Orientador: Paulo Eduardo Martins Ribolla / Banca: Henrique M. C. da Silveira / Banca: Alexandre A. Peixoto / Resumo: Estudos recentes vêm elucidando a importância de uma diversidade de proteínas do intestino médio de insetos vetores, tanto nos processos de digestão como em respostas imunológicas e interações parasita-hospedeiro. Este trabalho teve como objetivo analisar a expressão de genes do intestino médio de Anopheles aquasalis, um importante vetor de malária no Brasil, a partir de clones sequenciados de bibliotecas de cDNA de machos e fêmeas alimentados apenas com sacarose. Nas fêmeas, pôde-se notar a grande predominância de serino proteases, proteínas ligantes de quitina e fatores relacionados à imunidade. Os machos também apresentaram diversos peptídeos de defesa imune, porém apenas uma protease digestiva e uma glicosidase. Alguns genes foram selecionados das bibliotecas para estudo de suas expressões durante a vida de An. aquasalis. Tripsina 1, peritrofina 1 e quinurenina 3-monooxigense tiveram seus níveis de expressão aumentados 6h após a ingestão de sangue, analisados através de qRT-PCR. No entanto, o silenciamento desses genes não resultou em alterações na longevidade de fêmeas adulta. O gene da serpina foi expresso em todas as fases do desenvolvimento do mosquito, exceto em ovos; e o gene da cecropina foi expresso em trato digestivo e carcaça de machos e fêmeas, principalmente após alimentação de açúcar ou sangue. Considerando que a ingestão de alimentos é a principal porta de entrada a microorganismos durante a vida adulta destes mosquitos, a presença de diversos produtos antimicrobianos, bem como a precoce expressão de peritrofina, outra proteína relacionada com a proteção do trato digestivo, mostrou que An. aquasalis está bem preparado imunologicamente contra esses microorganismos. Esta proteção está envolvida com o hábito alimentar desta espécie e pode também estar associada à sua baixa capacidade vetorial com relação aos plasmódios. / Abstract: The importance of midgut proteins of Anopheles aquasalis has been elucidated both in digestion process as in immune responses and parasite-host interactions. This project targeted to analyze the midgut genes expression from An. aquasalis, an important malaria vector in Brazil, selecting clones from midgut cDNA library of female and male mosquitoes fed only on sugar. Serine proteases were predominant in females besides chitin binding proteins and immunity factors. Male mosquitoes also showed immune defense peptides, however only one digestive protease and one glucosidase. Some genes were selected from these libraries to expression study during mosquito development stages. Trypsin 1, peritrophin 1 and kynurenine 3-monoxygenase expression were up regulated at the midgut 6h after blood feeding, analyzed by real time PCR. Nevertheless, the gene silencing did not change the survivorship of adult females. Serpin gene was expressed in all mosquito development stages but eggs; cecropin gene was expressed in midgut and carcass from male and female, mainly after sugar or blood feeding. Considering the alimentation is the main entrance way of pathogens, the presence of antimicrobial peptides as the early peritrophin expression showed that An. aquasalis is immunologically adapted against these microorganisms. This protection is involved in feeding behavior of this specie and can be also related to its low Plasmodium vector capacity. / Mestre

Este trabalho consistiu em observações de campo e de laboratório, sendo que a primeira foi desenvolvida na localidade da Fazenda Folha Larga, município de Cananéia, Estado de São Paulo. As capturas foram feitas nos ambientes extra e peridomiciliares com isca humana em horário de pico máximo de atividade hematófaga de Anopheles (Kerteszia) cruzii. Resultados preliminares obtidos em campo objetivaram determinar a paridade, enquanto que sob condições laboratoriais procurou-se conhecer a duração do ciclo gonotrófico, ambos considerados parâmetros implicados na determinação da capacidade vetora de An. cruzii. Assim pois, das 631 fêmeas dissecadas, 90,49 por cento revelaram-se nulíparas e somente 9,50 por cento uníparas, mostrando que a maior parte da população era constituída de indivíduos jovens e sem haver diferença significativa quanto a proporção de fêmeas uníparas nos diversos ambientes. No laboratório trabalhou-se com 1.158 larvas dessa espécie, oriundas de aproximadamente 600 bromélias. O período de desenvolvimento larvário foi de aproximadamente 40 dias. Desse material, 283 fêmeas foram alimentadas com sangue humano, sendo que 50 por cento conseguiu o seu desenvolvimento folicular completo com um único repasto sangüíneo. Outras 25 fêmeas, não receberam alimentação sangüínea e, quando dissecadas, todas apresentaram o seu primeiro folículo no estágio II de Christophers e Mer, provavelmente pela falta de estímulo sangüíneo. Além do mais, determinou-se que o tempo médio entre a alimentação sangüínea e a oviposição foi de 6 a 7 dias. Em vista disso, as observações preliminares, possibilitam considerar esta espécie com razoável capacidade vetora, apesar da baixa longevidade, pois esta é provavelmente, compensada pela alta densidade. / This study consisted on field and laboratory observations. The former was developed at Folha Larga Farm., Cananéia County, São Paulo State. Collections were made in extra and peridomiciliary environments with human bait during the maximum peak of Anopheles cruzii blood feeding activity. Preliminary results obtained in the field, led to the determination of parity, and those developed in the laboratory led to the determination of the gonotrophic cycle length, both considered as involved parameters in the determination of vector capacity. From 631 females dissected, 90.49 per cent were nulliparous, and 9.0 per cent uniparous, showing that the greater part of the population was composed of young females. No significant difference between the proportion of uniparous females in the extra and peridomiciliary environment was found. At the laboratory 1,158 larvae o f An.cruzii from 600 bromelia were examined. The period of larval development was about 40 days. From this material, 283 females were fed with human blood, 50 per cent of them reached complete follicular development witb a single bloodmeal, showing the first follicular in the Christophers and Mer\'s stages ll, probably because of the lack of blood stimulus. Besides, was determined that the average time between bloodmeal and oviposition was 6 to 7 days. As a result of these preliminary observations was possible to consider this species as a vector with positive capacity, inspite of its low longevity, that could be probably compensated by its high density.

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Almeida.pdf: 2582224 bytes, checksum: 619daa8290bd853bcd6681edd48defdf (MD5) Previous issue date: 2017-07-28 / FAPEAM - Fundação de Amparo à Pesquisa do Estado do Amazonas / The species of the family Lamiaceae are known in Brazil for being very aromatic and for presenting bioactive properties as antioxidant, bactericide, fungicide and insecticide. In this work was analyzed Hyptis dilatata Benth, found in areas of cerrado in Brazil. The chemical components, the biological activity of the essential oil and extracts of leaves and flowers, considering dry and rainy periods, of specimens of the Serra do Tepequém, in Roraima-Brazil, were studied. The essential oils were extracted from the leaves collected at different times (morning, afternoon and night) and from the flowers in the dry and rainy periods. In relation to the hexane and ethanolic extracts from leaves and flowers, these were extracted from specimens collected during the dry and rainy season. The results of the phytochemical profile, derived from the analysis of the hexane and ethanolic extracts of leaves and flowers, presented different secondary metabolites, being identified coumarin, flavonoids, phenols, saponins, alkaloids, among others. The chemical characterization of the essential oils of leaves and flowers showed as main substances α-pinene, β-pinene, limonene, 3-carene, β-caryophyllene, fenchone and camphor. The essential oil extracted from the flowers in the rainy and dry period inhibited the enzyme acetylcholinesterase, respectively, 93.4% and 92.4%, whereas the essential oil extracted from the leaves, in the dry period and in the morning, showed greater inhibition (96.4%). In the analysis of the cytotoxicity activity performed with larvae of Artemia salina, the essential oil extracted from the leaf collected in the rainy period in the morning, and the essential oil extracted from the flower, collected in the rainy season, presented higher toxicity. The ethanolic extracts of the leaf and the hexane extracts of the flower, both collected during the rainy season, were also more toxic. In the antimicrobial assays, flower oils collected in the rainy and dry period demonstrated greater inhibition against the bacteria Staphylococcus aureus and Bacillus cereus. The Candida albicans yeast only showed inhibition with the essential oils of the flower collected in the dry period. In the rainy season only the essential oil extracted from the leaf collected in the morning and afternoon, showed inhibition against the bacteria S. aureus and B. cereus. Regarding the dry period, the essential oil that showed the highest inhibition in B. cereus bacteria was collected in the afternoon. The hexane extracts of the flower collected in the rainy season showed greater inhibition of S. aureus and in the leaves collected in the rainy season. This extract showed greater toxicity with the bacterium Salmonella typhymurium. In the test against yeast C. albicans, the most toxic hexanic extract was that of the leaf collected in the dry period. In this work the essential oils and extracts of H. dilatata were also tested in larvicidal activities of Aedes aegypti, Anopheles darlingi and adulticides with Ae. aegypti. The essential oil with higher toxicity in the larvicidal activities of Ae. aegypti were extracted from the leaf collected in the rainy season, at night time and from the flower in the rainy season. Only the hexane extracts extracted from the flowers showed toxic activity on the larvae of Ae. aegypti. In larvae of An. darlingi, toxicity predominated with the essential oils of the flowers extracted in the dry period and in the essential oil of the leaf extracted in the same period, in the morning. In the adulticidal assays of Ae. aegypti, leaf oil collected in the rainy season at night showed a mortality of 54.4% to 91.9%, at concentrations of 62.5 μg/mL -1 and 1000 μg/mL-1 respectively in the range of 90 minutes of exposure. The knockdown effect (mosquito immobility) was observed in the test for the development of the product prototype with leaf essential oil, collected in the rainy season and in the morning. For that, the insecticidal activity against the mosquito Ae. aegypti, in 20 minutes at the start of the test, with more than 50% of mosquitoes in knockdown state. / As espécies da família Lamiaceae são conhecidas no Brasil por serem bastante aromáticas e por apresentarem propriedades bioativas como antioxidante, bactericida, fungicida e inseticida. Neste trabalho foi analisada a espécie Hyptis dilatata Benth, encontrada em áreas de cerrado no Brasil. Estudou-se os componentes químicos, a atividade biológica do óleo essencial e dos extratos de folhas e flores, considerando os períodos seco e chuvoso, de espécimes da Serra do Tepequém, em Roraima-Brasil. Os óleos essenciais foram extraídos das folhas coletadas em diferentes horários (manhã, tarde e noite) e das flores nos períodos seco e chuvoso. Em relação aos extratos hexânicos e etanólicos, oriundos das folhas e flores, estes foram extraídos de exemplares coletados nos períodos seco e chuvoso. Os resultados do perfil fitoquímico, originados a partir da análise dos extratos hexânicos e etanólicos das folhas e flores, apresentaram diferentes metabólitos secundários, sendo identificados cumarina, flavonóides, fenóis, saponinas, alcalóides, entre outros. A caracterização química dos óleos essenciais das folhas e flores mostrou como substâncias majoritárias α-pineno, β-pineno, limoneno, 3-careno, β-cariofileno, fenchona e cânfora. O óleo essencial extraído das flores, no período chuvoso e seco, inibiu a enzima acetilcolinesterase, respectivamente, 93,4% e 92,4%, enquanto que o óleo essencial extraído das folhas, no período seco e pela manhã, apresentou maior inibição (96,4%). Na análise da atividade de citotoxidade, realizadas com larvas de Artemia salina, o óleo essencial extraído da folha coletada no período chuvoso, pela manhã, e o óleo essencial extraído da flor, coletada no período chuvoso, apresentaram maior toxicidade. Apresentaram também maior toxicidade os extratos etanólicos da folha e os extratos hexânicos da flor, ambas coletadas no período chuvoso. Nos ensaios antimicrobianos, os óleos das flores coletadas no período chuvoso e seco demonstraram maior inibição contra as bactérias Staphylococcus aureus e Bacillus cereus. A levedura Candida albicans só apresentou inibição com os óleos essênciais da flor coletada no período seco. No período chuvoso, somente o óleo essencial extraído das folhas coletadas no horário da manhã e tarde, mostraram inibição contra as bactérias S. aureus e B. cereus. Em relação ao período seco, o óleo essencial que apresentou maior inibição contra bactérias B. cereus foi coletado no horário da tarde. Os extratos hexânicos da flor coletada no período chuvoso mostraram maior inibição contra S. aureus e nas folhas coletadas no período chuvoso. Este extrato demonstrou maior toxicidade com a bactéria Salmonella typhymurium. No teste contra a levedura C. albicans, o extrato mais tóxico foi o hexânico da folha coletada no período seco. Neste trabalho também foram testados os óleos essenciais e extratos de H. dilatata em atividades larvicidas de Aedes aegypti, Anopheles darlingi e teste adulticidas com Ae. aegypti. O óleo essencial com maior toxicidade nas atividades larvicidas de Ae. aegypti foram extraídos da folha coletada no período chuvoso, no horário da noite e da flor no período chuvoso. Somente os extratos hexânicos extraídos das flores apresentaram atividade tóxica sobre as larvas de Ae. aegypti. Nas larvas de An. darlingi, a toxicidade predominou com os óleos essenciais das flores extraídas no período seco e no óleo essencial da folha extraído no mesmo período, no horário da manhã. Nos ensaios adulticida de Ae. aegypti, o óleo das folhas coletadas no período chuvoso, no horário da noite, apresentou mortalidade entre 54,4% a 91,9%, nas concentrações de 62,5 μg mL-1 e 1000 μg mL-1, respectivamente, no intervalo de 90 minutos de exposição. No teste para o desenvolvimento do protótipo do produto com o óleo essencial da folha, coletada no período chuvoso e no horário da manhã, foi observada a ação do efeito knockdown (imobilidade do mosquito). Para tanto, considerou-se a atividade inseticida contra o mosquito Ae. aegypti, em 20 minutos no início do teste, com mais de 50% dos mosquitos em estado de knockdown.

The vectors of avian malaria (Haemosporida) are an understudied component of wildlife disease ecology. Most studies of avian malaria have focused on the secondary bird hosts. This imbalance leaves a significant gap in our knowledge and understanding of the insect hosts. This study investigates the diversity of malaria parasites carried by mosquitoes (Diptera, Culicidae) in the state of Mississippi. Using PCR techniques, haemosporidian infection rates were determined and parasites were identified in a phylogenetic context to those previously annotated. A total of 27,157 female mosquitoes representing 15 species were captured. Five of those species tested positive for malaria parasites with an overall infection rate of 4 per 1000 mosquitoes infected. Mosquitoes were shown to harbor Plasmodium and Haemoproteus parasites. Surprisingly, a unique lineage of parasites was discovered in Anopheles mosquitoes potentially representing a new genus of haemosporidian parasites, reinforcing the need to continue investigating this diverse group of parasites.

No description available.

MicroRNAs (miRNAs) are non-coding RNAs that often play a fundamental role in gene regulation. Currently, hundreds to over a thousand miRNAs are predicted to be present in many eukaryote species, with many to be discovered; the functions of most are unknown. While much attention has gone towards model organisms, a much greater depth of understanding remains to be gained for the miRNAs of many organisms directly important to humans. There are few verified miRNAs for any mosquito species, despite the role of mosquitoes in many of humanity’s worst diseases. Anopheles gambiae and Aedes aegypti, carriers of malaria and dengue, respectively, are responsible for over a million deaths a year. To date, there are sixty-six microRNAs in An. gambiae in miRBase, a central repository for miRNA sequences. Many of these are based on homology to primarily Drosophila miRNAs. While sequence conservation suggests an important function for these miRNAs, expression has not been experimentally verified for most mosquito miRNAs. Using small RNA cloning and northern blots, I discovered and analyzed 27 different microRNAs in aged female An. stephensi mosquitoes, the age group responsible for transmission of malarial parasites. Three of these miRNAs are only found in mosquitoes (miR-1889, -1890, and –1891). Cloning and northern analysis revealed an abundance of a miRNA that is linked to longevity in flies, miR-14, across different life stages of mosquitoes. It was also shown that miR-989 was expressed almost exclusively in the adult ovary and its expression fluctuated in response to bloodfeeding, suggesting a possible role in reproduction, an area of great importance to controlling mosquito populations. Building upon the above cloning experiment, a later high-throughput sequencing effort uncovered 98 miRNA precursors from Ae. aegypti. There are a total of 13 novel miRNAs that have not been found in other organisms by bioinformatic predictions or experiments. These “mosquito-specific” miRNAs may play a role in processes such as blood-feeding or vector-host interactions. A detailed examination of the expression of eight of these miRNAs was conducted in An. gambiae, An. stephensi, Ae. aegypti, and T. amboinensis to determine their expression profile, conservation, and provide hints to their function. My work revealed conserved and sometime stage-specific expression profiles of some of the mosquito-specific miRNAs. I also provided evidence for three lineage-specific miRNAs that may shed light on the divergence of different mosquito lineages. Extending the finding that miR-989 may be involved in mosquito reproduction, we conducted a detailed analysis of its evolution, expression, possible targets and regulation. miR-989 is conserved in holometabolous insects. miR-989 expression in female An. stephensi and Ae. aegypti dramatically rises following pupal emergence until strong signal is observed, until a blood meal is taken. Expression remains quite strong then begins a steep decline in expression at 32-40 hours post blood meal (PBM), and even by 96 hours PBM, remains weak. Bioinformatic predictions of miR-989 targets coupled with a PCR-based approach uncovered three potential target leads, though preliminary results were artifacts. Although the miR-989 post-emergence expression profile correlates with the expression of Juvenile Hormone, a key reproductive hormone in mosquitoes, no observable induction occurred when abdominal ligation samples were administered methoprene, a JH analog. However, methoprene impacted a number of other miRNAs, with up to a 3.87 fold induction (miR-1891), and a 3.15 fold suppression (miR-9a) of signal. Subsequent northern analysis provided visual confirmation of observable fold changes for miR-1891 and miR-9a, but not for miRNAs that showed changes below two fold. This analysis provides a foundation to study Juvenile Hormone regulation of miRNAs in mosquitoes. In summary, we have expanded the understanding of microRNAs in mosquitoes. An improved understanding of mosquito physiology can assist in efforts to control mosquito-borne infectious diseases. / Ph. D.

Many insects rely on olfaction as their primary method of interaction with their environment. One of the best examples of this is the olfactory driven host-seeking behavior displayed by female mosquitoes. Although mosquitoes are capable of extracting blood from a variety of hosts many mosquito species show marked preferences for particular host species. Mosquitoes displaying preference for humans above bovines are more likely to be disease vectors. Therefore understanding the molecular basis of this preference is important for public health. These differences may be the result of genetic variations in olfactory signaling components such as mosquito odorant receptors. This hypothesis is supported by several lines of evidence including the highly divergent and lineage-specific nature of this receptor family. Likely these differences are subtle and will be identified in highly focused studies. Even closely related sibling species of mosquitoes can display large behavioral differences. In our current study I have studied several aspects of both Anopheles and Aedes genus odorant receptors with emphasis on comparing receptors in species that are part of the Anopheles genus. The first goal of this project was to study the insect odorant receptor family for potential sites of heterodimer formation. Numerous studies have shown that insect odorant receptors are involved in detection of odorants. More recent studies have demonstrated that odorant receptors are also involved in protein trafficking and in forming cation channels. Both of these activities involve heterodimer formation between odorant receptors that bind odorants and those that are part of the Or83b subfamily. There is little informaiton on how heterodimers are formed and where within the protein heterodimer sites exist. The C-terminal region has been implicated as sites for such heterodimer formation. A hidden markov model based program, Multiple em for motif elicitation (MEME), was used to uncover three motifs in the C-terminus of the odorant receptor peptides from Anopheles gambiae, D. melanogaster, and Apis mellifera. Previous studies have shown that insect odorant receptors are highly divergent between different insect lineages suggesting conservation of these motifs is functionally important. I propose that these motifs are involved in receptor-receptor protein interactions, contributing to the heterodimer formation between Or83b subfamily members and other odorant receptors.The next goal was to identify odorant receptors in closely related mosquito species and compare and contrast them. This was accomplished by using public sequence data of An. gambiae and BAC library screening to identify orthologous gene clusters in An. stephensi and An. quadriannulatus. Although I have identified many different odorant receptor genes the chapter in this dissertation discusses my work with the Or2 gene cluster. Multi-species comparison of these orthologous regions in An. gambiae, An. quadriannulatus, and An. stephensi revealed highly conserved gene structure among the OR genes and the discovery of the An. stephensi Or10x gene (AsOr10x), which is present only in An. stephensi. AsOr10x showed a different expression pattern than AsOr2 and AsOr10, the other members of this gene subfamily in An. stephensi. Therefore AsOr10x might be adapting or has adapted a new function. Analysis of the phylogeny and physical location of all known members of the Or2/Or10 gene subfamily in Anopheles, Aedes, and Culex mosquitoes suggest that a few events of gene duplication and loss resulted in the current gene distribution. The final focus of this project was to develop a method to study the function of mosquito odorant receptors. There is currently no in vivo system to study mosquito odorant receptors, and experimental systems pioneered in D. melanogaster are not transferable to mosquitoes. I decided to employ a reverse genetics strategy involving the silencing of three Aedes aegypti odorant and gustatory receptors of known or suspected function. These gustatory receptors are members of a small subfamily that encode olfactory and not taste receptors. As a preliminary step the expression profiles of these three genes and an additional gustatory receptor were determined using non-quantitative and quantitative RT-PCR. We found that the putative CO₂-detecting gustatory receptors are expressed in Ae. aegypti larvae, and hence these larvae may respond to CO₂, an observation that has not been reported previously. The purpose of silencing these receptors is to generate a loss-of-function behavior phenotype that will allow for inference of receptor function. Recombinant Sindbis viruses were used to knockdown mRNA levels of these receptors. GFP-expressing recombinant Sindbis viruses were shown to infect chemosensory tissue. Additional viruses containing fragments of receptor genes were found capable of lowering odorant and gustatory receptor mRNA levels. Infected mosquitoes displayed varying levels of gene knockdown with one virus generating supression of mRNA levels to 15.0% of normal. These mRNA levels may not be low enough to generate an unambiguous phenotype. Future experimentation is focused on developing more effective recombinant viruses and identifying characteristics of viruses more effective in receptor gene knockdown. A safe and effective behavior assay setup is needed to test the behavioral responses of these infected mosquitoes. In this study I outline a preliminary behavior assay that is being developed and optimized. When established it will provide a powerful tool in the study of both basic mosquito behavior and phenotype screening of recombinant Sindbis virus-infected mosquitoes. / Ph. D.

The emergence of pesticide-resistant mosquitoes and drug-resistant parasites in the last twenty years has made control of malaria more difficult. One novel strategy to better control malaria is the development and release of transgenic mosquitoes whose enhanced immunity prevents transmission of the parasite to the mammalian host. One candidate effector gene is Anopheles stephensi nitric oxide synthase (AsNOS), whose inducible expression and subsequent synthesis of nitric oxide (NO) limits Plasmodium development in A. stephensi. In mammals, one of the most potent physiological regulators of NOS gene expression and catalytic activity is transforming growth factor-β (TGF-β). Moreover, human TGF-β can activate Drosophila melanogaster Smads, the proteins responsible for TGF-β signal transduction. We have determined that following a bloodmeal, active human TGF-β1 (hTGF-β1) persists in the midgut of A. stephensi for up to 48 hours. My data demonstrate that the midgut epithelium recognizes hTGF-β1 as an immunomodulatory cytokine. Specifically, induction of AsNOS by hTGF-β1 occurs in the midgut within minutes of bloodfeeding. Moreover, hTGF-β1 limits development of the human malaria parasite Plasmodium falciparum in the midgut. In other experiments, provision of the AsNOS catalytic inhibitor L-NAME partially reverses the effect of hTGF-β1 on Plasmodium development. These results suggest that AsNOS is a target of hTGF-β1 signaling and additional effectors that impact parasite development may be regulated by hTGF-β1 as well. The fact that hTGF-β1 signals mosquito cells to limit malaria parasite development suggests that there is an endogenous TGF-β signaling network in place. An analysis of the A. gambiae genome database revealed the presence of six TGF-β ligands, including gene duplication in the 60A gene, the first evidence of ligand gene duplication outside of chordates. In addition to five receptors, three Smads were identified in the A. gambiae genome predicted to support TGF-β/Activin- and BMP-like signaling. Midgut epithelial cells and an immunocompetent A. stephensi cell line express all three Smads, confirming that a signaling pathway is in place to support signaling by divergent exogenous and endogenous TGF-β superfamily proteins. The results presented here provide the first evidence of immunological crosstalk between divergent free living hosts of a single parasite. Further, these results imply that the interface between mammals and the mosquitoes that feed on them provide a unique opportunity for circulating molecules in the blood, including TGF-β and other cytokines, to alter the mosquito immune response. / Master of Science

Une distribution nationale de moustiquaires imprégnées d'insecticide à longue durée d'action à destination des populations vulnérables du Niger a été effectuée fin 2005. Déjà montrée lors d'études pilotes à l'échelle du village, l'efficacité de cet outil dans le contrôle du paludisme restait à évaluer à l'occasion de vastes programmes opérationnels qui se multiplient en Afrique. Peu d'études des populations de vecteurs ont été publiées dans ce cadre. Nous avons mis en place un suivi entomologique au niveau de sites sentinelles répartis dans la zone Sahélienne du Niger, ayant couvert trois saisons de transmission, dont une avant intervention considérée comme période contrôle. Les paramètres entomologiques de la transmission ont été déterminés pour An. gambiae s.l., et la distribution spatiale des deux principaux vecteurs, An. gambiae et An. arabiensis, a été précisée. Le suivi temporel a mis en évidence une baisse globale du niveau de transmission de P. falciparum, probablement entrainée par la forte hausse d'utilisation de moustiquaires imprégnées. Cependant la hausse de la résistance des populations aux pyréthrinoïdes semble avoir été rapidement amorcée faisant craindre à moyen terme une perte d'efficacité de cet outil central des stratégies de lutte contre le paludisme. L'étude de la structure génétique des populations d'An. gambiae et d'An. arabiensis à l'aide de marqueurs microsatellites a montré une homogénéité génétique dans l'espace, entre les villages, même séparés par plusieurs centaines de kilomètres, ainsi que dans le temps, entre la saison de transmission 2005 contrôle et la saison 2006 après distribution. Ces résultats ont suggéré qu'au cours de la première année d'intervention, la couverture en moustiquaires imprégnées atteinte n'a pas eu d'effet de masse suffisant pour entrainer une baisse de la diversité génétique ou une modification des fréquences alléliques des populations. La faible différenciation spatiale observée pourrait être expliquée par des échanges de gènes importants à l'intérieur de la zone d'étude, hypothèse appuyée par l'expansion rapide de la mutation kdr dans l'ensemble des sites où An. gambiae est présent. L'évaluation rigoureuse de tels programmes de contrôle permettra d'améliorer les outils de contrôle et par exemple de préserver l'efficacité des pyréthrinoïdes, seule classe d'insecticides actuellement disponible pour l'imprégnation des moustiquaires.

En Afrique de l'Ouest, le moustique An. gambiae s.s vecteur majeur du paludisme est subdivisé en deux formes moléculaires, M et S, génétiquement et écologiquement différenciées. La forme moléculaire M se développe préférentiellement dans des collections d'eau pérennes en zone aride, généralement d'origine anthropique, permettant sa présence tout au long de l'année alors que la forme S se reproduit principalement dans des gîtes temporaires de savane humide dépendant des précipitations et disparaît en saison sèche. Cette subdivision génère des profils de dynamique de transmission palustre différents en fonction des zones où ces formes sont implantées. Dans ce contexte, cette thèse a pour objectif l'étude des facteurs écologiques de différenciation entre M et S, en se focalisant notamment sur leur écologie larvaire, afin de mieux appréhender leur distribution actuelle et future. L'étude de la distribution des populations naturelles de ces vecteurs dans une zone d'endémie palustre au Burkina Faso a permis de mettre en évidence que les niches écologiques de ces deux formes sont en étroite corrélation avec la temporalité des milieux aquatiques et la complexité des écosystèmes qu'ils hébergent. La forme M apparaît clairement liée aux habitats permanents anthropiques et à la structure des communautés qu'ils soutiennent alors que la forme S ainsi que l'espèce jumelle An. arabiensis sont associées aux habitats simples et temporaires, majoritairement retrouvés en zone rurale de savane.Cette distribution des deux formes le long d'un gradient d'hydropériode est en accord avec les interactions dominantes et les adaptations qu'elles induisent afin de pouvoir exploiter ces milieux. La forme S, associée aux milieux temporaires, s'est révélée plus compétitive que la forme M en diminuant son temps de développement larvaire en présence de compétiteurs (forme M). L'étude de la pression de sélection due à la prédation, interaction dominante dans les milieux permanents, démontre que la forme M est moins susceptible que la forme S. L'analyse du comportement larvaire a permis de mettre en évidence des différences entre ces deux formes, notamment l'existence d'un comportement plus plastique chez la forme M qui réduit son activité en présence d'un prédateur. Ce mécanisme est une des adaptations qui a favorisé le succès d'An.gambiae dans les milieux permanents.Notre approche, basée sur l'écologie larvaire des formes M et S d'An. gambiae nous a permis de mieux comprendre les processus par lesquels ces vecteurs ont évolué et se sont adaptés à différents contextes écologiques. Ces adaptations reflètent la spécialisation de ces deux formes dans leur milieu respectif et permettent en partie d'expliquer la ségrégation écologique observée sur le terrain. L'amélioration de nos connaissance sur la bio-écologie de ces vecteurs est primordiale afin d'en apprécier le potentiel évolutif dans le contexte actuel des changements globaux. / In West Africa, the main Malaria vector, the mosquito Anopheles gambiae is actually subdivided into two molecular forms named M and S, which can be genetically and environmentally differentiated. The M form preferentially breeds in permanent freshwater collections mainly resulting from human activity and is reproductively active all year round, whereas the S form thrives in temporary breeding sites and is present during the rainy season only. This subdivision generates different dynamics of Malaria transmission in areas where these forms are found. In this context, this thesis aims to study the ecological factors of differentiation between M and S, focusing on their larval ecology to better understand their current and future distribution.The study of the distribution of natural populations of these vectors in an endemic area in Burkina Faso has provided evidence that the ecological niches of these forms are closely correlated with the degree of temporality and the community complexity of aquatic ecosystems. The M form is clearly linked to permanent anthropogenic habitats and the structures they support, while the S form and its sibling species An. arabiensis are associated with simple and temporary habitats, mostly found in rural savannas.The distribution of the two forms along a hydroperiod gradient is consistent with the dominant interactions and adaptations they induce in order to be able to exploit their environments. In relation to temporary habitat, the S form was more competitive than the M form by reducing its larval development time in the presence of competitor (M form). The study of selection pressure due to predation, dominant interaction in permanent habitat, shows that the M forms suffer lesser predation rate than the S form. Analysis of larval behavior highlighted differences between these two forms, such as the existence of a more plastic behavior in the form M, which reduced its rate of activity in predator presence. This mechanism is one of the adaptations that have facilitated the success of An. gambiae in permanent aquatic habitats.Our approach, based on the larval ecology of M and S forms of An. gambiae has enabled us to better understand the processes by which these vectors have evolved and adapted to different ecological contexts. These adaptations reflect the specialization of these two forms in their respective habitats and can partially explain the ecological segregation observed in the field. Improving our knowledge on bio-ecology of these vectors is essential to appreciate their evolutionary potential in the current context of global change.