Les maladies transmises par les moustiques représentent une menace permanente en santé publique. La principale mesure pour protéger les populations contre les piqûres infectieuses repose actuellement sur l’utilisation d’insecticides, mais celle-ci est menacée par la propagation des mécanismes de résistance au sein des populations de moustiques. Dans ce contexte, les répulsifs représentent un outil de choix pour réduire à la fois les nuisances provoquées par les moustiques et le risque d’infection. Parmi eux, le DEET (N, N-diéthyl-3-méthylbenzamide) a prouvé une efficacité remarquable aussi bien lors d’expérimentations en laboratoire que sur le terrain. Malgré cela, les répulsifs ont toujours été utilisés de manière empirique, et leur mode d’action reste très mal connu. Le but de ce projet de thèse a été : i) de participer à l’évaluation du potentiel d’intégration du répulsif DEET dans la lutte antivectorielle, ii) de mesurer l’impact de paramètres physiologiques du moustique d’intérêt épidémiologique tels que l’expérience, l’âge et l’infection sur l’efficacité des méthodes de lutte, répulsifs et insecticides pyréthrinoïdes et iii) de quantifier l’impact des répulsifs et insecticides sur les traits d’histoire de vie des moustiques. Les expériences ont été réalisées sur Anopheles gambiae et Aedes albopictus, respectivement vecteurs de l’agent du paludisme et d’arbovirus tels que le virus de la dengue. Nos résultats ont permis de démontrer que l’état physiologique du moustique influe sur l’efficacité des méthodes de lutte ; premièrement, les moustiques porteurs de la mutation kdr ayant obtenu un repas de sang en contact avec de la perméthrine ne sont plus irrités par ce composé lors d’une seconde exposition, dans les conditions testées et à une dose recommandée pour l’imprégnation de moustiquaires. Au contraire, un repas de sang obtenu en présence DEET n’a pas affecté l’efficacité de ce composé à inhiber un repas de sang à l’exposition suivante. Ensuite, le DEET s’est montré plus efficace chez les moustiques âgés que chez les moustiques jeunes, et les résultats sont similaires chez les deux espèces et indépendants du statut de résistance aux insecticides. D’autre part, l’efficacité de la deltaméthrine et du DEET ne sont pas modifiés lorsque les moustiques sont infectés par le parasite Plasmodium falciparum. Cependant, une augmentation de la mortalité a été observée chez les femelles anophèles infectées au stade sporozoites lors de certaines expérimentations, indépendamment du traitement chimique de la moustiquaire, et suggère un coût de l’infection « réplicat-dépendant ». Enfin, le DEET génère un impact au long terme sur la fécondité et la fertilité des moustiques, effets qui ne sont pas observés dans notre protocole pour la perméthrine. Ces résultats donnent donc des pistes pour redéfinir les priorités dans les programmes de lutte, afin de cibler en premier lieu les moustiques les plus à même de transmettre des pathogènes. Ils soulignent également la nécessité de considérer à la fois l’état physiologique du moustique et l’impact sur le long terme des insecticides et répulsifs lors de l’évaluation des outils de lutte. / Mosquito-borne diseases are a permanent public health threat. The main way to protect populations against infectious bites relies on the use of insecticides, but their efficacy is threatened by the spread of resistance mechanisms among vector populations. In this context, repellents are a promising tool for reducing both mosquito nuisance and infection risk. Among them, DEET (N, N-diéthyl-3-méthylbenzamide) has proven great efficacy both in laboratory and field experiments. Despite this, repellents, are still used from an empirical way and their mode of action remains poorly understood. The aim of the present project was to: i) participate to the evaluation of the potential of integration of the DEET repellent in mosquito control, ii) measure the impact of mosquito physiological parameters of epidemiological relevance such as experience, age and infection, on the efficacy of repellents and insecticides, and iii) quantify the impact of repellents and insecticides on mosquito life-history traits. Experiments were performed on Anopheles gambiae and Aedes albopictus, vectors of the malaria parasite and of arboviruses such as dengue, respectively. Our results allowed to demonstrate that mosquito physiological state influences the efficacy of control tools; first, after successfully obtaining a blood meal in contact with permethrin, mosquitoes carrying kdr mutation are no longer inhibited by this compound at the subsequent exposure, at the recommended dose for net impregnation and under our experimental design. On the contrary, a first blood meal obtained in presence of DEET does not affect its efficacy at the following exposure. Then, DEET appeared to be more efficient against old mosquitoes that against younger ones, and results are consistent in the two species, independently of the insecticide resistance status. Besides, efficacy of DEET and deltamethrin is not modified when mosquitoes are infected with Plasmodium falciparum. However, an increased mortality was observed in anopheles when infected with transmissible stages (i.e. sporozoites), independently of chemical exposure, which suggest a cost of infection and seems to be “replicate-dependent”. Finally, DEET produces a long-term impact on mosquito fecundity and fertility, which is not observed with permethrin under our protocol. These results give insights to redefine the priorities in mosquito control programs, in order to specifically targeting mosquitoes that are the most susceptible to transmit pathogens. These observations also underline the need for considering both mosquito physiological state and the long-term impact of repellents and insecticides during the evaluation of control tools.

Virtually all organisms exhibit circadian rhythms, this includes mosquitoes. Many aspects of their biology are under the control of their circadian clocks like their vision, olfaction, host-seeking, mating, oviposition, metabolism, locomotion, and more. However, how the circadian clock regulates mosquito-host interactions and adapts to specific environmental conditions remains largely unknown, despite its importance to vector disease control. Here, we relied on a multidisciplinary, integrative, and comparative approach to shed light on the mechanisms underlying mosquitoes adaptations to various temporal niches. We use CRISPR/Cas9 to knockout timeless in Aedes aegypti and show this mutation causes an increase in their free running periods under continuous darkness conditions. External factors can also influence a vector's activity pattern like climate, light pollution, as well as host preference and availability. To investigate the influence these factors have on activity patterns, we compare the activity patterns of multiple lab rear strains of Aedes albopictus as well as two field collected populations of Aedes japonicus. Our results suggest host availability and light pollution could cause the differences in activity profiles that we observed. With vision playing an important role in both circadian rhythms, host seeking, and threat detection, we compared a nocturnal mosquito (Aedes japonicus) and a diurnal mosquito (Aedes aegypti). We introduced a looming visual stimulus in an LED arena and found Aedes aegypti to be more responsive to the looming stimulus than Aedes japonicus. Finally, the underlying mechanisms that mediate this threat detection and escape behavior were examined, revealing a possible candidate for a giant fiber neuron in Aedes aegypti. Overall these results provide novel insights into mosquito biology that have the potential to be applied towards vector control methods. / Doctor of Philosophy / Most living things, including mosquitoes, follow a daily pattern called a circadian rhythm. This rhythm affects many parts of their lives including their vision, smell, and activity. Understanding how their circadian clock functions can help us control populations of mosquitoes and prevent the transmission of diseases they may carry. By studying different species and populations of mosquitoes in the lab and from the wild, we have found that factors like light pollution and host availability could affect when mosquitoes are active. In addition to these results we have also demonstrated that mosquitoes that are active during the day and mosquitoes that are active during the night will respond differently to a looming visual stimulus. We examined the brain of the mosquitoes that were most responsive to the looming stimulus and found a system of large neurons that could potentially be responsible for eliciting their escape behaviors. Overall these results provide new and important information about mosquito biology and can be applied to mosquito control.

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Agradecemos a compreensão. on 2016-06-23T13:03:25Z (GMT) / Submitted by RAFAEL PIOVEZAN null (piovezan.rafael@gmail.com) on 2016-06-24T10:26:33Z No. of bitstreams: 1 Piovezan, Rafael.pdf: 4696151 bytes, checksum: 994b7f23d9372dfc37c189de81feee65 (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-06-24T14:24:34Z (GMT) No. of bitstreams: 1 piovezan_r_dr_rcla.pdf: 4696151 bytes, checksum: 994b7f23d9372dfc37c189de81feee65 (MD5) / Made available in DSpace on 2016-06-24T14:24:34Z (GMT). No. of bitstreams: 1 piovezan_r_dr_rcla.pdf: 4696151 bytes, checksum: 994b7f23d9372dfc37c189de81feee65 (MD5) Previous issue date: 2016-06-13 / As atividades de controle da dengue são preconizadas pelo Ministério da Saúde e pelas Secretarias de Estado da Saúde. A Superintendência de Controle de Endemias no Estado de São Paulo é o órgão responsável pelo acompanhamento e auxílio, em algumas situações, nas atividades de combate à dengue. A cargo dos municípios paulistas fica a execução das ações. Nesse contexto, é de amplo conhecimento técnico que atividades de vigilância epidemiológica e entomológica se mostram fundamentais para a redução dos riscos causados pela transmissão de agentes patogênicos, através dos mosquitos, à sociedade. Com esse intuito, diversos trabalhos foram executados no município de Santa Bárbara d'Oeste, interior do Estado de São Paulo, com o objetivo de melhorar a compreensão da dinâmica da dengue. Foram realizados dois estudos sobre o perfil epidemiológico da dengue, o primeiro analisou a epidemia ocorrida durante o ano de 2010, e o segundo apresentou análises espaciais sobre a doença no período de 1995 a 2010. No aspecto entomológico, três trabalhos foram executados. O primeiro verificou a distribuição de culicídeos no bairro Cruzeiro do Sul, que apresenta características rurais, ainda que em transição para a condição urbana. No segundo trabalho foram analisados os resultados obtidos através de dois métodos de aplicação de inseticida, conhecidos como nebulização leve e nebulização com equipamento pesado acoplado a veículo. E o último trabalho apresentou resultados sobre as coletas realizadas em cinco bairros rurais do município através da instalação de larvitrampas. Os resultados obtidos nos estudos sobre as epidemias demonstraram que, durante o ano de 2010, os homens foram mais acometidos por dengue e a faixa etária com maior número de casos foi entre 15 e 19 anos. Os sintomas mais comuns relatados foram febre, mialgia, cefaléia e artralgia. Também verificou-se que duas áreas do município apresentaram maior risco com relação a dengue e que o histórico das epidemias apresentou como ponto de início da transmissão a zona leste da cidade. Nas pesquisas com enfoque entomológico, foi possível verificar cinco espécies de mosquitos capturadas nas armadilhas durante a pesquisa no bairro rural Cruzeiro do Sul, e seis espécies identificadas nas pesquisas realizadas em diferentes áreas rurais do município. Especificamente para o bairro Cruzeiro do Sul, a comparação entre esses dois trabalhos desenvolvidos demonstrou que Aedes albopictus sofreu redução em sua freqüência, enquanto Aedes aegypti ampliou o número de ocorrências nessa área. Nos dois estudos realizados, o primeiro apenas no Cruzeiro do Sul e o segundo em cinco localidades rurais distintas, o índice de diversidade de Simpson obteve resultados maiores nos locais onde a presença de Ae. aegypti era menos freqüente. No trabalho sobre a comparação dos métodos de nebulização foi possível observar que o número de ovos coletados nas armadilhas foi menor onde se utilizou o equipamento de nebulização pesado. Também se observou diferença no número de pacientes acometidos pela dengue entre as duas áreas estudadas. Diante dos resultados obtidos, conclui-se que os métodos apresentados para estabelecimento do perfil epidemiológico da doença na cidade e as pesquisas que permitiram identificar a dinâmica das espécies de mosquitos em áreas rurais, são importantes indicadores que devem ser considerados nos trabalhos de controle, assim como as autoridades competentes devem executar os métodos mais efetivos de combate ao vetor frente à complexidade e à importância que as doenças relacionadas ao Ae. aegypti impõem à sociedade. / The activities related to the control of dengue fever are recommended by the Ministry of Health and by the Health Secretariat of the State - this latter under the responsibility of the Superintendence of the Control of Endemic Diseases of the State of São Paulo. The execution of the relevant actions is the responsibility of the municipalities of the State. Within this context, both epidemiological and entomological surveillance are fundamental for the reduction of the risks arising from the transmission of diseases by mosquitoes to society. Various studies were undertaken in Santa Bárbara d'Oeste municipality in the interior of São Paulo State, for the purpose of understanding the dynamics of dengue fever better. Two studies were conducted on the epidemiological profile of dengue fever, the first of which analyzed the epidemic which occurred during 2010, and the second presented an analysis of the disease during the period from 1995 to 2010. Three studies were undertaken focusing on the entomological aspect. The first to ascertain the distribution of Culicidae in the Bairro Cruzeiro do Sul suburb, which presents rural characteristics but which is transitional to an urban environment. In the second study the results obtained by means of two methods of the application of insecticide, known as light nebulization and nebulization with heavy equipment coupled to the vehicle, were analyzed. The last study presented the results obtained from the collections undertaken in five rural districts of the municipality by means of the installation of larvitraps. The results obtained by the studies of the epidemics demonstrated that, in 2010, men contracted dengue fever more often than women and that the greatest number of cases occurred in young people between 15 and 19 years of age. The most commonly reported symptoms were fever, myalgia, headache and arthralgia. It was also discovered that two areas of the municipality presented greater risk of dengue fever, and that the history of the epidemics presents the eastern zone of the city as the district in which the epidemic broke out. In the research undertaken with entomological focus, it was ascertained that five mosquito species were captured in the traps during the project in the rural district of Cruzeiro do Sul, and six species were identified in the research undertaken in the various rural areas. Specifically for the Bairro Cruzeiro do Sul suburb, a comparison of the two studies undertaken shows that Aedes albopictus became less frequent, while Aedes aegypti increased its occurrences in this area. In the two studies undertaken in the various rural areas, Simpson’s diversity index obtained the highest results at those sites where the presence of Ae. aegypti was less frequent. In the study comparing the methods of nebulization it was observed that the number of eggs collected on the traps was smaller where the equipment for heavy nebulization was used. A difference between the numbers of patients suffering from dengue fever between these two areas was also observed. In the light of the results obtained it is concluded that the methods presented for the establishment of the epidemiological profile of the disease in the city as also the research which permitted the identification of the dynamics of the mosquito species in the rural areas are important indicators which should be borne in mind in undertaking control actions and also that the competent authorities should make use of the most effective methods to combat the vector in the light of the complexity and gravity of the pathogens which Ae. aegypti transmits to society.

Blood-feeding mosquitoes, by transmitting parasites and viruses to their hosts, kill several hundred thousand people every year. Mosquito populations are currently developing raising levels of insecticide resistance, and there is a need for a better understanding of their behavior so that new control solutions can be imagined, and existing ones can be improved. There has been a vast number of studies examining the host seeking behavior of mosquitoes, however there is a lack of knowledge concerning how mosquitoes are evading the threats their hosts pose via their defensive behaviors. Female mosquitoes are indeed in this unique position where their fitness and reproduction depend on them being able to locate hosts as well as evade them. In order to do this, they rely on sensory cues that they must be able to continuously re-evaluate during host tracking to potentially decide to quickly escape at any point during these interactions. Host seeking is mediated by multiple sensory modalities such as vision, olfaction, and thermosensation. However, it is not clear whether mosquitoes may also be using some of these same cues to identify that their host is turning into a threat. Focusing solely on visual cues in the context of escape behavior, we used a looming stimulus to elicit escape responses from the Yellow Fever mosquito, Aedes aegypti. A virtual reality environment was adapted from previous work, to display the looming stimulus while the mosquito is in a variety of conditions (i.e., landed, in free or tethered flight). Results from these experiments allowed us to characterize the escape responses of mosquitoes, by determining the angles and distances to the stimuli that will most likely trigger an avoidance response. / Master of Science in Life Sciences / Mosquitoes are the deadliest animals in the world because of the several diseases they can transmit. Females are the only ones that bite, since they require a blood meal in order to produce offspring. The task of obtaining this blood meal from a mobile, and defensive host can be extremely dangerous. The females must find a host, approach it, land, feed, and flee without being killed so they can reproduce. At any point, the host might detect the mosquito and try to kill it, typically by swatting at it. For this reason, mosquitoes have evolved the ability to navigate in close proximity to the host, while assessing their level of defensiveness and avoiding these threats. Insects, in general, are well known to display escape behaviors in response to visual, predator-like, looming stimuli. However, in spite of great epidemiological importance, very little is known about the mechanisms that allow mosquitoes to evade their predators, as well as swatting from their hosts. Mosquitoes use visual, olfactory, and thermal cues to track their hosts but what kinds of sensory cues are being used to avoid threats? As a first step towards bridging this knowledge gap, we analyzed the behavioral responses of the Yellow Fever mosquito, Aedes aegypti, to looming visual stimuli. For this, we used a virtual-reality environment designed for mosquitoes, where we displayed looming squares to mosquitoes in a variety of conditions (for example: landed, in flight). Results from these experiments allowed us to characterize the escape responses of mosquitoes, by determining the angles and distances to the stimuli that will most likely trigger an avoidance response. Ultimately, better understanding mosquito vision in the context of their escape behavior, can help us improve the design of control tools, such as traps, to increase their efficiency.