Aedes aegypti is an important vector of the viruses that cause dengue fever, dengue hemorrhagic fever, and yellow fever. In depth genetic studies of vector species have been made possible due to the availability of genome sequences and techniques for producing stably transformed mosquitoes. These resources have also contributed to the establishment of new genetics-based approaches to the control of vector borne disease. Genetic studies of Ae. aegypti have benefited from the ability to drive targeted transgene expression, however a ubiquitous inducible promoter has not been identified in this mosquito. The Drosophila melanogaster heat shock 70 promoter has been shown to drive inducible expression in heterologous systems; however, DmHsp70 possesses significant basal activity in Aedes aegypti. This study characterized the sequence and expression of the heat shock 70 genes of Aedes aegypti. AaHsp70 genes were found to be organized in two clusters, each comprised of three divergent pairs. AaHsp70 genes exhibited robust expression upon heat shock in larvae, pupae, and adults as well as in heads, salivary glands, midguts and ovaries. Genomic regions upstream of AaHsp70 genes were found to drive heat-inducible expression of a reporter in both cell and embryo assays. Deletion analysis of AaHsp70-derived promoters yielded two ~1.5 kb genomic fragments that maintained robust heat inducibility in these systems. Aedes aegypti were transformed with AaHsp70-luciferase gene cassettes using the transposable element Mos1. AaHsp70-luciferase transcripts accumulated specifically after heat shock, and displayed a pattern of rapid induction and decay similar to endogenous AaHsp70 genes. Heat-induced expression of luciferase was observed in transgenic larvae, pupae and adults as well as heads, midguts and ovaries but not salivary glands, with levels varying between transgenic strains. The effect of heat shock on the endogenous RNAi pathway as well as the effect of blood feeding on the expression of AaHsp70 genes was investigated, though reproducible results could not be obtained using the assays employed. In conclusion, the heat shock 70 gene family of Aedes aegypti was identified and characterized. The AaHsp70 promoters described could be valuable for gene function studies as well as for the precise timing of the expression of anti-pathogen molecules. / Ph. D.

Juvenile hormone (JH) plays pivotal roles in the development and reproduction of insects. Efforts to characterize the mechanisms of JH regulation are complicated due to JH pathways often being intertwined with those of 20-hydroxyecdysone (20E). Upon adult emergence, female Aedes aegypti enter a period of development during which they gain competence for mating, bloodfeeding, and egg production. JH levels rise dramatically and peak during the first 2-3 days post-emergence and remain relatively high until a bloodmeal is consumed, while 20E titers remain very low throughout the entire stage. Thus, post-emergence development offers a unique opportunity to study the effects of JH in the absence of 20E. In this study, four potential JH response genes were identified in newly emerged females. One such gene, AaKr-h1, is a homologue of Kr-h1, a zinc-finger transcription factor which has been characterized in Manduca sexta, Drosophila melanogaster, Tribolium castaneum, and Apis mellifera, and is involved in a diverse range of JH-regulated pathways. AaKr-h1 demonstrated a dose-dependent transcriptional response to JHIII as well as two JH mimics in abdominal ligation assays. The findings of this study indicate that Kr-h1 may be regulated by JH independently of any 20E regulation and suggests a fundamental, conserved role for Kr-h1 in JH-regulated pathways. / Master of Science in Life Sciences

Mosquito surveillance is an integral part of understanding mosquito-borne disease, including and not limited to the La Crosse virus. The primary container-inhabiting Aedeni vectors include Aedes triseritatus, Aedes albopictus, and Aedes japonicus. To better understand the efficacy of gravid traps and oviposition traps as surveillance methods for these mosquitoes, field studies were conducted in three different habitat types. Ae. triseriatus, Ae. albopictus, Ae. japonicus, and Aedes aegypti eggs were described with the aid of color images and SEM. All eggs were broadly cigar shaped with Ae. triseriatus and Ae. japonicus eggs being dull or matte black while Ae. albopictus and Ae. aegypti eggs were shiny jet black. Ae. triseriatus eggs were larger, lighter in color, and have a rougher appearance when compared to Ae. japonicus. Ae. albopictus and Ae. aegypti can be differentiated by the distinct presence of a micropylar collars in Ae. aegypti. Ovitrap and gravid trap efficacy for the surveillance of Ae. triseriatus, Ae. albopictus, and Ae. japonicus were measured in three different habitats on two different sites. Both sites contained the same 2ha habitats with varying degrees of forest canopy disturbance. Ae. triseriatus was the most abundant mosquito on all sites. Ovitraps and gravid traps were efficient in collecting Ae. triseriatus, while ovitraps were not efficient in collecting Ae. japonicus and Ae. albopictus. / Master of Science in Life Sciences

A dengue é considerada uma das mais importantes arboviroses reemergentes em termos de morbidade, letalidade e implicações econômicas e ocorre em todas as regiões tropicais e subtropicais do planeta. tem demonstrado tendência ascendente, devido as suas condições ambientais, climáticas e a ineficiência das políticas públicas. No município em estudo nos últimos dez anos a cidade sofreu várias epidemias de dengue com aumento dos casos graves, hospitalizações e óbitos. Esta pesquisa teve como objetivo analisar a distribuição espacial e a evolução temporal da incidência da dengue e sua correlação com variáveis entomológicas e climáticas em um município brasileiro de tríplice fronteira, no período de agosto de 2006 a julho de 2016. O estudo foi desenvolvido na cidade de Foz do Iguaçu. Foram feitas as análises descritivas, espaciais, temporais e analíticas, seguidas de testes de regressão linear simples para avaliar a correlação entre as variáveis, utilizando o programa estatístico \"R\". Foram avaliados a correlação entre a variáveis incidência da dengue e fatores climáticos e entomológicos para os anos epidêmicos e não epidêmicos em relação ao mesmo mês, um, dois e três meses antes da ocorrência da incidência da doença. Os dados foram coletados de fontes secundárias dos Sistemas de Informações em Saúde, disponibilizados pela Secretaria Municipal de Saúde. A base territorial geográfica utilizada foi a unidade de estrato. Os resultados mostraram que os anos epidêmicos apresentou 60% do período estudado. A incidência da doença nos anos epidêmicos foi alta, ultrapassando a 1000 casos/100 mil/habitantes e acometeu todas as regiões da cidade. Os resultados dos testes de regressão, apresentaram correlação negativa da dengue com índice de infestação Predial, para os anos epidêmicos, e positiva para os anos não epidêmicos com intervalo de dois e três meses. houve correlação positiva da dengue para os anos de epidemia com o índice de infestação do mosquito adulto, com intervalo de três meses, com a umidade relativa do ar em período simultâneo, dias de chuvas, média da temperatura média e média da temperatura máxima, com intervalo de um e dois meses, pluviosidade com intervalos de um, dois e três meses. Sendo assim pode inferir que o índice e infestação predial é fraco em estimar o risco de transmissão da doença, embora seja preconizado pelo Ministério da Saúde para esse fim. O clima foi um dos principais fatores a responderem pela incidência da dengue. O intervalo de três meses, em relação ao início das epidemias, mostrou-se o período mais oportuno para realizar as ações de controle do vetor. Conclui-se que os resultados desta pesquisa podem contribuir proporcionado a precocidade da informação e viabilizando a tomada de decisão em tempo oportuno de forma a focalizar as áreas de maior risco e desta forma otimizar o serviço de campo e a prevenção das doenças causadas pelo Aedes aegypti / Dengue is considered one of the most important reemerging arboviruses in terms of morbidity, lethality and economic implications and occurs in all tropical and subtropical regions of the planet. has shown an upward trend due to its environmental, climatic conditions and the inefficiency of public policies. In the city under study in the last ten years the city suffered several epidemics of dengue with an increase in severe cases, hospitalizations and deaths. The objective of this research was to analyze the spatial distribution and temporal evolution of dengue incidence and its correlation with entomological and climatic variables in a Brazilian municipality of triple border, from August 2006 to July 2016. The study was developed in the city of Foz do Iguaçu. Descriptive, spatial and temporal analyzes were performed, followed by simple linear regression tests to evaluate the correlation between the variables, using the statistical program \"R\". The correlation between the variables dengue incidence and climatic and entomological factors for the epidemic and non-epidemic years in relation to the same month, one, two and three months before the occurrence of the disease was evaluated. Data were collected from secondary sources of the Health Information Systems, made available by the Municipal Health Department. The geographic territorial base used was the stratum unit. The results showed that the epidemic years presented 60% of the studied period. The incidence of the disease in the epidemic years was high, surpassing 1000 cases / 100 thousand / inhabitants and affected all regions of the city. The results of the regression tests showed a negative correlation of dengue with Predial infestation index for the epidemic years and positive for the non - epidemic years with two - and three - month intervals. there was a positive correlation between dengue in the epidemic years and the adult mosquito infestation index, with a three-month interval, with the relative humidity of the air in the same period, rainy days, average temperature and mean maximum temperature, with interval of one and two months, rainfall with intervals of one, two and three months. Therefore, it can be inferred that the index and land infestation is weak in estimating the risk of transmission of the disease, although it is recommended by the Ministry of Health for this purpose. The climate was one of the main factors responding to the incidence of dengue. The three-month interval, in relation to the beginning of the epidemics, proved to be the most opportune period to carry out the vector control actions. It is concluded that the results of this research can contribute to the precocity of information and enable decision making in a timely manner in order to focus on the areas of greatest risk and thus optimize the field service and prevention of diseases caused by Aedes aegypti

Les zoonoses émergentes sont en augmentation au cours des dernières décennies. Ainsi, il y est urgent de comprendre les mécanismes de cette émergence, en particulier d’étudier les facteurs écologiques sous-jacents qui déterminent les transferts de ces maladies de l’animal à l’homme.Originaire d’Asie, le moustique tigre Aedes albopictus est arrivé au Brésil dans les années 80 et s’est établi dans 60% des villes brésiliennes. Les forêts brésiliennes sont considérées comme un point chaud de la biodiversité mondiale, abritant des centaines d'arbovirus zoonotiques. Ces forêts que, par fois, cercles des grandes agglomérations urbaines, souffrent de la transformation du paysage réalisé par l’homme, un terreau idéal pour l'apparition de zoonoses. Cette espèce représente une préoccupation majeure pour la transmission d’arbovirus épidémiques (Dengue, Chikungunya, Zika) mais également une menace potentielle pour l’émergence de maladies zoonotiques en raison de sa présence dans les interfaces ville / forêt, de son comportement alimentaire opportuniste et de sa compétence vectorielle pour nombreux virus. Ainsi, Ae. albopictus pourrait potentiellement jouer le rôle de vecteur pont pour le transfert en milieu urbain de arbovirus zoonotiques qui circulent dans les forêts brésiliennes. Dans cette étude, nous évaluons le potentiel de cette espèce à servir de vecteur de pont entre la faune et l'homme. Pour cette proposition (i) Nous avons effectué une recherche et une analyse bibliographiques portant sur trois éléments clés permettant d’évaluer la capacité de ce moustique à assurer le transfert interspécifique des arbovirus dans les zones sylvatiques : 1) la capacité à exploiter les sites de reproduction larvaire naturels, 2) le comportement alimentaire et 3) la compétence vectorielle pour les dif férentes arbovirus. (ii) Nous avons étudié sur le terrain la colonisation, la dispersion, le comportement trophic et l'impact sur la biodiversité d'Ae. albopictus dans l'interface ville / forêt au Brésil. Ce travail de terrain a été réalisé à partir de dix écosystèmes de fragments de forêts dans trois Biomes au Brésil: 1) Biome Amazonia dans la réserve forestière d'Adolpho Ducke à Manaus; 2) Biome Mata Atlantica dans la réserve forestière de Pedra Branca à Rio de Janeiro, dans les fragments de forêt urbaine à Salvador, Serra, Belo Horizonte et dans les fragments de forêt rurale à Domingos Martins, Simonésia, Casimiro de Abreu, Marica-RJ; Biome Cerrado dans la forêt de Morro dos Macacos à Goiania. Dans cette thèse nous avons confirmé qu’Ae. albopictus a la capacité de coloniser des sites de reproduction naturels avec un comportement alimentaire opportuniste et une claire préférence pour les sources de sang humain, suivi par d'autres mammifères et oiseaux. Nous observons que le processus de colonisation et de dispersion est limité à la bordure de la forêt avec un impact sur la composition des espèces dans ces environnements. Nous avons observé également que ce moustique est capable de transmettre 13 arbovirus dans notre recherche bibliografic, mais aucun arbovirus n’a été détecté chez des moustiques prélevés lors du travail de terrain. Globalement, nos résultats confirment et estiment le rôle potentiel d’Ae. albopictus comme vecteur pont des maladies zoonotiques aux interfaces forêt / ville au Brésil. Ces travaux ouvrent un domaine de recherche dans lequel de nouvelles investigations pourraient évaluer le risque potentiel de propagation des maladies zoonotiques des zones forestières aux zones urbaines, dans le but de limiter les futures émergences virales. / Zoonotic emerging diseases are increasing during the last decades. There is an urgent need to understand the mechanisms of this emergence, in particular to study the underlying ecological factors determining spill-over events, the transfer from animals to humans. Being native from Asia, the tiger mosquito Aedes albopictus arrived to Brazil in the 80’s being nowadays established in 60% of brazilian cities. Brazilian forests are considered a hotspot of wildlife biodiversity, harbouring hundreds of zoonotic arboviruses, suffering human landscape transformation and surrounding large urban cities, an optimal breeding ground for the emergence of zoonotic diseases. This species represents a major concern for the transmission of epidemic arboviruses (dengue, chikungunya, Zika) but also a potential threat for the emergence of zoonotic diseases due to its presence in urban/forest interfaces, its opportunistic feeding behavior and its vector competence to transmit numerous viruses. Thus, Ae. albopictus might potentially participate as a bridge vector for the transfer to urban environments of zoonotic arboviruses that are circulating on Brazilian forests. In this study we evaluate the potential of this species to act as a bridge vector between wildlife and humans. For this propose (i) we performed a bibliographic research and analysis focusing on three key components for assessing the ability of a mosquito to ensure interspecies transfer of arboviruses in sylvatic areas: 1) the capacity to exploit natural larval breeding sites, 2) blood-feeding behaviour and 3) the vector competence for arboviruses. (ii) We investigated from fieldwork the colonization, dispersion, host feeding and potential impact of biodiversity patterns of Ae. albopictus in the urban/forest interface in Brasil. This field work was realized from ten forest-fragments ecosystems in tree Brazilians Biome: Biome Amazonia in Adolpho Ducke forest reserve-Manaus; Biome Mata Atlantica in Pedra Branca forest reserve- Rio de Janeiro, in the urban forest fragment Salvador, Serra, Belo Horizonte and in the rural forest fragment Domingos Martins, Simonésia, Casimiro de Abreu, Marica-RJ; Biome Cerrado in Morro dos Macacos forest – Goiania. This work confirmed that Ae. albopictus has the capacity to colonize natural breeding sites with the opportunistic feeding behavior with a preference for human blood sources, followed by other mammals and birds. We observe that the colonization and dispersion process is limited to the edge forest with an impact in species composition in this environments. We observed that this mosquitos is vector competence for 13 arbovirus from bibliographic recherché, but no arbovirus was detected from fildework collected mosquitos. Globally our results confirm and estimates the potential role of Ae. albopictus to act as a bridge vector of zoonotic diseases at the forest/urban interfaces in Brazil. This work opens a research area in which further investigations may assess the potential spill-over risk of zoonotic disease from forested to urban areas with the aim to mitigate potential future viral emergences.

Lipases hidrolisam triacilgliceróis a glicerol e ácidos graxos. Neste trabalho foram estudadas as lipases digestivas no mosquito Aedes aegypti (larva e adulto) e no carrapato Amblyomma cajennense, vetores de Dengue e da Febre Maculosa. As lipases estudadas pertencem a família das lipases neutras, são estáveis em ampla faixa de pH e em altas temperaturas. Estas enzimas apresentam maior eficiência hidrolítica sobre substratos de cadeias carbônicas curtas (butirato e heptanoato). As lipases digestivas nas duas fases de desenvolvimento do mosquito são distintas cinéticamente. O estudo combinado do genoma de Ae. aegypti e PCR comprovaram a expressão diferencial destas enzimas constituintes de uma família multigênica envolvida na expressão de lipases digestivas provavelmente reguladas pela dieta. / Lipases hidrolyse triacylglycerol to glycerol and fat acids. In the present work we studied the digestive lipases from Aedes aegypti (larvae and adults) and Amblyomma cajennense. Studied lipases belong to the neutral lipase family, stable in a large range of pH and to high temperatures. These enzymes have a higher catalytic efficiency upon short substrates like butyrate and heptanoate. The digestive lipases from both larval and adult mosquitoes are kinetically distinct. Combined study of Ae.aegypti genome and PCR showed the differential expression of lipases which belong to a multigenic family of enzymes probably regulated by diet.

Os mosquitos pertencentes à família Culicidae (Diptera) desempenham importante papel como vetores de patógenos causadores de doenças que acometem o homem em várias partes do mundo (MARCONDES, 2001), causando um grande impacto socioeconômico e de saúde pública nas áreas em que ocorrem. Aspectos biológicos e comportamentais dos mosquitos também contribuem decisivamente para sua atuação como transmissor de patógenos que atingem as populações humanas. O repasto sanguíneo é um processo fisiológico fundamental no ciclo de vida destas espécies. Por outro lado, a alimentação sanguínea infectada causa mudanças no metabolismo das fêmeas recémingurgitadas, desencadeando a expressão diferencial de genes envolvidos em respostas imunológicas, como também de genes envolvidos em outros processos fisiológicos. Como um dos interesses de nosso grupo é estudar as alterações sofridas pelo inseto vetor após a infecção por patógeno, nesse trabalho o ponto de partida para esse estudo foi a escolha dos genes DV431431, DV340891 e DV411647 que nas análises in silico apresentaram expressão diferencial mediante a infecção por vírus da Dengue ou Plasmodium gallinaceum. Após experimentos de RT-PCR em tempo real, foi mostrado que apenas o gene DV411647 apresentou expressão elevada 48 horas após a alimentação sanguínea infectada tanto por P. gallinaceum como por vírus dengue. Na busca por uma função predita desse gene em mosquitos, nós utilizamos a ferramenta Editseq através do software DNAstar para traduzir o peptídeo que apresentou maior número de aminoácidos, e confrontamos através de Blastp com o banco de dados GenBank, obtivemos uma provável desidrogenase/redutase de cadeia curta (AAK55494). Experimentos de Western blot evidenciaram que esta proteína esta presente no intestino médio das fêmeas 72 horas após a alimentação com sangue infectado por vírus dengue tipo 2. / Mosquitoes belonging to the family Culicidae (Diptera) play an important role as vectors of disease-causing pathogens that affect humans in various parts of the world (Marcondes, 2001), causing a great socioeconomic impact and public health areas in which they occur. Biological and behavioral aspects of mosquito also contribute decisively to its role as a transmitter of pathogens that affect human populations. The blood meal is a fundamental physiological process in the life cycle of these species. On the other hand, the infected blood meal causes changes in the metabolism of newly engorged females, triggering the differential expression of genes involved in immune responses, as well as genes involved in other physiological processes. As one of the interests of our group is to study the changes undergone by the insect vector after infection with the pathogen, in this work the starting point for this study was the selection of genes DV431431, DV340891 and DV411647 that the in silico analysis showed differential expression by Dengue virus infection of Plasmodium gallinaceum.Após or RT-PCR experiments in real time, it was shown that only the gene DV411647 had high expression 48 hours after the blood meal infected by both P. gallinaceum as for dengue virus. In the search for a predicted function of this gene in mosquitoes, we use the tool Editseq by DNAstar software to translate the peptide with the highest number of amino acids, and confronted by blastp with the GenBank database, we obtained a probable dehydrogenase / reductase short chain (AAK55494). Western blot experiments showed that this protein is present in the midgut of females 72 hours after feeding with blood infected with dengue virus type 2.

A crescente demanda por combustíveis provenientes de fontes renováveis, em função de questões ambientais e possível escassez dos derivados de petróleo, juntamente com a obrigatoriedade de sua adição no óleo diesel empregado atualmente, levou a uma \"explosão\" do número de empresas que produzem biodiesel. Este produto é obtido pela transesterificação de gorduras animais e óleos vegetais na presença de catalisador, processo que resulta um único sub-produto, a glicerina, a qual teve um aumento de produção na medida do aumento de produção do biodiesel. Uma forma de contribuir para diminuir os problemas advindos do acúmulo da glicerina é utilizá-la como base para formular meios de fermentação e obter produtos de interesse sócio-econômico. Um exemplo é o bioinseticida produzido pela bactéria Bacillus thuringiensis para o controle do mosquito transmissor do vírus da dengue. Este produto apresenta várias vantagens quando comparado aos inseticidas químicos, pois impede que os insetos adquiram resistência, não polui e é seletivo, não oferecendo riscos de intoxicação para os insetos benéficos e animais vertebrados. Cabe salientar que as toxinas produzidas por esta bactéria são letais também para larvas dos mosquitos transmissores da malária e da elefantíase. Assim, no presente trabalho propõe-se obter um bioinseticida com princípio ativo produzido por Bacillus thuringiensis var. israelensis, empregando o resíduo da fabricação de biodiesel a partir de óleo vegetal. Para tanto, o trabalho compreendeu três fases: 1. Definição de um tratamento para o resíduo que proporcionasse o crescimento da bactéria e a produção de toxina. 2- Estudo do processo fermentativo com o objetivo aumentar a atividade larvicida do meio fermentado em frascos (definição da composição do meio e de condições de fermentação) e 3. Estudo da transferância de oxigênio em diferentes fases do desenvolvimento da bactéria, em biorreator. O estudo em frasco foi realizado em Erlenmeyer de 1.000 mL, provido ou não de chicanas, com 200 mL de meio. O estudo da transferência de oxigênio foi realizado em fermentador Bioflo III, (New Brunswick Sci. Co.) de 1,25 L. Com os resultados obtidos, foi possível concluir que o melhor tratamento para o resíduo em estudo, consistiu do abaixamento do pH até 4 com ácido fosfórico, seguido de decantação por 24 horas. Com este tratamento foi possível obter uma fonte de carbono mais viável enocomicamente e, pelos estudos de otimização de processo, foi possível determinar a melhor composição de meio de cultura (10 g/L de glicerina tratada, 6 g/L de extrato de levedura, 3 g/L de (NH4)2SO4, 0,12 g/L de CaCl2.2H2O, 1,5 g/L de MgSO4.72H2O, 0,09 g/L de MnSO4, 1,5 g/L de K2HPO4 e 1,5 g/L de KH2PO4) e a melhor condição de aeração (manter a concentração de oxigênio dissolvido em 45% da saturação durante todas as fases do desenvolvimento da bactéria). Desta forma, foi possível produzir um meio fermentado 583 vezes mais potente que o ensaio controle empregando glicerol PA. / The increasing demand for fuels originated from renewable sources, due to environmental issues and possible scarce of petroleum products, together with the mandatory of its inclusion in diesel oil currently used, generated a significant increase in the number of biodiesel producting companies. This product is obtained through transesterification reaction of animal fat and vegetable oils in the presence of a catalyzer, process that results in a single by-product, the glycerin, which had an increase of its production as biodiesel production increases. A way to contribute for the reduction of problems generated by the excess of glycerin is to use it in the composition of fermentation medium and obtain products that are demanded by society. An example is the bioinsecticides produced by the bacteria named Bacillus thuringiensis, used in the controlling of mosquito vectors of the dengue viruses. This product presents several advantages when compared to chemical insecticides because it prevents insects resistance, it does not pollute the environment and it is selective, which means it does not create risks of intoxication for other insects and vertebrate animals. It is important to mention that toxins produced by these bacteria are lethal as well to the larvae of malaria and elephantiasis mosquito vectors. Thus, in this work it is intended to obtain a bioinsecticide containing active ingredient from Bacillus thuringiensis var. israelensis by using the biodiesel production waste from vegetable oils. In order to achieve that, this work consisted of three steps: 1. Definition of a treatment for the waste of biodiesel to provide the growth of Bacillus thuringiensis and its toxin production; 2. Study of the fermentative process to increase larvicidal activity of the fermentation broth in flasks (definition of the medium composition and the fermentative conditions) and 3. Study of the oxygen transference in bioreactor in diferent development phases of the bacteria. The study in flask was made in a 1000 mL Erlenmeyer flask, with and without baffles, and 200 mL of medium. The study of oxygen transference was realized in a 1,25 L bench fermentor (New Brunswick Sci. Co./Bioflo III). With the results obtained it was possible to conclude that the best treatment to the waste of biodiesel consisted of decreasing pH until 4 with phophoric acid, followed by 24 hours of decantation. With this treatment it was possible to obtain a promising carbon source more and, by the process optimization studies, it was possible to determine the best composition of culture medium (10 g/L of treated glycerin; 6 g/L of yeast extract; 3 g/L of (NH4)2SO4; 0,12 g/L of CaCl2.2H2O; 1,5 g/L of MgSO4.7H2O; 0,09 g/L of MnSO4; 1,5 g/L of K2HPO4 and 1,5 g/L of KH2PO4) and the best aeration condition (mantaining the oxygen concentration at 45% of saturation during all phases of bacterial development). So, it was possible to obtain a fermented broth 583 times more potent than the control test using pure glycerol.

Com o aumento progressivo do número de casos de infecção por diferentes arbovírus, por exemplo, dengue, zica e chikungunya, faz-se necessário o desenvolvimento de novas técnicas para o combate a esses arbovírus. A manipulação genética possibilitou a obtenção de mosquitos geneticamente modificados que sejam capazes de suprimir a população selvagem ou impedir a transmissão de agentes etiológicos gerando doenças. O estudo teve como objetivo o estabelecimento de linhagens para supressão populacional de Aedes aegypti. Esta construção apresenta esterilidade condicionada à presença ou ausência de antibiótico no meio em que esses mosquitos se desenvolvem durante a fase larval. Dessa forma, sem a necessidade de se utilizar radiação para obter insetos estéreis, é possível melhorar a qualidade dos machos adultos liberados e aumentar a competitividade dos mesmos em competir por fêmeas selvagens e adicionalmente gerar o desejado quadro de supressão populacional. / The increasing number of infection cases by arboviruses, for instance Dengue, Zika and Chikungunya, it is necessary to develop new techniques to fight against these arboviruses. Genetic manipulation allowed the production of genetically modified mosquitoes that are capable of suppressing the wild population or prevent pathogens transmission and avoid disease development. The project proposes to establish lines for population suppression of Aedes aegypti, one of the main vectors of those diseases. This construction presents the conditional sterility due to the presence or absence of antibiotics in the environment where these mosquitoes are developed during the larval stage. Thus, without the need to use radiation for sterile insects, it is possible to improve the quality of the released adult males and enhance the competitiveness thereof in competing with wild females and additionally generate population suppression.

O mosquito Aedes (Stegomyia) aegypti Linnaeus, 1762 é o principal vetor do vírus do dengue sorotipo 1-4 (DEN 1-4) e da febre amarela. Por não haver vacina disponível, a redução da transmissão da dengue só pode ser alcançada mediante o controle do vetor. Entre as medidas de controle, os órgãos responsáveis utilizam-se de compostos químicos, principalmente organofosforados. Além disso, devido ao grande incomodo causado pelo Aedes, a população faz uso de inseticidas domésticos, a base de piretróides, na tentativa de eliminar e ou repelir o mosquito. As repetidas aplicações destes inseticidas e seu uso contínuo possibilitam o desenvolvimento de resistência em populações de mosquitos, processo resultante do efeito seletivo de exposição a dosagens que matam os indivíduos suscetíveis, sobrevivendo os resistentes, que transferem essa capacidade a seus descendentes. Dentre os mecanismos de resistência, a redução da sensibilidade do sítio alvo é dada por mutações pontuais no sitio de ação dos inseticidas. Tais mutações podem levar a uma substituição de aminoácidos na molécula alvo e a uma diminuição da afinidade do inseticida com essa molécula. No caso dos piretróides a mudança estrutural na molécula formadora do canal de sódio (Nav), sítio alvo deste inseticida, é a causa da resistência tipo knockdown resistence (kdr), um mecanismo de resistência bastante conhecido. Em Aedes aegypti, a associação entre a presença da mutação V1016I e o fenótipo de resistência a piretróide já foi verificada em algumas regiões Brasileiras, utilizando primers alelo-específicos. A mutação I1011M também está associada à resistência a piretróides e já foi descrita no Brasil. O objetivo deste estudo foi determinar a frequência destas mutações que levam às substituições V1016I e I1011M no AaNav de populações de Aedes aegypti no estado de São Paulo e avaliar a evolução desta frequência no período de dez anos. Para isso, indivíduos coletados em 2001 e 2011 tiveram o DNA extraído e primers alelo específicos foram utilizados para a realização de PCR a fim de verificar a presença da mutação. Houve um aumento significativo do alelo 1016 Ile nas populações estudadas, comparando-se os anos 2001 e 2011. Entretanto para o alelo 1011 Met, somente a população de Santos apresentou essa diferença significativa. Este aumento na frequência destas mutações pode ter sido ocasionado pela utilização de inseticidas domésticos à base de piretróides, uma vez que os órgãos de controle interromperam a utilização desses compostos / The mosquito Aedes (Stegomyia) aegypti Linnaeus, 1762 is the main vector of the virus dengue serotypes 1-4 (DEN 1-4) and the yellow fever virus. As there is not vaccine available, the reduction of the transmission of dengue can only be achieved by controlling the vector. Among the control measures, the responsible agencies use chemical compounds, organophosphate mainly. Furthermore, due to the big nuisance caused by Aedes, the population makes use of domestic insecticide, pyrethroids based, trying to eliminate or repel the mosquito. The repeated applications of these insecticides and the continuous use of them enable the resistance development in the mosquitoes population. This process is the result of selective effect of exposure to dosages that can kill the susceptible individuals, and then the surviving individuals transfer these characteristics to their descendents. Among the mechanism of resistance, the reduction of sensibility of the target site is given by punctual mutation (SNIP) in the action site of the insecticides. This mutation can lead to a substitution of amino acids in the target molecule and a reduction of affinity of the insecticide with this molecule. In the case of pyrethroids, the structural change in the forming molecule of the sodium channel (Nav), target site of this insecticide, is the cause of knockdown resistence (kdr) type. In Aedes aegypti, the association between the presence of the mutation V1016I and the pyrethroid resistance phenotype has been previously verified in many Brazilian regions, using Allele Specific primers The mutation I1011M is also associated with the pyrethroids resistance and it has been described in Brazil. The objective of the this study was to determine the frequency of the mutations that leads to the substitutions V1016I e I1011M in AaNav of the Aedes aegypti population in the state of Sao Paulo and to evaluate the evolution of this frequency in the period of ten years. Therefore, collected individuals in 2001 and 2011 had their DNA extracted and specific primers were used for PCR, in order to verify the presence of the mutation. There was a significant raise of allele 1016 Ile in the studied population, comparing the years 2001 and 2011. Nevertheless for allele 1011 Met, only the population of Santos showed this significant difference. This raise in the frequency of these mutations may have been caused by the utilization of domestic insecticides pyrethroid based, whereas the control agencies interrupted the utilization of this compost