Orientador: Ricardo Luiz Dantas Machado / Banca: Mauro Toledo Marrelli / Banca: Carlos Eugênio Cavasini / Resumo: Responsável por 300 a 500 milhões de novas infecções e 1,5 a 3 milhões de mortes por ano no mundo inteiro, a malária continua sendo a principal doença parasitária. Se importante é o combate contra os parasitos, importante também é o conhecimento dos aspectos de incidência, distribuição, especificidade de hospedeiros, além do conhecimento de fatores biológicos e moleculares que determinam a distribuição destes. Neste cenário, um dos principais parâmetros analisados para o controle e monitoramento da malária é a detecção de espécies de Plasmodium nos vetores capazes de infectarem humanos. Embora existam diversas metodologias com este fim, a maioria delas apresenta algumas limitações. O objetivo do presente estudo foi desenvolver um método que permita a identificação do P. falciparum, do P. malariae e das variantes do P. vivax no vetor. Uma PCR foi padronizada, utilizando iniciadores contra regiões específicas do gene CS. A PCR-RFLP foi utilizada para distinguir as variantes do P. vivax. A eficiência da metodologia desenvolvida foi comparada com uma nested-PCR, utilizando Anopheles infectados experimentalmente. Um total de 90 mosquitos foram infectados artificialmente com P. vivax (n = 30), P. falciparum (n = 30) e P.malariae (n = 30). Estes mosquitos infectados, juntamente com outros 30 não infectados foram avaliados em relação a identificação do Plasmodium por nested PCR e com o CS-PCR. A nested PCR para o P. vivax, P. falciparum e P. malariae identificou 19, 16 e 21 mosquitos positivos, respectivamente; enquanto o CS-PCR detectou em 17, 14 e 16 mosquitos, respectivamente. A comparação entre os dois métodos revelou uma boa concordância (κ = 0,723, 0,867 e 0,657, respectivamente, para P. vivax, P. falciparum... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Malaria remains the most serious vector-borne disease, affecting some 300-500 million people annually, causing 1.5-3 million deaths. Is important the fight against the parasites, but it is also important the knowledge of the incidence, host specificity, molecular and biological factors determining the distribution of these parasites. In this scenario, identification of the human-specific Plasmodium species in the mosquito host is an essential component for planning and monitoring of malaria control operations. However, most of the detection methods show some potential limitations. The objective of this study was development an effective assay for detecting Plasmodium falciparum, Plasmodium malariae and Plasmodium vivax variants in Anopheles mosquitoes. A PCR was development targeting the CS gene. A PCR-RFLP was used to distinguish the P. vivax variants VK210, VK247 and P. vivax-like. The new PCR assay was compared against a nested PCR using artificially infected Anopheles mosquitoes. A total of 90 mosquitoes were artificially infected with P. vivax (n = 30), P. falciparum (n = 30) and P. malariae (n = 30). These infected mosquitoes along with another 30 unfed mosquitoes were checked for the identification of Plasmodium by nested PCR and with the CS-PCR. Nested PCR for P. vivax, P. falciparum and P. malariae detected positive infection in 19, 16 and 21 mosquitoes respectively; whereas CS-PCR detected in 17, 14 and 16 mosquitoes, respectively. The comparison revealed a close agreement between the two assays (κ = 0.723, 0.867 and 0.657, respectively for P. vivax, P. falciparum and P. malariae groups). Subsequently, PCR-RFLP efficiently discriminate P. vivax variants... (Complete abstract click electronic access below) / Mestre

INTRODUÇÃO: O subgrupo Anopheles strodei é pouco estudado apesar de sua importância epidemiológica potencial. Espécies desse subgrupo foram encontradas naturalmente infectadas por parasitos que causam malária em humanos, Plasmodium falciparum, Plasmodium vivax e Plasmodium malariae, no Brasil. O subgrupo Anopheles strodei compreende oito espécies: An. rondoni (Neiva & Pinto), An. albertoi Unti, An. arthuri Unti, An. strodei Root, An. strodei CP, e três outras espécies que foram propostas por Bourke et al. (2013), mas não foram descritas: An. arthuri B, An. arthuri C e An. arthuri D. OBJETIVOS: A definição e delimitação precisa de espécies que atuam como vetores de agentes infecciosos é um dos objetivos da entomologia da saúde pública. Os objetivos deste estudo foram: 1) Estabelecer as relações filogenéticas entre espécies do Subgrupo Strodei; 2) Estimar a distribuição espacial potencial das espécies do Subgrupo Strodei; 3) Confirmar a presença de quatro espécies sob o nome An. arthuri. MÉTODOS: Sequências de DNA de um gene mitocondrial (fragmento de 658 pares de bases do código de barras do gene COI, citocromo oxidase subunidade I) e de três genes nucleares codificadores de proteínas (White, CAD e CAT) foram empregadas para estabelecer as relações filogenéticas potenciais entre as espécies que compõe o subgrupo An. strodei. As análises filogenéticas foram conduzidas utilizando abordagem Bayesiana das sequências de DNA dos quatro genes. Para estabelecer a distribuição espacial potencial das espécies, utilizou-se abordagem de máxima entropia de nichos ecológicos. Para isso as localidades das coletas, juntamente com os dados climáticos e geográficos foram introduzidos no programa MAXENT. RESULTADOS: Os resultados das análises filogenéticas demonstraram e, portanto, confirmaram o monofiletismo do Subgrupo Strodei, a presença de pelo menos sete espécies sob o nome An. strodei, ou seja, corroborou a validade de An. albertoi, An. arthuri, An. strodei, An. strodei CP, além das espécies denominadas, preliminarmente, como An. arthuri B, An. arthuri C e An. arthuri D. Portanto, como definida atualmente, An. arthuri não representa grupo monofilético, pois inclui táxons que deverão ser formalmente descritos em estudos futuros. CONCLUSÃO: As distribuições potenciais de espécies do Subgrupo Strodei foram propostas pela primeira vez. Cinquenta e cinco sequências do gene nuclear CAT e outras 46 sequências do gene nuclear CAD únicas foram recentemente caracterizadas para espécies do Subgrupo Strodei de Anopheles (Nyssorhynchus), confirmando a presença de pelos menos sete espécies, além de An. rondoni que não foi alvo deste estudo, mas de outros anteriores que confirmaram a validade da mesma. / Introduction Anopheles strodei sensu lato is an understudied subgroup of potential epidemiological importance, having been found naturally infected in Brazil with Plasmodium falciparum, Plasmodium vivax and Plasmodium malariae. An. strodei s.l. is currently composed on 8 species: An. albertoi Unti, An. CP Form, An. rondoni (Neiva & Pinto), An. strodei Root, An. arthuri Unti and three other unnamed species that have been proposed by Bourke et al. (2013): An. arthuri B, An. arthuri C and An. arthuri D. Objectives As delineating species accurately is an essential goal of public health entomology, the objectives of this study were to: 1) Determine the phylogenetic relationships within the Strodei Subgroup and reaffirm or reject the hypothesis of the 3 new species (An. arthuri B, An. arthuri C and An. arthuri D) 2) Address the potential spatial distribution of species of the An. strodei subgroup to provide support for the candidate species in the Strodei Subgroup Methods Bayesian inference, which included DNA sequences of one mitochondrial and three nuclear protein coding genes: CO1, white, CAD and CAT, was used to determine the phylogenetic relationship within the group. To propose a species distribution, collection localities, along with climatic and geographic data were input into MAXENT. Results When analyzing the four molecular markers employed, support was found for allopatry in the Strodei Subgroup. The paraphyletic clade of An. arthuri was supported. Conclusion Potential species distributions of the Strodei Subgroup were addressed for the first time. Fifty-five unique CAT sequences and 46 unique CAD sequences were newly characterized.

Problemas de saúde pública advindos da proliferação de vetores de agentes patogênicos são frequentemente relatados após a construção de grandes barragens nas regiões tropicais. Porto Primavera é um dos últimos grandes projetos hidrelétricos implementados no Brasil, abrangendo uma faixa de cerca 200 km do Rio Paraná, entre os Estados de São Paulo e Mato Grosso do Sul. Resultados preliminares de projetos de pesquisa desenvolvidos na região desde 1997 apontam para um aumento na densidade de Anopheles darlingi, coincidindo com uma intensificação dos fluxos e atividades humanas na área, como turismo, pesca e transporte. Diante disso, a preocupação com a ocorrência de surtos de malária nas localidades à margem do lago motivou a busca de um maior conhecimento sobre as populações destes vetores. O objetivo desta pesquisa foi investigar a associação entre a abundância das formas imaturas e adultas de An. darlingi com variáveis do microhabitat e da ecologia da paisagem (respectivamente), após a formação do reservatório. Em relação ao microhabitat, foram selecionados o pH, condutividade, o sombreamento, a presença de cianofíceas filamentosas, detritos flutuantes, matéria orgânica galhos ou raízes e a composição das assembléias de macrófitas aquáticas. Quanto à ecologia da paisagem, foram analisados o comprimento da borda abrigo-criadouro e o tamanho das áreas dos habitats através das técnicas de sensoriamento remoto e SIG. Para a verificação de associações entre variáveis do microhabitat e imaturos de An. darlingi foi empregada a análise de correspondência canônica e para as métricas da paisagem e formas adultas, a regressão linear simples. As formas imaturas de An. darlingi apresentaram associação positiva com todas as variáveis do microhabitat, exceto com a abundância de cianofíceas filamentosas. As associações mais fortes se deram na seguinte ordem: abundância de Wolffia sp. e profundidade no primeiro eixo canônico e presença de material flutuante tipo II, pH e grupo de macrófitas e componentes da superfície no segundo eixo. Foi encontrada associação positiva entre abundância de espécimes adultos de An. darlingi e o comprimento da borda fragmento florestal-coleção hídrica, mas não em relação às áreas dos fragmentos florestais. De acordo com estes resultados, novos referenciais teóricos podem ser aplicados no mapeamento do risco da transmissão da malária em escala regional e na vigilância e controle entomológicos em nível local. / Public Health problems raised up from vector-borne diseases proliferation are frequently related after the construction of big dams in tropical regions. Porto Primavera is one of these greats hydroeletric projects stabilished recently in Brazil, occuping aproximately 200 km of Paraná river between São Paulo and Mato Grosso do Sul States. Preliminary results on developping investigations up to 1997 showed an Anopheles darlingi increase simultaneously the intensification of human moviment and activities in region, such as turism, fishing and transport. In face to the worry about the possibility of occurrence of malaria outbreaks in localities around the Lake raised the motivation to investigate a better knowledge about the populations of this vectors. In general, the objective was to investigate the association between the abundances of immatures and adult forms of Anopheles darlingi with microhabitat variables and landscape ecology respectively, after filling the reservoir. In relation to microhabitat variables was selected: pH, condutivity, shadow, deep, presence of filamentous cianoficeas, floating debris, and composition of aquatic macrophytes. In relation to landscape ecology it was analysed the perimeter of the florested shelters and breeding sites-edge and the area of florestal fragments, based on remote sensing and GIS technologies. Cannonical Correspondance analysis was the method to verify the association between microhabitat variables and immatures abundance. A large distribution of An. darlingi was noted because was find in all surveyed locations. Immature forms it was be positively associated with all microhabitat variables except filamentous cianoficea. The strongly association were in order: Wolffia sp. and deep to the first cannonical axis and floating debris of Type II, pH and macrophytes groups and surface components for the second. The Simple Linear Regression was the method to verify associations between landscape ecology variables and adult forms was find positive association between the abundance of An. darlingi and the perimeter of the florested shelters and breeding sites-edge, but not to the area of florestal fragments. According to this results, new theoretical knowledges are able to apply in the risk maps elaboration for malaria transmission in regional scale and to enthomological vigilance and control in local scale.

A Malária autóctone de Mata Atlântica é caracterizada por baixa incidência de casos com pouca ou nenhuma manifestação clínica e baixa parasitemia, tendo como principal agente etiológico o Plasmodium vivax (ou plasmódios muito semelhantes a este). O principal mosquito envolvido na transmissão é o Anopheles (Kerteszia) cruzii, cujas formas imaturas se desenvolvem na água acumulada nas imbricações das folhas de bromélias, vegetal muito abundante neste bioma. As formas adultas de An. cruzii tendem a viver e se alimentar com maior frequência na copa das árvores, comportamento conhecido como acrodendrofilia, no entanto, em diversas situações esta espécie tem sido observada em elevada densidade no estrato inferior da floresta. Evidências sugerem que a malária possa ser transmitida de forma zoonótica nestas áreas, uma vez que primatas das famílias Atelidae e Cebidae são encontrados portando plasmódios muito próximos geneticamente aos que infectam humanos e An. cruzii pode se alimentar do sangue de ambos os hospedeiros. O presente estudo teve como objetivo investigar a influência da composição e configuração da paisagem sobre as variações na abundância e no comportamento acrodendrófilo de An. cruzii e como estas variações atuam sobre a dinâmica de transmissão de malária na Mata Atlântica, considerando um cenário de transmissão zoonótica. Coletas entomológicas mensais foram conduzidas no período de março de 2015 a abril de 2017 em duas unidades de conservação situadas no município de São Paulo, ambas com histórico de malária autóctone. Em cada área foram selecionados pontos de coleta com diferentes graus de interferência antrópica e os mosquitos foram coletados utilizando armadilhas do tipo CDC e Shannon. Métricas de composição e configuração da paisagem foram mensuradas para cada ponto de estudo e modelos estatísticos foram utilizados para avaliar a relação entre estas métricas e variações na abundância e acrodendrofilia de An. cruzii. Foi proposto um modelo matemático de transmissão zoonótica que permitiu observar, por meio de simulações, como a dinâmica de transmissão da malária é influenciada por variações na abundância e acrodendrofilia do vetor. Os resultados obtidos sugerem que a perda de vegetação natural e aumento das áreas de borda levam a uma redução na abundância de An. cruzii mas favorecem uma maior atividade desta espécie próximo ao solo. As análises baseadas no modelo teórico corroboram com observações anteriores, apontando que as variações na acrodendrofilia do vetor podem ter um papel importante na dinâmica de transmissão de plasmódios entre símios e humanos na Mata Atlântica. Em condições nas quais An. cruzii tende a se alimentar quase exclusivamente na copa das árvores ou quase exclusivamente próximo ao solo a transmissão zoonótica parece não se sustentar, ainda que a abundância do vetor seja elevada. De outra forma, condições nas quais An. cruzii se desloque com maior frequência entre os estratos copa e solo tendem a favorecer a transmissão zoonótica, sendo que quanto maior o deslocamento menor é a abundância relativa do vetor necessária para que um símio infectado gere mais do que um caso novo na população de humanos suscetível ou vice-versa. / The autochthonous Malaria of the Atlantic Forest is characterized by a low incidence of cases with little or no clinical manifestation and low charge of parasites in the blood stream, being Plasmodium vivax (or plasmodiums very similar to it) the main etiologic agent. The main mosquito involved in transmission is the Anopheles (Kerteszia) cruzii, whose immature forms develop in the water held in bromeliads, which are very abundant plants in this biome. The adult forms of An. cruzii prefer to live and feed on the treetops, a behavior known as acrodendrophily, however, in several situations this species has been observed in high density near the ground of the forest. Some evidences suggest that malaria can be transmitted zoonotically in these areas, since monkeys of Atelidae and Cebidae families are found carrying plasmodiums very close to those that infect humans and An. cruzii can feed on the blood of both hosts. This study aimed to investigate the influence of the landscape composition and configuration on the variations in the abundance and acrodendrophic behavior of An. cruzii and how these variations affect the dynamics of malaria transmission in the Atlantic Forest, considering a zoonotic transmission scenario. Monthly entomological collections were conducted from March 2015 to April 2017 in two conservation units located in the municipality of São Paulo, Brazil, where human and simian malaria occurs. In each area, collection points with different degrees of anthropic intervention were selected and mosquitoes were collected using CDC and Shannon traps. Metrics of landscape composition and configuration were measured for each study point and generalized linear models were used to evaluate the relationship between these metrics and variations in abundance and acrodendrophily of An. cruzii. It was proposed a mathematical model of zoonotic transmission that allowed to observe, through simulations, how the dynamics of malaria transmission is influenced by variations in the abundance and acrodendrophily of the vector. The results suggest that loss of natural vegetation and increase in edge effect lead to a reduction in the abundance of An. cruzii but favor a higher activity of this species near the ground level. The analyzes based on the theoretical model corroborate previous observations, pointing out that the variations in the acrodendrophily of the vector may play an important role in the dynamics of plasmodium transmission between monkeys and humans in the Atlantic Forest. In situations in which An. cruzii seeks to feed almost exclusively on the treetops or in situations where it feeds almost exclusively near the ground, the zoonotic transmission does not seem to occur, even in situations of high abundance of the vector. Otherwise, conditions in which An. cruzii moves more frequently between canopy and ground level tend to favor zoonotic transmission, and the more vertical movement occurs, the lower is the needed relative abundance of the vector for an infected monkey to generate more than a new case in the susceptible human population or vice versa.

La gestion de la résistance aux insecticides et plus particulièrement aux pyréthrinoïdes chez les vecteurs du paludisme est un enjeu majeur pour conserver les acquis de la lutte antivectorielle contre le paludisme. Parmi les mécanismes de résistance, de nombreux travaux ont porté sur les mutations de la cible insecticides (kdr) ou encore les enzymes qui métabolisent les molécules insecticides. La résistance cuticulaire chez Anopheles gambiae a jusqu'à ce jour au mieux été suspectée mais pas démontrée. Dans ce cadre, le but de ce travail était donc de déterminer i) s’il y a une implication de la cuticule dans le phénotype résistant d’Anopheles gambiae aux pyréthrinoïdes ; ii) de déterminer les principaux acteurs de cette résistance. Nous avons construit une souche d’Anopheles gambiae présentant un phénotype résistant (MRS) mais ne portant pas la mutation kdr. Notre approche a été d’étudier l’expression des gènes, mais aussi celle des protéines de la cuticule et celles intervenant dans la détoxication. L’ultrastructure de la cuticule, sa composition en chaines hydrocarbonées ainsi que sa perméabilité à la deltaméthrine ont aussi été testés. Nous avons montré que la cuticule est impliquée dans le phénotype résistant plus particulièrement en réduisant significativement la pénétration de la deltaméthrine dans le corps de l’insecte. La structure même de la cuticule est profondément modifiée chez MRS. Nos résultats indiquent l’implication des membres de la famille CPAP3 dans la résistance cuticulaire et probablement des chaines hydrocarbonées. Des enzymes métaboliques sont également impliqués. Au cours de ce travail, nous avons démontré l’existence de la résistance cuticulaire chez An.gambiae, ce mécanisme agit de concert avec les mécanismes de détoxication. La caractérisation des différents acteurs nous permettra surement de trouver de nouvelles cibles pour la lutte anti-vectorielle.Mots clés : Résistance, cuticule, An.gambiae, deltaméthrine, ultrastructure, perméabilité, protéines / The management of Anopheles insecticide resistance, especially to pyrethroids, is a key challenge to preserve success of vector control against malaria. Among resistance mechanisms target site mutation (kdr) and metabolic resistance are well known. Cuticular resistance in malaria vectors has been overlooked, just suggested but not established so far. The aim of this PhD work was to demonstrate i) the involvement of cuticle in pyrethroid resistance in An. gambiae; ii) to determine which components act on this mechanism. To achieve this, we built An.gambiae strain resistant to pyrethroids and free of kdr mutation (MRS). We studied both cuticular and metabolic genes and proteins expressions by transcriptomic and proteomic approaches. Cuticle ultrastructure and biochemical composition were also investigated. At least, cuticle permeability to deltamethrin was also assessed. Our results showed that cuticle is involved in resistant phenotype in An.gambiae. MRS cuticle reduces insecticide uptake in the mosquito’s body linked to an increasing cuticle thickness in MRS. Expression and chemical studies revealed that CPAP3 family and epicuticular hydrocarbons takes part in this process. This work also suggested that metabolic enzymes act together with cuticular mechanism to resistant phenotype. How the different components interacts could improve our knowledge of resistance and bring new target for vector control.Keywords: Resistance, cuticle, An.gambiae, deltamethrin, ultrastructure, permeability, proteins

La zone transfrontalière entre la Thaïlande et le Myanmar (ZTTM) est une zone d'endémicité palustre où l'intensité de la transmission est faible, saisonnière et instable. Par conséquent, le paludisme est symptomatique dans toutes les classe d'âge et responsable d'une mobi-mortalité élevée, en particulier chez les femmes enceintes et les enfants. Les résistances de Plasmodium falciparum aux médicaments antipaludiques constituent le principal obstacle à la lutte contre le paludisme dans la région. L'apparition de souches de P. falciparum résistantes aux derniers médicaments encore efficaces (notamment les résistances à l'artémisinine) est particulièrement inquiétante car elle menace les acquis fragiles toutefois très importants en terme de contrôle du paludisme (dans la ZTTM, l'incidence du paludisme a été divisée par 20 au cours des trente dernières années passant 2 cas/personne/an dans le milieu des années 1980 à <0.1 cas/personne/an après 2010). L'objectif principal de cette thèse est de d'évaluer l'impact d'une stratégie d'élimination de P. falciparum basée sur des moyens médicamenteux (accès universel au diagnostic et traitement précoce et élimination du réservoir asymtpomatique, généralement sous-microscopique, par le traitement de masse)sur les indicateurs entomologiques de la transmission du paludisme. Les objectifs secondaires sont multiples et visent à mieux décrire et comprendre les modalités et la dynamique de la transmission du paludisme. / The Thai-Myanmar border is an area of low, seasonal and unstable malaria transmission. Consequently, the infection is symptomatic in all age groups and malaria morbidity and mortality is high especially in pregnant women and children.Antimalarial drug resistances in Plasmodium falciparum constitute the main obstacle to tackle malaria in this area. In the absence of alternative antimalarial drugs, the development of artemisinin resistance in Plasmodium falciparum is a major threat to global public health as it could impair the important yet labile progress made to reduce malaria endemicity (on the TMB, malaria incidence drastically decrease from 2 cases / person / year in 1980 to 0.1 cases / person / year after 2010. The primary objective of this work is to evaluate the impact of antimalarial-based elimination strategy relying on universal coverage with early diagnosis and treatment and elimination of malaria transmission hotspots (villages were the prevalence of asymptomatic infection, usually submicroscopic)with mass drig administration. Secondary objectives aim at describing and understanding the modalities and dynamic of malaria transmission in the studied area.

La lutte anti-vectorielle manque d‘efficacité quand elle ne cible pas de façon optimale les lieux et les périodes où les vecteurs prolifèrent. Des outils tels que la télédétection ou les systèmes d‘information géographique permettent d‘accéder et de traiter un large panel de données environnementales. La mise en relation des données télédétectées avec des observations entomologiques peut contribuer à améliorer les connaissances sur l‘écologie des vecteurs et permettre de développer des modèles prédictifs de la dynamique de leurs populations. Aucun modèle de ce type n‘est actuellement disponible en Guyane pour assister les autorités chargées de la lutte anti-vectorielle. L‘objectif général a été d‘identifier les déterminants environnementaux de la distribution et de la densité des anophèles, moustiques responsables de la transmission du paludisme, pour l‘implémentation d‘outils prédictifs de la situation entomologique. Une attention particulière a été portée sur l‘utilisation des données issues de l‘imagerie satellitaire. Un premier travail de caractérisation de la dynamique spatiotemporelle de la faune anophélienne a été réalisé dans la région de Saint-Georges de l‘Oyapock. Cette étude a confirmé l‘impact des facteurs environnementaux, en particulier de l‘occupation du sol et de la pluviométrie, sur la distribution et la densité des moustiques anophèles. La seconde partie des travaux a eu pour objectif la modélisation et la cartographie dynamique des densités d‘Anopheles darlingi, le principal vecteur du paludisme en Guyane. Parmi un panel de variables environnementales télédétectées et de paramètres météorologiques, six prédicteurs ont été identifiés. L‘implémentation de ces prédicteurs dans un modèle statistique et son extrapolation a permis de produire des cartes hebdomadaires prédictives des densités d‘An. darlingi à Saint-Georges de l‘Oyapock. La troisième partie des travaux a visé à actualiser les connaissances sur la faune anophélienne dans la région de Cayenne. A partir de données télédétectées, un modèle prédictif de la présence d‘An. aquasalis a été implémenté. Les résultats, dans leur ensemble, mettent en évidence l‘intérêt de l‘imagerie satellitaire pour la surveillance et le contrôle des vecteurs du paludisme en Guyane. Des outils prédictifs et d‘aide à la décision à destination des acteurs de la lutte anti-vectorielle sont proposés. Dans un contexte de besoin de nouvelles stratégies et méthodes pour améliorer la surveillance entomologique, ces travaux sont d‘une utilité majeure pour les autorités de santé et répondent pleinement aux objectifs du plan quadriennal de lutte contre le paludisme publié en 2015 par l‘Agence Régionale de Santé de Guyane. / Vector control is often seen as inefficient because it is not targeted to sites and times when mosquitoes are abundant. Tools such as remote sensing and geographic information systems can access and process a wide range of environmental data. The linking of remote sensing data with entomological observations can establish knowledge on vectors ecological preferences and be used to develop predictive models of mosquitos‘ dynamics. No model of this type is currently available in French Guiana to assist vector control strategies. The general aim of the thesis was to identify environmental determinants of the distribution and the density of anopheles mosquitoes for the implementation of predictive tools. The research was conducted on two study sites: Saint-Georges de l‘Oyapock and Cayenne region. First, a characterization of the spatiotemporal dynamics of the anopheles fauna in Saint-Georges de l‘Oyapock was conducted. This preliminary study confirmed the impact of environmental factors, especially land cover and rainfall, on the distribution and the density of anopheles mosquitoes. The second part of the work was aimed modeling and dynamic mapping of Anopheles darlingi densities, the main malaria vector in French Guiana. Among a panel of remotely sensed environmental variables and meteorological parameters, six accurate predictors have been identified. The implementation of these parameters in a statistical model and its extrapolation allowed producing predictive maps of An. darlingi densities in the municipality of Saint-Georges de l‘Oyapock. The third part of the work has updated knowledge on the anopheles fauna of Cayenne region. Also, based on remotely sensed environmental data, a predictive model of the presence of An. aquasalis was implemented. Outcomes of this thesis highlight the interest of satellite imagery to monitor and control malaria vectors in French Guiana. This work offers predictive and decision support tools to assist vector control actions. In a context of need of new strategies and methods to improve entomological surveillance, this thesis is a major benefit for health authorities and fully meets the objectives of the quadrennial malaria control plan published in 2015 by the regional health agency of French Guiana.

Le paludisme est une maladie provoquée par un protozoaire du genre Plasmodium sp. qui est transmis lors de la piqure par des moustiques du genre Anopheles. Actuellement, le contrôle de la maladie repose essentiellement sur la lutte contre les vecteurs avec notamment l’utilisation d’insecticides en imprégnation sur les moustiquaires ou en aspersion sur les murs à l’intérieur des habitations. Dans certaines régions, l’efficacité des insecticides est menacée par des mécanismes de résistance aux insecticides qui se développent dans les populations de vecteurs. Alors, que les mécanismes de résistance physiologique font l’objet de nombreuses recherches et sont maintenant assez bien connus, les modifications comportementales qui peuvent également conférer ou participer à la résistance à ces outils de lutte sont encore très peu étudiées. L’objectif de cette thèse est donc d’apporter des éléments nouveaux pour la compréhension des interactions entre insecticides, mécanismes de résistance physiologique et comportement. Durant cette thèse, nous nous sommes concentrés sur les mutations de cible L1014F et G119S des gènes codant respectivement pour le canal sodium voltage dépendant et pour l’acétylcholinestérase conférant une résistance croisée aux pyréthrinoïdes et organochlorés pour la première et aux carbamates et organophosphorés pour la seconde. Nous avons étudié leur influence sur le comportement de recherche de l’hôte et le système olfactif périphérique en présence ou non d’insecticides. Nos résultats ont permis de mettre en évidence des interactions complexes entre les insecticides présents dans l’environnement et les gènes de résistance sur le système olfactif et sur le comportement. Ces informations sont cruciales pour l’étude de l’impact de ces interactions sur l’efficacité des stratégies mis en place pour lutter contre la transmission du paludisme. / Malaria is caused by a protozoan belonging to the genus Plasmodium sp. transmitted to humans by Anopheles mosquitoes. Malaria control mainly relies on vector control strategies such as insecticide-treated bed nets or insecticide residual sprayings. In some areas, the effectiveness of insecticides is threatened by insecticide resistance mechanisms spreading in vector populations. Physiological resistance mechanisms have been deeply investigated whereas behavioural modulations conferring or involved in resistance have been overlooked. The objective of this thesis is to provide new insights to better understand the interactions between insecticide, physiological-resistance mechanisms and behaviour. In this thesis we focused on target-site mutations L1014F and G119S on genes respectively coding for voltage gated sodium channel and acetyl-cholinesterase conferring crossed resistance to organochlorides and pyrethroids for the former and crossed resistance to organophosphates and carbamates for the latter. We studied their impact on the host seeking behaviour and the peripheral olfactory system in the presence or absence of insecticides. Our results highlight the complex interactions between insecticides in environment and resistance mechanism on the olfactory system and on behaviour. That could lead to a better resistance management and an improvement in the use of insecticides. This knowledge is essential in order to decipher with the impact of such interactions of the efficacy of vector control tools scaled up to fight against malaria.

0009014A Msc thesis Science Animal plant and environmental sciences / Anopheles arabiensis is considered an important vector of human malaria in the southern African region where the disease is the major cause of morbidity and mortality. Gene flow plays an important role in malaria control with the spread of insecticide resistance. The main objectives of this study were to (i) measure the genetic variability within and between five populations (Botswana, Namibia, South Africa, Swaziland and Zimbabwe) of wild An. arabiensis and (ii) estimate the level of gene flow between natural populations across the Southern-limits of An. arabiensis. A total of 1225 An. arabiensis specimens were identified out of 1300 mosquitoes collected from 2000-2003 with the sample sizes ranging from 180-292 per country. Variation at four microsatellite markers was investigated on non-denaturing polyacrylamide gels. The results showed fewer variations between populations (2.96%) than within populations (82.60%) suggesting considerable homogeneity. However, estimates of gene flow (Nm) calculated from mean FST and RST statistics were relatively low, 1.14 and 1.19 respectively, suggesting somewhat restricted gene flow between populations. The occurrence of gene flow within subpopulations of An. arabiensis in Zimbabwe but not in South Africa is interesting with regard to the spread of insecticide resistance in Zimbabwe. The results presented here are obviously subject to the limitations inherent in manual, silver staining method of analysing microsatellite DNA markers. It is possible that a different set of results would be obtained if an Automated Sequencing Analyzer were used. ii

A malária é uma doença grave, cujos vetores são mosquitos do gênero Anopheles. Esse gênero é composto por cerca de 500 espécies e aproximadamente 45 são responsáveis pela transmissão do parasito em todo o mundo. Alguns destes vetores são componentes de complexo de espécies crípticas como Complexo Albitarsis e Complexo Triannulatus. O Complexo Albitarsis é formado por quatro espécies: An. albitarsis s.s, An. albitarsis B, An. marajoara e An. deaneorum. O Complexo Triannulatus é composto por três espécies: An. triannulatus, An. halophylus e An. triannulatus C. Algumas técnicas moleculares são utilizadas para auxiliar na distinção destas espécies crípticas. Dessa forma, DNA de An. albitarsis s.l e An. triannulatus s.l., capturados na Usina de Porto Primavera, foram amplificados para ITS2 e para o gene ND4 do DNAmt. Todas as amostras foram submetidas à técnica de RFLP. Algumas amostras foram seqüenciadas diretamente ou clonadas para posterior seqüenciamento a fim de se confirmar a espécie. Das amostras do Complexo Albitarsis, 62,85% foram identificadas como An. deaneorum para ITS2-RFLP. As restantes foram amplificadas para ND4-RFLP e foram identificadas como An. albitarsis s.s. Observou-se, no entanto, que as amostras do Complexo Albitarsis não exibiram similaridade total com as depositadas no GeneBank sendo que para ITS2-PCR estas foram identificadas como An. albitarsis s.s. e An. deaneorum, enquanto que para o gene ND4 todas foram identificadas como An. albitarsis B. O ITS2-RFLP para o An. triannulatus s.l demonstrou polimorfismos entre as espécies. No seqüenciamento, estas amostras foram similares àquelas depositadas no GeneBank. O emprego de marcadores moleculares podem, em parte, auxiliar na distinção de espécies crípticas, porém outros marcadores devem ser avaliados a fim de se elucidar a identificação do Complexo Albitarsis. / Malaria is a severe disease whose vectors are mosquitoes belonging to the genus Anopheles. This genus contains 500 species of which approximately 45 are responsible for the worldwide transmission of these parasites. Some of these vectors belong to cryptic species such as those of the Albitarsis and Triannulatus Complex. The Albitarsis Complex is composed of four species: An. albitarsis s.s., An. albitarsis B, An. marajoara and An. deaneorum. The Triannulatus Complex contains three species: An. triannulatus, An. halophylus and An. triannulatus C. We used molecular techniques to differentiate these cryptic species. Thus, DNA of An. albitarsis s.l and An. triannulatus s.l, captured at the Porto Primavera Dam had their ITS2 and their mtDNA, ND4 genes amplified. All samples were analyzed by the RFLP technique. Some samples were directly sequenced while others were cloned for subsequent sequencing for species confirmation. Within the Albitarsis Complex, 62.85% were identified as An. deaneorum by RFLP-ITS2. The remaining were amplified by RFLP-ND4 and identified as An. albitarsis s.s. However the results of sequencing of the samples of the Albitarsis Complex did not overlap entirely with those deposited in the GeneBank since those amplified by RFLP-ITS2 were identified as An. albitarsis s.s. and An. deaneorum while those obtained by RFLP-ND4 were identified as An. albitarsis B. Also RFLP-ITS2 of A. triannulatus s.l. contained polymorphic regions among different species. By sequencing, these samples were similar to those deposited in the GeneBank. The use of molecular markers did, in some instances help to distinguish species within cryptic complexes, however other markers need to be evaluated to elucidate further identification of the Albitarsis Complex.