incluant les psoques et les moustiques. / Vector control is one of the most important aspects of medical entomology and requires accurate identification of vectors. Within the past decade, the MALDI-TOF MS technique has proven its potential as a fast and effective tool for identification of adult blood-sucking arthropods. From then on we were interested in the development of an identification protocol of aquatic stages of mosquitoes by MALDI-TOF MS. On the other hand, the detection of a pathogen in an arthropod does not necessarily mean its ability to transmit. Incrimination of an arthropod as vector follows certain rules ranging from suspicion to demonstrate its vector competence in the laboratory. To better understand the epidemiology of R. felis we first participated in an investigation conducted in Reunion, testing fleas, the only biological vectors known to date. We demonstrated the potential role of the mosquito particularly Anopheles gambiae, in the transmission of R. felis. Finally, we used the MALDI-TOF MS for the determination of the Anopheles gambiae infection status to R. felis. We also offer a probable transmission cycle of R. felis to man including psocids and mosquitoes.

Philosophiae Doctor - PhD / The discovery of microRNAs (miRNAs) is one of the most exciting scientific breakthroughs in the last decade. miRNAs are short RNA molecules that do not encode proteins but instead, regulate gene expression. Over the past several years, thousands of miRNAs have been identified in various insect genomes through cloning and sequencing, and even by computational prediction. However, information concerning possible roles of miRNAs in mosquitoes is limited. Within this context, we report here the first systematic analysis of these tiny RNAs and their target mRNAs in one of the principal African malaria vectors, Anopheles funestus s.s. Firstly, to extend the known repertoire of miRNAs expressed in this insect, the small RNAs from the four developmental stages (egg, larvae, pupae and the adult females), were sequenced using next generation sequencing technology. A total of 98 miRNAs were identified, which included 65 known Anopheles miRNAs, 25 miRNAs conserved in other insects and 8 novel miRNAs that had not been reported in any species. We further characterized new variants for miR-2 and miR-927 and stem-loop precursors for miR-286 and miR-2944. The analysis showed that many miRNAs have stage-specific expression, and co-transcribed and co-regulated during development. Secondly, for a better understanding of the molecular details of the miRNAs function, we identified the target genes for the Anopheles miRNAs using a novel approach that identifies overlap genes among three target prediction tools followed by filtering genes based on functional enrichment of GO terms and KEGG pathways. We found that most of the miRNAs are metabolic regulators. Moreover, the results suggest implication of some miRNAs not only in the development but also in insect-parasite interaction. Finally, we developed the InsecTar database (http://insectar.sanbi.ac.za) for miRNA targets in the three mosquito species; Anopheles gambiae, Aedes aegypti, and Culex quinquefasciatus, which incorporates prediction and the functional analysis of these target genes. The proposed database will undoubtedly assist to explore the roles of these regulatory molecules in insects. This type of analysis is a key step towards improving our understanding of the complexity and regulationmode of miRNAs in mosquitoes. Moreover, this study opens the door for exploration of miRNA in regulation of critical physiological functions specific to vector arthropods which may lead to novel approaches to combat mosquito-borne infectious diseases.

This ethnographic study was conducted among the riverine people, also known as Ribeirinhos, in the state of Amazonas, Brazil, during four months of field research. The study focused on learning from Ribeirinhos’ experiences and practices of malaria. In this thesis, I argue that paying attention to Ribeirinhos’ experiences and diagnostic, treatment, and control practices of malaria can provide useful insights into blind spots in the current interventions to control the disease in Brazil. As this is a thesis by publications, the findings are presented in three manuscripts. The first manuscript focuses on how malaria is experienced by Ribeirinhos. It explores the embodiment of malaria, empirical strategies to distinguish it from other febrile sicknesses, misalignment between bodies and current biomedical diagnosis methods, “becomings” of bodies and experiences, and the vicissitudes of having the disease. The second manuscript examines experiences and treatment practices for vivax malaria highlighting the uses of pharmaceuticals, side effects of antimalarial drugs, and traditional treatments for malaria. The third manuscript describes Ribeirinhos’ perceptions of malaria-carrying mosquitoes, their everyday practices to manage these beings, and their experiences with control interventions, such as time monitoring recommendations (TMR), indoor residual spraying (IRS), and insecticide-treated nets (ITN). The three manuscripts clearly show that Ribeirinhos' lives are thoroughly entangled with Amazonian rivers and forests; malaria also takes part in these entanglements. Learning from their experiences and practices of malaria has provided information about the nuances, improvisations, and continuous negotiations required to coexist with the parasite and disease vectors.