O gene A1 da alfa amilase foi isolado de uma biblioteca genômica em lambda EMBL3, feita com DNA do díptero primitivo Anopheles merus (Díptera, Nematocera, Culicoidea). Ele foi parcialmente seqüenciado, caracterizado pelo padrão da clivagem das enzimas restritivas e clonado no plasmídeo pIBI24 por Pernasetti (1991 ). No presente trabalho relata-se sua expressão em: bactéria (Escherichia coli AD494), células de inseto ( Spodoptera frugiperda) e em levedura (Pichia pastoris), tendo-se observado que somente nas células de Spodoptera frugiperda a enzima é expressa com atividade. A expressão extracelular do gene A1 foi ensaiada em E.coli AD494 usando o vetor pRSETc. Para este fim a seqüência do gene contendo seu próprio peptídeo sinal, foi inserida em fase de leitura (\"frame\") nesse vetor. A expressão sem peptídeo sinal foi executada usando o vetor pAE2, uma construção derivada- do pRSETc. Numa outra construção, o gene A1 foi inserido em fase de leitura após a seqüência do promotor e do peptídeo sinal do gene A2 (alfa amilase de Bacillus stearothermophilus) utilizando-se o vetor pIBI24-Bst. As células de E.coli AD494 transformadas com estes construções expressaram a enzima sem atividade. A amilase A1 não foi secretada nem com seu peptídeo sinal nem com o da amilase A2. A expressão do gene A1 em levedura (Pichia pastoris) foi feita usando o vetor pPic9, que permite a expressão tanto intracelular quanto a secreção da proteína. O gene foi amplificado por PCR usando \"primers forward\" com o intuito de obter o gene com ou sem peptídeo sinal. O gene contendo o peptídeo sinal foi construído com diferentes seqüências tipo Kozak precedendo o códon ATG. Para a expressão extracelular usou-se o peptídeo sinal do fator alfa do pPic9. Os resultados mostraram que a enzima é expressa mas que permanece dentro da célula sem atividade. As mesmas construções do gene A1 feitas para Pichia foram inseridas em Baculovírus para transfectar células de Spodoptera frugiperda. Neste sistema a amilase foi expressa com atividade principalmente no sobrenadante das culturas transformadas com construções usando o gene contendo o seu próprio peptídeo sinal assim com o peptídeo sinal da amilase de Zabrotes subfasciatus. O gene da alfa amilase de Bacillus stearothermophilus foi expresso no sistema Baculovírus-Spodoptera e na levedura Pichia, usando o gene contendo o peptídeo sinal da amilase de Z. subfasciatu. A proteína foi secretada e tinha atividade em ambos os sistemas. / The alpha amylase A1 gene was isolated trom a genomic library of lambda EMBL3 made with DNA from the primitve Díptera Anopheles merus (Díptera, Nematocera, Cuclicoidea). This gene was partialy sequenced, characterized by standard restriction enzyme cleavage, and cloned into the pIBI24 plasmid by Pernasetti (1991). Here we present the expression of this gene in: bacteria (Escherichia coli AD494), insect cell (Spodoptera frugiperda) and yeast (Pichia pastoris). It was shown that only in Spodoptera frugiperda cells the protein display enzymatic activity. The presence of the A1 product in the extracelullar culture media was tested in Escherichia coli AD494 using the vector pRSETc. To this end, the gene sequence containing its own signal peptide was inserted in frame into the vector. Expression without a signal peptide was conducted using the pAE2 vector, a construct derived from pRSETc. In another construct, the A1 gene was inserted in frame after the promoter sequence and the signal peptide of the A2 gene (alpha amylase Bacillus stearothermophilus) using the pIBI24-Bst vector. Although the E.coli AD494 cells transformed with these constructs expressed the enzyme, no enzyme activity was detected. A1 was not secreted, either with its own signal peptide or with that of amylase A2. Expression of the A1 gene in yeast (Pichia pastoris) was conducted using the pPic9 vector, wich allows intracellular expression or secretion of this protein. The gene was amplified by PCR with forward primers, so as to amplyfy the gene sequence with or without the signal peptide. The gene containing the signal peptide was constructed with different Kozak sequences preceeding the ATG codon. For extracellular expression, the signal peptide of the pPic9 alpha factor was used. The results showed that the enzyme is expressed , but remained within the cell and do not display activity. The same constructs of the A1 gene made for P. pastoris were inserted in Baculovirus to infect Spodopera frugiperda cells. In this system, the amylase was successfully expressed and display enzymatic activity, mainly in the extracellular supernatant, using constructs of the gene with own signal peptide or with the signal peptide for amylase of Zabrotes subfasciatus. The gene A2 was expressed in the Baculovirus/Spodoptera system and the yeast P. pastoris using constructs containing the signal peptide of alpha amylase of Z. subfasciatus, the protein was secreted with activity.

Introdução: Assentamentos rurais são recorrentes arranjos sociais em expansão na Região Amazônica, que geram perturbações no ambiente natural e na respectiva ecologia de mosquitos vetores envolvidos na transmissão de malária. Objetivos: Elaborar revisão das espécies do gênero Anopheles que foram registradas na Região Amazônica. Para a pesquisa quantitativa, estabelecer potenciais fatores dos ambientes e das características das águas de diversos habitats larvais que possam estar associadas com a presença de espécies de anofelíneos, através de coletas em criadouros de diferentes graus de perturbação ambiental. Métodos: para a revisão das espécies que foram registradas na Amazônia, foram empregados termos de busca, utilizando sete bases bibliográficas de referência. Para os estudos em campo, foram realizadas capturas de larvas juntamente com estimação e mensuração de algumas condições dos habitats larvais, para comparação dos parâmetros analisados entre ambientes naturais e artificiais. Resultados: a revisão mostrou maior quantidade de artigos que notificaram a ocorrência do subgênero Nyssorhynchus, seguido pelos subgêneros: Anopheles, Stethomyia, Kerteszia e Lophopodomyia. O Anopheles darlingi se mostrou a espécie com maior ocorrência nos artigos levantados, muito pelo seu caráter epidemiológico. Importantes vetores auxiliares pertencentes ao subgênero 6 Nyssorhynchus também foram registrados em grande quantidade. Os resultados das análises conduzidas com os dados das capturas em campo demonstraram que as forças de transformação do ambiente natural favorecem o desenvolvimento de habitats larvais de espécies do subgênero Nyssorhynchus, incluindo o An. darlingi, além de um importante vetor secundário, o An. triannulatus. Discussão e Conclusão: projetos de assentamentos na Amazônia brasileira registram índices significativos de casos de malária, devido à ocupação de áreas naturais pelo homem. Este estudo mostrou que o subgênero Nyssorhynchus possui amplo registro na literatura de inquéritos entomológicos do gênero na floresta tropical equatorial, indicando que o agrupamento pode ser considerado endêmico às fronteiras de transformação na região, tendo assim importante papel na manutenção da transmissão da malária. Os estudos de campo apresentaram evidências de que formação de habitats artificiais favorecem formas iniciais de vida dos mosquitos com histórico vetorial para a região. Esses habitats larvais podem ser respostas ao uso do solo e das consequentes alterações provocadas no ambiente natural pelas atividades humanas. As modificações do ambiente natural selecionam e propiciam a formação de habitats para as espécies do Nyssorhynchus. Este agrupamento taxonômico engloba espécies que são consideradas vetores para a malária na região Neotropical. / Introduction: Rural settlements are recurrent social arrangements expanding in the Amazon region, which generate disturbances in the natural environment, affecting the mosquitoes ecology involved in malaria transmission. Objective: A systematic review of Anopheles species were recorded in the Amazon region. For the qualitative research, were establish potential factors of the environment and the characteristics of the waters of several larval habitats that may be associated with the presence of species of Anopheles mosquitoes, by surveys of the samples presents in larval habitats within environmental disturbance gradient. Methods: for the review of the species, were used search terms, in seven bibliographic databases of reference. For studies in the field, larvae were trapped along estimation and measurement of some conditions of the larval habitats, to compare the parameters analyzed between natural and artificial environments. Results: The review showed greater amount of articles that reported the occurrence of Nyssorhynchus subgenre, followed by: Anopheles, Stethomyia, Kerteszia and Lophopodomyia. The Anopheles darlingi proved the species with the highest occurrence in the papers showed, due to its epidemiological importance. Important auxiliary vectors belonging to the subgenus Nyssorhynchus were also recorded in large scale. The results of the qualitative shown that the natural 8 environment transformation forces favor the development of larval habitats of the species of subgenus Nyssorhynchus, including An. darlingi and an important secondary vector, An. Triannulatus. Discussion and Conclusion: settlement projects in the Brazilian Amazon recorded significant rates of malaria cases. This study showed that, the Amazon basin an important determinant of this phenomenon is the formation of artificial habitats that are occupied by early forms of life of mosquito vectors. These larval habitats may be answers to land use and consequent changes in the natural environment caused by human activities. Modifications to the natural environment select and provide the formation of habitats for species in Nyssorhynchus. This taxonomic group includes species that are considered vectors for malaria in the Neotropics. Discussion and Conclusion: settlement projects in the brazilian Amazon recorded significant rates of malaria cases. This study showed that the review of the literature shows Nyssorhynchus subgenre ample record in entomological surveys in the equatorial rainforest, indicating that the group may be considered endemic to the borders of transformation in the region, and thus important role in the maintenance of malaria transmission. Field studies showed evidence that formation of artificial habitats favor early forms of the mosquito vector, with history for malaria in the region. These larval habitats may be answers to land use and consequent changes in the natural environment caused by human activities. Modifications to the natural environment select and provide the formation of habitats for species in Nyssorhynchus.

Au cours de ma thèse, j’ai développé et mis en place de nouvelles méthodes utilisant les récentes technologies du séquençage à très haut débit, des outils bioinformatiques et du «reciprocal allele-specifique RNA interference » (rasRNAi) dans l’objectif d’identifier les facteurs génétiques et non génétiques responsables de la résistance du moustique Anopheles gambiae aux parasites du paludisme murin Plasmodium berghei. J’ai mis en place une stratégie d’identification des polymorphismes dans les lignées résistantes et sensibles afin de sélectionner des marqueurs génétiques pour de futures analyses génétiques et lister les gènes polymorphiques. J’ai contribué à l’élaboration de nouvelles sondes ARN double-brin (dsRNAs) allèle spécifique pour la méthode du rasRNAi en identifiant le processus de découpage du dsRNA injecté chez des moustiques par l’analyse des petits ARNs séquencés issus du dsRNA injecté. J’ai élaboré un pipeline pour identifier la composition du microbiote des lignées sensibles et résistantes dans le but de les comparer. / During my PhD, I developed and implemented new methods and tools using the latest technologies of the Next Generation Sequencing, bioinformatics tools and the « reciprocal allele-specific RNA interference » (rasRNAi) method with the aim of identifying genetic and non-genetic factors responsible for the resistance of the mosquito Anopheles gambiae to the mouse malaria parasites Plasmodium berghei. I have implemented a strategy for identifying polymorphisms in the resistant and susceptible lines to (1) select genetic markers for future genetic analysis and (2) list the polymorphicgenes. I contributed to the development of a new allele-specific dsRNA probe for the rasRNAi method by identifying how mosquitoes process the injected dsRNA by the analysis of sequenced small RNAs from the injected dsRNA. I developed a pipeline to identify the microbiota composition in susceptible and resistant lines in order to compare them.

Estudo entomológico foi realizado nos limites extremos do município de São Paulo, que possuem cobertura vegetal significativa em forma de Unidades de Conservação representadas ao norte, pelo Parque Estadual da Cantareira (PEC), onde epizootias e casos humanos de febre amarela têm ocorrido nos últimos anos, e ao sul, pela Área de Proteção Ambiental (APA) Capivari Monos onde ocorrem casos assintomáticos de malária-bromélia. Mosquitos foram coletados no período de fevereiro de 2015 a abril de 2017, em sete ambientes com feições periurbana, intermediária e silvestre. Armadilhas CDC foram instaladas na copa e ao nível do solo, com objetivo de verificar a estratificação vertical das espécies de mosquitos. Aspiração e armadilha de Shannon complementaram as coletas de fêmeas ingurgitadas para verificação do comportamento alimentar por técnica de PCR. Nove oligonucleotídeos do citocromo B mitocondrial foram utilizados para este propósito. Riqueza, abundância, composição e equabilidade foram utilizados para verificar a diversidade dos ambientes. No total foram obtidos 10.070 espécimes de culicídeos adultos distribuídos em 95 espécies/categorias taxonômicas distribuídas em dezesseis gêneros. Na APA, foram identificadas 75 espécies/categorias taxonômicas e 7.472 espécimes e desse total 67% foram mais frequentes na copa. As espécies Culex (Culex) nigripalpus e Anopheles (Kerteszia) cruzii, ambas ocorrendo com maior frequência no estrato copa em todos os ambientes pesquisados. No PEC, 58 espécies/categorias taxonômicas e 2.598 espécimes foram coletados e 55% foi coletado no estrato solo. Wyeomyia (Prl.) confusa (solo), Culex (Mel.) vaxus (copa) e Limatus durhamii (solo) foram as mais abundantes. Com relação aos hábitos alimentares 386 amostras foram obtidas, Anopheles cruzii representou 70% (270), se alimentando principalmente em hospedeiro humano no estrato solo. A presença de culicídeos vetores evidenciam a alta importância epidemiológica dos ambientes estudados merecendo atenção das autoridades em saúde publica, dada a interação entre as espécies de mosquitos e suas fontes de repasto sanguíneo. / An entomological study was carried out in the extreme limits of the municipality of São Paulo, which have significant vegetation cover in the form of Conservation Units, represented in the north, by the State Park of the Cantareira (PEC), where epizootics and cases human of Yellow Fever have been occurred in the last years, and in the South by the Environmentally Protected Area Capivari Monos, where asymptomatic cases of malaria-bromelia have been occurred. Mosquitoes were collected from February 2015 to April 2017 in seven environments with peri-urban, intermediate and wild features. CDC Traps were installed in the canopy and at ground level, with the objective of verifying the vertical stratification of mosquito species. Aspiration and Shannon traps complemented the collection of engorged females to verify the feeding behavior by PCR technique. Nine mitochondrial cytochrome B oligonucleotides were used to this propose. Richness, abundance, composition and equability were used to verify the diversity of environments. In total, 10,070 adult culicidis were collected, comprising 95 species / taxonomic categories distributed in sixteen genera. At APA, there were 75 species / taxonomic species and 7,472 specimens and a total of 67% were more frequent in the canopy. The species Culex (Culex) nigripalpus and Anopheles (Kerteszia) cruzii, occurred more frequently and canopy stratum in all the environments surveyed. In the PEC, 58 species / taxonomic categories and 2,598 specimes were collected and 55% were collected in the ground strata. Wyeomyia (Prl.) confusa (ground), Culex (Mel.) vaxus (canopy) and Limatus durhamii (ground) were more abundant. Regarding the feeding habits, 386 samples were obtained, Anopheles cruzii represented 70% (270), feeding mainly on human host in the ground strata. The presence of vector culicids evidences the high epidemiological importance of the studied environments, deserving attention of the Public Health Authorities due to the presence and interaction of mosquito species and their sources of blood feeding.

Les femelles moustiques peuvent être vectrices de nombreux agents infectieux (virus, protozoaires, helminthes) pour l'Homme, qui peuvent être la cause de maladies graves comme le paludisme et la dengue. Ces maladies menacent respectivement 50 et 40% de la population mondiale ; le paludisme étant responsable de près d'un million de décès par an. Les méthodes de lutte anti-vectorielle destinées à limiter les populations vectrices et stopper la transmission de maladies, se heurtent au développement incessant de résistances de la part des moustiques et des agents infectieux vis-à-vis des traitements employés. Bien que certaines régions du monde aient réussi à stopper efficacement la transmission de certaines de ces maladies, une grande partie des régions tropicales reste menacée. De plus l'expansion rapide de certaines espèces vectrices, telles qu'Aedes albopictus, accroît les risques sanitaires dans de nouvelles régions du globe. La technique de l'insecte stérile (TIS), qui a permis l'éradication ou la suppression des populations de nombreux insectes nuisibles aux cultures et à l'Homme, représente un moyen de lutte prometteur contre les moustiques. Cette technique s'appuie sur le lâcher en masse de mâles stérilisés par rayonnements ionisants qui, en transférant un sperme stérile aux femelles sauvages, vont permettre une diminution progressive de la population cible. Suite à l'épidémie de chikungunya à l'île de la Réunion en 2005 et face aux menaces permanentes de recrudescence de la dengue et du paludisme, les services de lutte anti-vectorielle réunionnais mettent en place d'importants moyens de lutte contre les populations de moustiques concernées. Toutefois, ces mesures ne permettant pas une diminution durable des densités de vecteurs, une étude de faisabilité est en cours quant à l'utilisation de la TIS pour diminuer et contrôler les populations d’Aedes albopictus, vecteur de la dengue et du chikungunya, et d’Anopheles arabiensis, vecteur du paludisme.Ce travail de thèse s'inscrit dans le cadre du projet TIS Réunion, dans le but d'étudier la biologie et le comportement des souches destinées aux lâchers de mâles stériles. Dans un premier temps, cette étude s'intéresse à la comparaison entre les souches d'élevage d’Anopheles arabiensis et les souches sauvages, ainsi qu'aux modalités de stérilisation des mâles de la souche à sexage génétique. Une seconde partie est consacrée à l'étude de l'effet de l'irradiation sur les mâles d’Aedes albopictus, en étudiant plus particulièrement leur stratégie de reproduction, leur capacité d'insémination en laboratoire, ainsi que leur compétitivité sexuelle et longévité face aux mâles sauvages en conditions semi-contrôlées. / Mosquito females are potential vectors of numerous pathogens (viruses, protozoa, helminths), which can cause serious diseases such as malaria and dengue in humans. These two infectious diseases are threatening 50 and 40% of the world population respectively. Malaria is responsible for nearly one million deaths per year, and is considered by many experts as the most important insect-transmitted disease. Antivectorial control methods, intended to limit the vector populations and to stop the disease transmission have to face many challenges such as the development of mosquitoes' and pathogens' resistance to the treatments employed to control them. Although various regions of the world have succeeded in efficiently stopping the transmission of some diseases, most of the tropical regions remain under threat. In addition, the rapid expansion of some vector species, such as Aedes albopictus, increases the risks in previously safe areas of the world. The sterile insect technique (SIT) has allowed the eradication or suppression of various insect pest populations threatening crops, animal, and human health, and could offer a promising control tool against mosquitoes. The classical SIT relies on the mass releases of males sterilized by ionizing radiation;they transfer sterile sperm to wild females, which results in a progressive reduction of the target population.Following the chikungunya outbreak in Reunion Island in 2005 and considering the constant threat of arecrudescence of dengue and malaria, the anti-vectorial services in Reunion Island are deploying important means to control the relevant mosquito populations. However, these measures do not confer a permanent, or long-lasting reduction of vector densities. A feasibility study is ongoing, evaluating the use of the SIT to diminish and control the populations of Ae. albopictus, a vector of dengue and chikungunya, and Anopheles arabiensis, a vector of malaria. This PhD work was developed in the context of the SIT Reunion project, with the aim of studying the biology and the behaviour of some strains intended for the sterile male releases. Firstly, this study endeavours to compare colonized and wild strains of An. arabiensis, and to determine the sterilisation procedures of the genetic sexing strain males. The second part of this work studies the effect of irradiation on male Ae. albopictus, and most notably their reproductive strategy, the insemination capacity in laboratory, and finally their sexual competitiveness and longevity against wild males under semi-field conditions.

Made available in DSpace on 2014-12-17T15:54:57Z (GMT). No. of bitstreams: 1 GlauciaOF_DISSERT.pdf: 4217190 bytes, checksum: 41f7567f8c73fdeb4a77a570f0e13c41 (MD5) Previous issue date: 2011-02-24 / The scarcity of occurrence records of culicid species, transmitting insects of etiologic agents for arboviruses, protozoan diseases and philarioses, and lack of information about their interactions with the forest environment make the Caatinga one of the most unknown biomes in Brazil. The aim of this study was to identify culicid species in a conservation unit located in a preserved area of the Caatinga and the possible associations with local abiotic factors, as well as characterize the natural and artificial ecotopes present in the Caatinga of Rio Grande do Norte state. The Ecological Station of Serido (ESEC-Serid?) is a Caatinga Conservation Unit located in a rural area of Rio Grande do Norte. The hot and dry climate of the Caatinga, along with a short rainy season, favor the adaptation and resistance of plants, resulting in completely distinct landscapes over the course of the year. The study took place over the course of one year, with monthly collections between 10h-12h, 14h-16h and 18h-20h. Adult mosquitoes were collected in a forest area using a manual suction trap with human bait as attractant at the three times and a Shannon trap for night collections. During the rainy season ovitraps were randomly distributed at different sites. The different capture sites were observed and photographed. Immature and adult insects captured were taken to the laboratory for identification. Of the 5081 insects collected in human-bait and Shannon traps, 75% were Mansonia wilsoni. In addition, Aedeomyia, Aedes, Anopheles, Coquillettidia, Culex, Haemagogus, Mansonia and Psorophora were identified. Of the 92 Haemagogus eggs found, 71 were collected on the banks of a reservoir. Mosquito larvae were found and photographed in an artificial breeding site of a neighboring rural area. Humidity had the greatest influence on culicid density in the Caatinga. Rain was important in the appearance of Haemagogus, Anopheles, Aedes and Psorophora. Temperature had a negative influence on Anopheles triannulatus. The occurence of important vector species of diseases in a little known preserved area of the Caatinga as well as environmental and sociocultural questions are factors that favor the emergence of transmitting insects, making these areas potential subject to the resurgence of diseases / A raridade de registro de ocorr?ncia de esp?cies de culic?deos, insetos veiculadores de agentes etiol?gicos de arboviroses, protozooses e filarioses, aliada ? inexist?ncia de informa??es sobre as intera??es destes com o ambiente silvestre contribui para tornar a Caatinga um dos Biomas mais desconhecidos do Brasil. Este estudo teve como objetivo identificar esp?cies de culic?deos em uma ?rea preservada de Caatinga, as poss?veis associa??es destes com fatores abi?ticos locais e a caracteriza??o dos ec?topos naturais e artificiais existentes na Caatinga do Serid? potiguar. A Esta??o Ecol?gica do Serid? (ESEC-Serid?) ? uma Unidade de Conserva??o da Caatinga situada em uma ?rea rural no interior do Estado do Rio Grande do Norte. O clima quente e seco com um curto per?odo chuvoso favorece a adapta??o de vegetais, resultando em paisagens distintas ao longo do ano. O estudo ocorreu ao longo de um ano, com coletas mensais, nos intervalos entre 10h-12h, 14h-16h e 18h-20h. Os mosquitos adultos foram coletados em ?rea de mata com um sugador manual com atrativo humano nos tr?s hor?rios, uma armadilha Shannon tamb?m foi utilizada nas coletas noturnas. Durante o per?odo chuvoso ovitrampas foram distribu?das aleatoriamente em diferentes pontos. Os diferentes locais de captura foram observados e fotografados. Os imaturos e adultos capturados foram levados ao laborat?rio para identifica??o. De 5081 f?meas coletadas em isca-humana e armadilha Shannon, 75% pertencem a esp?cie Mansonia wilsoni e foram identificados 8 g?neros Aedeomyia, Aedes, Anopheles, Coquillettidia, Culex, Haemagogus, Mansonia e Psorophora. Dos 92 ovos de Haemagogus encontrados, 71 foram ? margem do a?ude. Larvas de mosquitos foram encontradas e fotografadas em criadouro artificial de uma ?rea rural vizinha. O fator que mais influenciou na densidade de culic?deos na Caatinga foi a umidade. A chuva foi importante no aparecimento dos g?neros Haemagogus, Anopheles, Aedes e Psorophora. A temperatura influenciou negativamente Anopheles albitarsis. A ocorr?ncia de importantes esp?cies vetoras de doen?as em uma ?rea preservada e pouco conhecida da Caatinga associada ?s quest?es ambientais e socioculturais s?o fatores que favorecem o aparecimento de insetos transmissores, tornando essas ?reas potencialmente sujeitas ao ressurgimento de doen?as

Le paludisme reste l'une des plus redoutables maladies infectieuses avec plus de 200 millions d'infections et près de 430 000 décès chaque année, principalement des enfants de moins de 5 ans vivant en Afrique subsaharienne. L'espèce Plasmodium falciparum est responsable de la grande majorité de la mortalité. Le contrôle de l'endémie palustre reste encore aujourd'hui un problème majeur de santé publique, notamment à cause des résistances aux antipaludiques développées par les parasites. L'apparition de ces résistances s'opère par la pression de sélection médicamenteuse, et leur diffusion progressive se fait principalement via le déplacement des hôtes infectés. Cependant, la dynamique d'émergence, de diffusion et de persistance des parasites résistants résulte d'interactions complexes entre les antipaludiques, l'Homme, le parasite et le vecteur. Le travail présenté ici participe à la démarche de lutte contre le paludisme en proposant tout d'abord un état des lieux de la résistance de Plasmodium aux antipaludiques utilisés au Cameroun, avec des outils moléculaires, phénotypiques et cliniques. Une deuxième partie explore, in vitro, les possibles conséquences d'une utilisation prolongée des dérivés d'artémisinine sur le phénotype de P. falciparum, alors que la résistance à cette molécule est déjà installée. Le modèle in vitro utilisé a permis de mettre en évidence un nouveau profil de pluri-résistance suite à des pressions continues à l'artémisinine. Enfin, une dernière partie de ce travail analyse le rôle du moustique dans l'épidémiologie des résistances et montre que la sporogonie favoriserait la diffusion des allèles minoritaires, résistants ou non, présents chez l'Homme. L'ensemble de ces travaux confirme la multiplicité des facteurs agissants sur la dynamique de résistance et la complexité de leurs interactions rendant toute prévision très spéculative. Même si une meilleure connaissance des phénomènes sociétaux, épidémiologiques, biologiques et pharmacologiques impliqués dans les résistances reste une priorité, la surveillance phénotypique et génotypique régulière sur le terrain apparait à ce jour, le meilleur outil pour adapter au mieux les stratégies de contrôle du paludisme. / Malaria remains one of the most terrible infectious diseases with more than 200 million infections and 430,000 deaths each year, mostly children under five years old in sub-Saharan Africa. Plasmodium falciparum is responsible for the vast majority of malaria mortality cases. Control of malaria still remains a major public health problem, in particular because of resistances to antimalarials that parasites developed. The apparition of these resistances is due to the drug pressure, and their progressive diffusion is mainly via the travelling of infected hosts. However, the dynamics of emergence, diffusion and persistence of resistant parasites result from complex interactions between the antimalarials, the Human, the parasite and the vector. The work presented here participates in the malaria control process by first proposing an inventory of Plasmodium resistance to antimalarials used in Cameroon, thanks to molecular, phenotypic and clinical tools. A second part explores the possible consequences of prolonged use of artemisinin derivatives on the P. falciparum phenotype, in areas where resistance to this molecule is already established. The in vitro model used showed that continuous artemisinin pressures induced a new pluri-resistance profile. Finally, a last part analyses the role of the mosquito in the epidemiology of resistances and shows that the sporogony favours the diffusion of minority alleles, resistant or not, presented in humans. All this work confirms the multiplicity of forces acting on the dynamics of resistances and the complexity of their interactions making any prediction very speculative. Even if better knowledge of the societal, epidemiological, biological and pharmacological phenomena involved in resistances is a priority, regular phenotypic and genotypic surveillance in the field remains the best tool for adapting malaria control strategies.

Introdução: Assentamentos rurais são recorrentes arranjos sociais em expansão na Região Amazônica, que geram perturbações no ambiente natural e na respectiva ecologia de mosquitos vetores envolvidos na transmissão de malária. Objetivos: Elaborar revisão das espécies do gênero Anopheles que foram registradas na Região Amazônica. Para a pesquisa quantitativa, estabelecer potenciais fatores dos ambientes e das características das águas de diversos habitats larvais que possam estar associadas com a presença de espécies de anofelíneos, através de coletas em criadouros de diferentes graus de perturbação ambiental. Métodos: para a revisão das espécies que foram registradas na Amazônia, foram empregados termos de busca, utilizando sete bases bibliográficas de referência. Para os estudos em campo, foram realizadas capturas de larvas juntamente com estimação e mensuração de algumas condições dos habitats larvais, para comparação dos parâmetros analisados entre ambientes naturais e artificiais. Resultados: a revisão mostrou maior quantidade de artigos que notificaram a ocorrência do subgênero Nyssorhynchus, seguido pelos subgêneros: Anopheles, Stethomyia, Kerteszia e Lophopodomyia. O Anopheles darlingi se mostrou a espécie com maior ocorrência nos artigos levantados, muito pelo seu caráter epidemiológico. Importantes vetores auxiliares pertencentes ao subgênero 6 Nyssorhynchus também foram registrados em grande quantidade. Os resultados das análises conduzidas com os dados das capturas em campo demonstraram que as forças de transformação do ambiente natural favorecem o desenvolvimento de habitats larvais de espécies do subgênero Nyssorhynchus, incluindo o An. darlingi, além de um importante vetor secundário, o An. triannulatus. Discussão e Conclusão: projetos de assentamentos na Amazônia brasileira registram índices significativos de casos de malária, devido à ocupação de áreas naturais pelo homem. Este estudo mostrou que o subgênero Nyssorhynchus possui amplo registro na literatura de inquéritos entomológicos do gênero na floresta tropical equatorial, indicando que o agrupamento pode ser considerado endêmico às fronteiras de transformação na região, tendo assim importante papel na manutenção da transmissão da malária. Os estudos de campo apresentaram evidências de que formação de habitats artificiais favorecem formas iniciais de vida dos mosquitos com histórico vetorial para a região. Esses habitats larvais podem ser respostas ao uso do solo e das consequentes alterações provocadas no ambiente natural pelas atividades humanas. As modificações do ambiente natural selecionam e propiciam a formação de habitats para as espécies do Nyssorhynchus. Este agrupamento taxonômico engloba espécies que são consideradas vetores para a malária na região Neotropical. / Introduction: Rural settlements are recurrent social arrangements expanding in the Amazon region, which generate disturbances in the natural environment, affecting the mosquitoes ecology involved in malaria transmission. Objective: A systematic review of Anopheles species were recorded in the Amazon region. For the qualitative research, were establish potential factors of the environment and the characteristics of the waters of several larval habitats that may be associated with the presence of species of Anopheles mosquitoes, by surveys of the samples presents in larval habitats within environmental disturbance gradient. Methods: for the review of the species, were used search terms, in seven bibliographic databases of reference. For studies in the field, larvae were trapped along estimation and measurement of some conditions of the larval habitats, to compare the parameters analyzed between natural and artificial environments. Results: The review showed greater amount of articles that reported the occurrence of Nyssorhynchus subgenre, followed by: Anopheles, Stethomyia, Kerteszia and Lophopodomyia. The Anopheles darlingi proved the species with the highest occurrence in the papers showed, due to its epidemiological importance. Important auxiliary vectors belonging to the subgenus Nyssorhynchus were also recorded in large scale. The results of the qualitative shown that the natural 8 environment transformation forces favor the development of larval habitats of the species of subgenus Nyssorhynchus, including An. darlingi and an important secondary vector, An. Triannulatus. Discussion and Conclusion: settlement projects in the Brazilian Amazon recorded significant rates of malaria cases. This study showed that, the Amazon basin an important determinant of this phenomenon is the formation of artificial habitats that are occupied by early forms of life of mosquito vectors. These larval habitats may be answers to land use and consequent changes in the natural environment caused by human activities. Modifications to the natural environment select and provide the formation of habitats for species in Nyssorhynchus. This taxonomic group includes species that are considered vectors for malaria in the Neotropics. Discussion and Conclusion: settlement projects in the brazilian Amazon recorded significant rates of malaria cases. This study showed that the review of the literature shows Nyssorhynchus subgenre ample record in entomological surveys in the equatorial rainforest, indicating that the group may be considered endemic to the borders of transformation in the region, and thus important role in the maintenance of malaria transmission. Field studies showed evidence that formation of artificial habitats favor early forms of the mosquito vector, with history for malaria in the region. These larval habitats may be answers to land use and consequent changes in the natural environment caused by human activities. Modifications to the natural environment select and provide the formation of habitats for species in Nyssorhynchus.

Made available in DSpace on 2014-06-12T15:05:33Z (GMT). No. of bitstreams: 2 arquivo1798_1.pdf: 723582 bytes, checksum: a74b99fbc6caba53d874185d4c81c04e (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2005 / Embora a malária seja uma doença endêmica no Estado de Roraima, o registro das espécies de anofelinos que ocorrem na região ainda é escasso. Nesse estudo investigou-se a ocorrência, abundância e distribuição das espécies do gênero Anopheles, seus principais criadouros, a influência das estações seca e chuvosa sobre as populações desses mosquitos, bem como, a freqüência horária da sua atividade hematofágica. Duas áreas com diferentes graus de impactação antrópica foram escolhidas no município de Cantá. Cinco pontos fisionomicamente distintos, compreendendo: peridomicílio, intermediário, extradomicílio, borda de mata primária e curral, foram selecionados como locais de coleta nas áreas A e B. Os mosquitos foram capturados durante coletas de 4 horas (18-22h) e 12 horas (18-06h) por três e dois dias respectivamente, nos meses de fevereiro, maio, agosto e novembro de 2004. Um total de 3.279 espécimes foram capturadas, compreendendo 11 espécies de anofelinos: A. albitarsis (41,8%); A. darlingi (24,8%); A. triannulatus (17,6%); A. nuneztovari (11,8%); A. oswaldoi (1,5%); A. evansae (1,4%); A. argyritarsis, A. braziliensis, A. intermedius, A. mediopunctatus e A. peryassui representaram menos de 1% da amostra. A. albitarsis (55,5%) e A. darlingi (22,7%) predominaram na área mais antropizada (área A), enquanto A. triannulatus (34,6%) foi mais abundante na área mais preservada (área B). A maior densidade ocorreu em maio (período chuvoso), enquanto as menores, apresentaram-se em fevereiro e novembro (período de estiagem). Nos 21 criadouros encontrados, registrou-se nove espécies de anofelinos, duas das quais, A. minor e A. strodei, não foram capturadas como adultos. A atividade hematofágica dos anofelinos, durante as coletas de 12h, iniciou-se no primeiro horário de coleta (18-19h) nas duas localidades, sendo esse o horário de pico na área B. Na área A o pico ocorreu das 19-20h. A única espécie que manteve atividade durante toda a noite foi A. darlingi, com pico das 21-22 horas. Esses resultados mostraram uma grande diversidade de anofelinos na área estudada cuja abundância pode ser influenciada por ações antrópicas no ambiente e variações climáticas. Indicam também que A. albitarsis e A. darlingi apresentam maior atividade hematofágica no peridomicílio, favorecendo a transmissão do parasito da malária na região

Les modèles de distribution d’espèces ont été identifiées comme pertinents pour cartographier et caractériser la qualitéd’habitat des moustiques vecteurs du paludisme, Anopheles, afin de participer à l’estimation du risque de transmission decette maladie et à la définition de stratégies de lutte anti-vectorielle ciblées. La transmission du paludisme dépend de laprésence et de la distribution des vecteurs, qui dépendent elles-mêmes des conditions environnementales définissant laqualité des habitats écologiques des Anopheles. Cependant, dans certaines régions, les données de captures d’Anophelessont rares, et rend difficile la cartographie de leurs habitats. De plus, le recueil de ces données est très souvent soumis à desbiais d’échantillonnage.Cette thèse fournit une solution à la cartographie des vecteurs du paludisme, en considérant deux aspects très peu étudiésdans la modélisation : le faible nombre de sites de présence disponibles et l’existence d’un biais d’échantillonnage. Uneméthode originale de correction de l’effet du biais d’échantillonnage est proposée puis appliquée à des données de présencedu principal vecteur du paludisme en Guyane, Anopheles darlingi. Un modèle de distribution d’An. darlingi a ensuite étéconstruit, permettant d’obtenir une carte de qualité d’habitat en cohérence avec la connaissance des entomologistes etfournissant des performances de prédiction élevées. La méthode de correction proposée a ensuite été comparée auxméthodes existantes dans un contexte applicatif caractérisé par la rareté des données d’occurrence de l’espèce et laprésence d’un biais d’échantillonnage. Les résultats montrent que la méthode développée est adaptée aux cas où le nombrede sites de présence est faible. Cette thèse contribue, d’une part, à combler les lacunes théoriques et d’applicabilité desméthodes actuelles visant à corriger l’effet des biais d’échantillonnage et, d’autre part, à compléter la connaissance sur ladistribution spatiale et la bioécologie du principal vecteur du paludisme en Guyane française. / Species distribution models are identified as relevant to map and characterize the habitat quality of Anopheles genusmosquitoes, transmitting malaria, and thus to both participate in the estimation of the transmission risk of this disease and inthe definition of targeted vector control actions. The malaria transmission depends on the presence and distribution of thevectors, which are themselves dependent on the environmental conditions that define the quality of the ecological habitats of the Anopheles. However, in some areas, Anopheles collection data remain scarce, making it difficult to model these habitats. In addition, the collection of these data is very often subjected to significant sampling biases, due, in particular, to unequal accessibility to the entire study area. This thesis provides a solution to the mapping of malaria vectors, considering two very few studied aspects in modeling: the low number of available presence sites and the existence of a sampling bias. An original method for correcting the effect of the sampling bias is proposed and then applied to presence data of Anopheles darlingi species - the main vector of malaria in South America - in French Guiana. Then, a distribution model of An. darlingi was built to obtain a map of habitat quality consistent with entomologists’ knowledge and providing high prediction performances. The proposed correction method was then compared to existing methods in an application context characterized by the scarcity of the species occurence data and the presence of a sampling bias. The results show that the developed method is adapted to cases where the number of sites of presence is low. This thesis contributes, on the one hand, to fill theoretical and applicability lacuna of current methods intended to correct the effect of the sampling bias and, on the other hand, to supplement the knowledge on both the spatial distribution and the bio-ecology of the main malaria vector in French Guiana.