Master of Science / Department of Biology / Rollie Clem / Apoptosis, or programmed cell death, is a crucial conserved process among organisms for deleting damaged unwanted cells, as well as for development and viral defense, and plays an important role in multiple diseases. Too much apoptosis may lead to Alzheimer’s disease, and too little may result in cancer. Therefore, the ability to understand this process is essential for improved medical knowledge today. Apoptosis has been explored in a number of species and pathways seem relatively conserved among most, with unique aspects contained in each, but little is known about apoptosis in mosquitoes. Improved knowledge and growing interest concerning apoptosis in mosquitoes is necessary considering the vast health effects seen across the globe as a result of diseases transferred by the mosquito vector. The Dengue virus mosquito vector Aedes aegypti was the focus here. A new player named defense repressor 1 was discovered in Drosophila melanogaster (DmDnr1), shown to play a role in apoptosis, and the homolog discovered in A. aegypti (AeDnr1). Silencing Dmdnr1 resulted in cells sensitized to apoptosis but was not enough to induce spontaneous apoptosis. In contrast, silencing Aednr1 in the A. aegypti cell line, Aag2, led to spontaneously induced apoptosis. This showed the importance of AeDnr1 as a member of the apoptotic pathway in this species. Epistasis experiments showed that apoptosis induced by silencing Aednr1 requires the initiator caspase Dronc and the effector caspase CASPS8, whereas apoptosis induced by silencing the inhibitor of apoptosis, Aeiap1, also requires Dronc but acts through the effector caspase CASPS7. Further epistasis experiments showed that apoptosis induced by silencing Aednr1 requires the IAP antagonist Mx, but not IMP. This showed for the first time a gene regulating upstream of an IAP antagonist. Biochemical studies showed that AeDnr1 regulates active CASPS8 but not CASPS7, and interacts with Mx and CASPS8 but not AeDronc, CASPS7 nor AeIAP1. Studies also showed Mx competes effectively with CASPS8 but not CASPS7 for AeIAP1 binding, and IMP competes effectively with CASPS7 but not CASPS8 for AeIAP1 binding. An improved apoptosis pathway for the mosquito A. aegypti emerged involving a potential feedback loop with explanations for the upstream IAP antagonist preference as well as the downstream effector caspase preference resulting from apoptosis induced by Aednr1 silencing. Through the discussed research, multiple unique findings resulted. Studying the mosquito model will allow us to find certain gene relations that are more difficult to uncover in the Drosophila model. Because Dnr1 is found in most systems, this improved pathway may shed light not only on a potential role of Dnr1 in apoptosis in insects but higher organisms as well.

Doctor of Philosophy / Department of Biology / Rollie J. Clem / Each year, over 500 million people are infected with mosquito-borne diseases, including malaria, yellow fever and dengue fever, which cause several million deaths, and long-term disability and suffering. This dissertation focused on the mosquito Aedes aegypti, a vector for dengue virus and yellow fever virus. Since Sindbis virus (SINV) is an arthropod-borne virus (arbovirus) that is vectored by A. aegypti and is well characterized at the molecular level, the SINV - A. aegypti model was used to determine whether apoptosis plays a role in the control of vector competency. In Chapter 2, the effects of inducing or inhibiting apoptosis on SINV replication were tested in mosquito cells. It was observed that recombinant SINVs expressing pro-apoptotic genes caused extensive apoptosis in mosquito cells, with decreased virus production after the cells underwent apoptosis. Infection of mosquito cells with SINV expressing the caspase inhibitor P35 inhibited actinomycin D-induced apoptosis, but had no observable effects on virus replication. This study was the first to test directly whether inducing or inhibiting apoptosis affects arbovirus replication in mosquito cells. Chapter 3 examined the effects of silencing apoptosis regulatory genes on SINV replication and dissemination in A. aegypti. Genes which either positively or negatively regulate apoptosis were silenced by RNA interference in mosquitoes, which were then infected with a recombinant SINV expressing green fluorescent protein (GFP). Reciprocal effects were observed on both the occurrence and intensity of expression of GFP in various tissues. These results suggest that systemic apoptosis positively influences SINV replication in A. aegypti. This was the first direct study to explore the role of apoptosis in determining mosquito vector competence for arboviruses. Finally, in Chapter 4, the mechanisms of apoptosis were explored in A. aegypti. Overexpression of IAP antagonists caused extensive cell death in mosquito cells, while silencing the expression of IAP antagonists attenuated apoptosis. The results showed that the IAP binding motif (IBM) of IAP antagonists was critical for their binding to AeIAP1. The IAP antagonists released initiator and effector caspases from AeIAP1 by competing for the binding sites and caused caspase-dependent apoptosis. These findings imply that the mechanisms of IAP antagonists regulating apoptosis are conserved between mosquitoes and the model insect where apoptosis has been mainly studied, Drosophila melanogaster.

Les maladies transmises par les arthropodes vecteurs sont responsables de centaines de milliers de cas d’infections humaines et de décès chaque année à travers le monde. Ces maladies, causées par des bactéries, virus ou parasites, parfois émergents ou réémergents, sont parfois peu connues ou sous-estimées. Les arthropodes peuvent être utilisés comme outil de suivi épidémiologique des micro-organismes qui leur sont associés et dont certains pourront être transmis à des hôtes vertébrés. L’identification des arthropodes reste cruciale dans les enquêtes entomologiques.Nous avons pu ainsi détecter de potentielles nouvelles bactéries dans les tiques de Tahiti et les triatomes de Guyane.Nous avons exploré le microbiote salivaire de près de mille moustiques de 3 pays différents par métagénomique 16S. Nous avons ainsi détecté un large nombre de bactéries pathogènes opportunistes mais aussi un très grand nombre de génotypes correspondant probablement à des espèces et genres bactériens nouveaux. Enfin notre axe majeur a été le développement de l’utilisation de la spectrométrie de masse MALDI-TOF en entomologie médicale. Pour pallier les limites des méthodes de référence d’identification des arthropodes existantes, nous avons validé l’utilisation de cet outil pour l’identification des moustiques (collectés sur terrain en Australie) et de puces (Espagne, Corse, Algérie). Nous avons également mis au point son utilisation pour l’identification de nouvelles familles d’arthropodes, comme les punaises de lits et les triatomes. Nous avons pu mettre en évidence, la capacité de la spectrométrie de masse MALDI-TOF pour différencier les anophèles infectés ou non par des plasmodies. / Vector-borne diseases are responsible for hundreds of thousands of cases of human infections and deaths each year worldwide. Generally, little is known about these diseases, caused by bacteria, viruses or parasites, sometimes emerging or re-emerging. Arthropods can be used as a tool for epidemiological monitoring of their associated microorganisms, some of which being able to be transmitted to vertebrate hosts. The identification of arthropods remains crucial in entomological investigations.We were able to detect potential new bacteria in ticks from Tahiti and triatomines in French Guiana.We explored the salivary microbiota of nearly a thousand mosquitoes from 3 different countries by 16S rRNA metagenomics. We have thus detected a large number of opportunistic pathogenic bacteria but also a very large number of genotypes probably corresponding to new bacterial species and genera. Finally, our major focus has been the development of the use of MALDI-TOF mass spectrometry in medical entomology. To overcome the limitations of existing arthropod identification reference methods, we validated the use of this tool for the identification of mosquitoes (collected in the field in Australia) and fleas (Spain, Corsica, Algeria). We have also developed its use for the identification of new families of arthropods, such as bed bugs (Cimicidae) and triatomines (Reduviidae). We were able to highlight the capacity of MALDI-TOF mass spectrometry to differentiate between anopheles infected or not by malaria parasites.

Os cimicídeos, conhecidos popularmente como percevejos-de-cama, são insetos hematófagos durante os estágios de ninfas e adulto. Algumas espécies apresentam marcante antropofilia e apesar de não transmitirem patógenos, podem causar algumas complicações a saúde humana, como alergias graves e anemia. O número de infestações nos últimos anos em muitos países tem aumentado significativamente e a relação com lugares de baixo IDH não é mais observada, inclusive na cidade de São Paulo. A precariedade nos estudos dificulta o conhecimento sobre as infestações. No presente estudo, teve-se o objetivo descrever infestações no estado de São Paulo e seus aspectos, e estudar a morfologia dos percevejos de cama encontrados, através de análise morfométrica. Foram coletadas e descritas cinco infestações, quatro na cidade de São Paulo e uma na cidade de Mirandópolis. Os resultados sugerem haver falta de conhecimento da população sobre esta praga urbana e comfirmam Cimex lectularius como principal espécie presente no estado de São Paulo, assim como sugere uma proximidade populacional das infestações dentro da cidade de São Paulo em relação a infestações de outras localidades. Porém os estudos devem ser estimulados no intuito de enriquecer o conhecimento sobre estes insetos, haja vista o grande fluxo de pessoas e a possibilidade de agravamento das infestações, uma vez que grandes eventos internacionais ocorrerão em capitais brasileiras. / The Cimicidae, commonly known as bedbugs are bloodsucking insects throughout all their life cycle. Some species show striking anthropophily and despite they dont transmit diseases they can cause some complications for human health, such as severe allergies and anemia. The number of infestations over the last few years in many countries has increased significantly compared to places with low HDI that is no longer observed, including São Paulo city. The lack of studies hinders knowledge about infestations. The present study aims to describe infestations in the state of São Paulo and its aspects, and to study the morphology of bedbugs found, through morphometric analysis. Five infestations in the city of São Paulo and one in the city of Mirandópolis were collected and described. The results suggest lack of knowledge of the population about this urban blight and confirm Cimex lectularius as the main species present in the state of São Paulo. Also suggests proximity of infestations within the city of São Paulo in relation to infestations of other localities. However, the studies should be encouraged in order to enrich the knowledge about these insects, considering the large flow of people and the possibility of aggravating the infestation, since large international events will occur in the largest Brazilian cities.

Leishmania braziliensis é o agente responsável pela maior parte dos casos de leishmaniose tegumentar e mucocutânea no Brasil. Entre as espécies incriminadas como vetoras, na região Sudeste encontram-se Nyssomyia intermedia e Nyssomyia neivai. A comprovação destas espécies como responsáveis pela transmissão de Leishmania sp. depende de vários fatores, entre estes, a competência vetorial, definida como a capacidade da espécie se infectar e repassar a infecção para um hospedeiro vertebrado. Objetivo: O presente estudo teve como objetivo avaliar experimentalmente a competência vetorial de Ny. intermedia e Ny. neivai para transmitir L. braziliensis. Métodos: Fêmeas de primeira geração de ambas as espécies foram alimentadas em hamster infectado com L. braziliensis e após a oviposição, desafiados a um segundo repasto sanguíneo em hamsters susceptíveis (limpos). Adicionalmente, foram obtidos dados do desenvolvimento imaturo das duas espécies. Resultados: em relação ao ciclo imaturo, observou-se que a maior mortalidade ocorreu no primeiro instar larval para ambas as espécies, 27,6% para Ny. intermedia e 53,9% para Ny. neivai. O quarto instar larval teve a maior duração, com mediana de 8 a 12 dias. Quanto à susceptibilidade à infecção das fêmeas colonizadas de ambas as espécies 0,89% das fêmeas de Ny. intermedia e 8,33 % das fêmeas de Ny. neivai apresentaram infecção por L. braziliensis. Nenhuma fêmea de Ny. intermedia realizou um segundo repasto sanguíneo, enquanto que 3 fêmeas de Ny. neivai realizaram. Destas, duas estavam infectadas e o hamster potencialmente infectado ficou em observação por dois meses. Amostras de tecido do hamster exposto demostraram, por meio de PCR, a presença de L. braziliensis. Conclusão: Nyssomyia intermedia apresentou desenvolvimento de formas flageladas porém não repassou a infecção a um hospedeiro suscetível. Nyssomyia neivai, além de se infectar com a L. braziliensis, também repassou a infecção a um hospedeiro suscetível, demostrando assim sua competência vetora. / Leishmania braziliensis is the agent related to cases of cutaneous and mucocutaneous leishmaniasis in Brazil. Among the sandflies involved in the Leishmania sp transmission in the Southeast Region, Ny. intermedia and Ny. neivai are suspected vectors. However, the incrimination of these species as vectors in the transmission of Leishmania sp. depends of several factors, including vector competence, defined as the ability of the species to develop the agent and transmit it to a vertebrate host. Objective: The present study aimed to demonstrate the vector competence to Ny. intermedia and Ny. neivai for L. braziliensis. Methods: To develop the experiments,specimens of both species were reared under laboratory conditions and the adults obtained were used to evaluate the vector competence. Thefemales were fed in infected hamsters and after oviposition, challenged to take a bloodmeal in suscetible hamsters (without infection). All females fed on infected hamster were dissected. The hamster bitted in the exposition were follow up for two months, after that, were euthanatized to evaluate the infection by PCR. Result: The highest immature mortality occurred in the first instar larval for both species, 27.6% for Ny. intermedia and 53.9% for Ny. neivai. The fourth larval instar had the longest duration, with a median varying between 8 to 12 days. About the females of Ny intermedia, only 0.89% of them develop the flagellates. For Ny. neivai, among 36 females feed on infected hamster, 2 were infected. None female of Ny intermedia take a second blood meal and 3 of Ny. neivai have had. The hamsters bitted by the infected females were follow up for 60 days. After that the hamster were euthanatized and the infection was observed. Conclusion: The vector competence of Ny. neivai to transmit Leishmania braziiensis was demonstrated. The susceptibility of Ny. intermedia to develop this leishmania was corroborated.

Os cimicídeos, conhecidos popularmente como percevejos-de-cama, são insetos hematófagos durante os estágios de ninfas e adulto. Algumas espécies apresentam marcante antropofilia e apesar de não transmitirem patógenos, podem causar algumas complicações a saúde humana, como alergias graves e anemia. O número de infestações nos últimos anos em muitos países tem aumentado significativamente e a relação com lugares de baixo IDH não é mais observada, inclusive na cidade de São Paulo. A precariedade nos estudos dificulta o conhecimento sobre as infestações. No presente estudo, teve-se o objetivo descrever infestações no estado de São Paulo e seus aspectos, e estudar a morfologia dos percevejos de cama encontrados, através de análise morfométrica. Foram coletadas e descritas cinco infestações, quatro na cidade de São Paulo e uma na cidade de Mirandópolis. Os resultados sugerem haver falta de conhecimento da população sobre esta praga urbana e comfirmam Cimex lectularius como principal espécie presente no estado de São Paulo, assim como sugere uma proximidade populacional das infestações dentro da cidade de São Paulo em relação a infestações de outras localidades. Porém os estudos devem ser estimulados no intuito de enriquecer o conhecimento sobre estes insetos, haja vista o grande fluxo de pessoas e a possibilidade de agravamento das infestações, uma vez que grandes eventos internacionais ocorrerão em capitais brasileiras. / The Cimicidae, commonly known as bedbugs are bloodsucking insects throughout all their life cycle. Some species show striking anthropophily and despite they dont transmit diseases they can cause some complications for human health, such as severe allergies and anemia. The number of infestations over the last few years in many countries has increased significantly compared to places with low HDI that is no longer observed, including São Paulo city. The lack of studies hinders knowledge about infestations. The present study aims to describe infestations in the state of São Paulo and its aspects, and to study the morphology of bedbugs found, through morphometric analysis. Five infestations in the city of São Paulo and one in the city of Mirandópolis were collected and described. The results suggest lack of knowledge of the population about this urban blight and confirm Cimex lectularius as the main species present in the state of São Paulo. Also suggests proximity of infestations within the city of São Paulo in relation to infestations of other localities. However, the studies should be encouraged in order to enrich the knowledge about these insects, considering the large flow of people and the possibility of aggravating the infestation, since large international events will occur in the largest Brazilian cities.

Leishmania braziliensis é o agente responsável pela maior parte dos casos de leishmaniose tegumentar e mucocutânea no Brasil. Entre as espécies incriminadas como vetoras, na região Sudeste encontram-se Nyssomyia intermedia e Nyssomyia neivai. A comprovação destas espécies como responsáveis pela transmissão de Leishmania sp. depende de vários fatores, entre estes, a competência vetorial, definida como a capacidade da espécie se infectar e repassar a infecção para um hospedeiro vertebrado. Objetivo: O presente estudo teve como objetivo avaliar experimentalmente a competência vetorial de Ny. intermedia e Ny. neivai para transmitir L. braziliensis. Métodos: Fêmeas de primeira geração de ambas as espécies foram alimentadas em hamster infectado com L. braziliensis e após a oviposição, desafiados a um segundo repasto sanguíneo em hamsters susceptíveis (limpos). Adicionalmente, foram obtidos dados do desenvolvimento imaturo das duas espécies. Resultados: em relação ao ciclo imaturo, observou-se que a maior mortalidade ocorreu no primeiro instar larval para ambas as espécies, 27,6% para Ny. intermedia e 53,9% para Ny. neivai. O quarto instar larval teve a maior duração, com mediana de 8 a 12 dias. Quanto à susceptibilidade à infecção das fêmeas colonizadas de ambas as espécies 0,89% das fêmeas de Ny. intermedia e 8,33 % das fêmeas de Ny. neivai apresentaram infecção por L. braziliensis. Nenhuma fêmea de Ny. intermedia realizou um segundo repasto sanguíneo, enquanto que 3 fêmeas de Ny. neivai realizaram. Destas, duas estavam infectadas e o hamster potencialmente infectado ficou em observação por dois meses. Amostras de tecido do hamster exposto demostraram, por meio de PCR, a presença de L. braziliensis. Conclusão: Nyssomyia intermedia apresentou desenvolvimento de formas flageladas porém não repassou a infecção a um hospedeiro suscetível. Nyssomyia neivai, além de se infectar com a L. braziliensis, também repassou a infecção a um hospedeiro suscetível, demostrando assim sua competência vetora. / Leishmania braziliensis is the agent related to cases of cutaneous and mucocutaneous leishmaniasis in Brazil. Among the sandflies involved in the Leishmania sp transmission in the Southeast Region, Ny. intermedia and Ny. neivai are suspected vectors. However, the incrimination of these species as vectors in the transmission of Leishmania sp. depends of several factors, including vector competence, defined as the ability of the species to develop the agent and transmit it to a vertebrate host. Objective: The present study aimed to demonstrate the vector competence to Ny. intermedia and Ny. neivai for L. braziliensis. Methods: To develop the experiments,specimens of both species were reared under laboratory conditions and the adults obtained were used to evaluate the vector competence. Thefemales were fed in infected hamsters and after oviposition, challenged to take a bloodmeal in suscetible hamsters (without infection). All females fed on infected hamster were dissected. The hamster bitted in the exposition were follow up for two months, after that, were euthanatized to evaluate the infection by PCR. Result: The highest immature mortality occurred in the first instar larval for both species, 27.6% for Ny. intermedia and 53.9% for Ny. neivai. The fourth larval instar had the longest duration, with a median varying between 8 to 12 days. About the females of Ny intermedia, only 0.89% of them develop the flagellates. For Ny. neivai, among 36 females feed on infected hamster, 2 were infected. None female of Ny intermedia take a second blood meal and 3 of Ny. neivai have had. The hamsters bitted by the infected females were follow up for 60 days. After that the hamster were euthanatized and the infection was observed. Conclusion: The vector competence of Ny. neivai to transmit Leishmania braziiensis was demonstrated. The susceptibility of Ny. intermedia to develop this leishmania was corroborated.

Investigou-se a existência de relação entre células normais de triatomíneos, formas evolutivas do T.cruzi e hospedeiros mamíferos. Hemócitos do vetor de espécies hematófagas não infectadas (Triatoma infestans, T. palidipennis, Dipetalogaster maximus, Rhodnius prolixus e Panstrongylus megistus) foram reconhecidos por soros de pacientes e camundongos infectados com Trypanosoma cruzi, por meio da técnica de imunofluorescência, enquanto que os hemócitos de um inseto fitófago, Diactor billineatus (ninfas e adultos) não foram reconhecidos pelos mesmos soros. Uma parcial reação cruzada foi observada entre hemócitos de T. infestans e soros de portadores de leishmaniose , o que não ocorreu com as amostras de portadores de doenças autoimunes, infecção com HIV e indivíduos normais. O tratamento de hemócitos com metaperiodato de sódio aboliu a fluorescência, mostrando que antígenos carboidratos estão envolvidos. O uso de lectinas marcadas revelou que N acetil-d-glicosamina, manose, glicose e βgal fazem parte da estrutura celular. O immunoblotting com extrato de hemócitos foi realizado com soros de pacientes chagásicos crônicos antes e depois do tratamento específico. Estes soros de ambos casos foram absorvidos com tripomastigotas e epimastigotas. Um número considerável de bandas desapareceu nos soros tratados com tripomastigotas, mas não com epimastigotas. Proteínas de 61-70, 41-50, 31-40 e 21-30 kDa foram as envolvidas no processo de absorção. Após a quimioterapia, as bandas de 121-130, 91-100, 81-90 e 61-60 kDa desapareceram nos soros não absorvidos. Um grupo de camundongos fêmeas A/Sn foi imunizado com hemolinfa livre de hemócitos (HL) e outro grupo com hemócitos (Hc) e desafiados com 5x102 tripomastigotas sangüícolas.(cepa Y). Picos de parasitemia dos camundongos HL e Hc foram observados um ou dois dias após o grupo controle. Apesar da parasitemia de ambos os grupos ter sido baixa, o grupo HL apresentou 43% de mortalidade, enquanto que o grupo Hc mostrou apenas 14 % de mortalidade. As citocinas IFNγ e TNFα estão envolvidas no processo de proteção,\' assim como óxido nítrico e anticorpos líticos. Os hemócitos induziram uma redução de IL-10, inibindo a resposta Th2. Desde que os hemócitos e tripomastigotas compartilham epítopos comuns, epítopos de hemócitos devem ser melhor estudados em virtude de seu potencial uso no imunodiagnóstico, imunoproteção e avaliação terapêutica. / Immunological relationship between normal triatomine cells, developmental life-cycle stages of T. cruzi and infected mammal hosts was investigated. Hemocytes from uninfected hematophagous vector species of triatomines (Triatoma infestans, T. palidipennis, Dipetalogaster maximus, Rhodnius prolixus and Panstrongylus megistus) were recognized by sera from patients and mice infected with Trypanosoma cruzi, by means of immunofluorescence assay (IFA), whereas hemocytes from a phytophagous bug, Diactor billineatus (nymphs and adults) were not recognized by the same sera. Hemocytes from T. infestans were partially recognized by leishmaniasis sera, but, not by samples from patients with autoimmune diseases, HIV positive sera. The treatment of hemocytes with sodium metaperiodate practically abolished the fluorescence staining, indicating the presence of carbohydrate antigens. These antigens were revealed be comprised of N acetyl-D-glucosamine, mannose and glucose and βgal, by means of fluorescent labelled lectins. Sera from chronic chagasic patients before and after the chemotherapy were reactive with hemocytes of P. megistus on immunoblotting, and the same sera previously absorbed with formaldehyde fixed cultured epimastigotes as well trypomastigotes. On immunoblotting a substantial number of bands diminished with sera absortion with trypomastigotes, but not with epimastigotes. Proteins of 61-70, 41-50, 31-40 and 21-30 kDa were the major proteins involved in the process of serum absorptions. After chemotherapy, protein bands of 121-130, 91-100, 81-90 and 51-60 kDa disappeared in unabsorbed sera. A group of female A/Sn mice were immunized with hemolymph free of hemocytes (MHL) and other group with hemocytes (MHC). Animais were challenged with 5x102 bloodstream trypomastigotes (Y strain). Parasitemia peaks of MHL and MHC groups were observed one or two days after the control group. In spite of the parasitemia for both groups was about 63% lower than the control group, the mortality for the MHL was 43%, differing from the MHC, which was 14% only. The Cytokines, IFNγ and TNFα, were shown to be involved in the protection of immunized mice, and also the nitric oxide and complement dependent lytic antibodies. Hemocytes showed to play a role in the reduction of the IL-10 production, inhibiting Th2 response. Thus, hemocytes and trypomastigotes share common epitopes, and these epitopes are potentially useful for immunodiagnosis, immunopratection, and evaluation of chemotherapic efficiency in Chagas\' disease control.

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.

Haematophagous parasites and disease vectors such as leeches, ticks, mites, lice, bed bugs, mosquitoes, and myiasis-causing fly larvae are common health problems in Lao Peoples Democratic Republic (Lao PDR). A main aim of my field work in Lao PDR in 2006-2010 was to document traditional knowledge among different ethnic groups about plants that people use to repel or to kill blood-feeding invertebrates. We carried out structured interviews in 66 villages comprising 17 ethnic groups, covering a range of ethnic group, throughout Lao PDR and recorded a total of 92 plant species - in 123 different plant-ectoparasite combinations - that are used as traditional repellents and/or as “pesticides” to kill "pest" invertebrates. Traditional use was confirmed in the scientific literature for 74 of these plant species, and for an additional 13 species based on literature on closely related species. We concluded that repellents and pesticides from many plant species are commonly used in the Lao countryside. We also investigated traditionally used Lao plants for their activity to repel or to kill certain disease vectors and parasites. Target organisms were mosquitoes (Diptera, Culicidae), fly larvae (Diptera, Cyclorrhapha) in fermented fish production, and terrestrial blood-sucking leeches (Hirudinea, Haemadipsidae). The potential mosquito repellent activities of essential oils of Croton roxburghii (Euphorbiaceae), Hyptis suaveolens (Lamiaceae), and Litsea cubeba (Lauraceae) were evaluated in the field near Vientiane. Oils at concentrations of 1.7-6.7 µg/cm2 were significantly repellent to Aedes, Armigeres and Culex attracted to human baits. The activities against fly larvae, infesting fermenting fish, of three plant species, Tadehagi triquetrum (Fabaceae), Uraria crinita (Fabaceae) and Bambusa multiplex (Poaceae) were investigated: When fresh material of the plants was added on top of fermenting fish infested with fly larvae significant proportions of the larvae were repelled or killed. The total protective effect, i.e., repellent and killing effect combined, of T. triquetrum, U. crinita, and B. multiplex was 60-83 %, 77-90 %, and 60-93 %, respectively. Field evaluation of the potential leech repellent activities of water extracts of Sapindus rarak (Sapindaceae), Catunaregam spathulifolia (Rubiaceae) and Vernonia elaeagnifolia, (Asteraceae) impregnated on stockings and worn by persons in two leech-infested biotopes revealed leech repellent activities of 82.6%, 62.6% and 63.0%, respectively. The corresponding repellencies of deltamethrin and diethyl-3-methyl-benzamide (DEET) were 73.1% and 88.4%, respectively. Identification of the active components in certain of the plants with the ultimate aim to develop more optimal, less costly repellents, insecticides, acaricides, and anti-leech compounds as alternatives to synthetic repellents and pesticides against blood-feeding insects, ticks, mites, and leeches is in progress.