Novas alternativas são necessárias para controlar o mosquito transmissor da dengue, como a manipulação genética. Baseada na técnica do inseto estéril (SIT) que compreende a esterilização, a criação em massa e a liberação de grandes números de insetos machos estéreis em uma área alvo, a tecnologia RIDL, baseada em SIT, compreende criação em massa e liberação de mosquitos machos que carregam gene letal para sua prole, neste caso a linhagem OX513A de Aedes aegypti é testada nesse projeto, através da avaliação em testes laboratoriais e em testes de campo com marcação liberação e recaptura, comparando esta linhagem com linhagens selvagens. Foi mensurada a competitividade, longevidade e dispersão. Os testes foram realizados no bairro de Itaberaba (Juazeiro/BA) e na Universidade de São Paulo. Além da avaliação foi realizado o monitoramento com armadilhas ovitrampa, captura de mosquitos adultos com aspirações em residências. E o desenvolvimento de um plano de comunicação para a sociedade. Os resultados apontam que a compatibilidade entre as linhagens (transgênica e selvagem) foi positiva e a competitividade não apresentou tendência entre as fêmeas de escolherem uma linhagem ou outra. Estatisticamente não há diferença entre o número de ovos e larvas (logo a fertilidade) entre a linhagem selvagem e transgênica. O monitoramento da área de estudo confirmou a presença de A. aegypti, e não foi capturado nenhum indivíduo de Aedes albopictus. Para avaliar a dispersão, os mosquitos machos transgênicos foram liberados no ambiente e esses apresentaram uma sobrevivência no campo de 2,3 dias e um raio de vôo de 80 metros do ponto de liberação. O índice de esterilidade relativa foi determinado baseado na competitividade e proporção de ovos fluorescentes encontrados. Foi possível estabelecer uma produção em massa para realizar a fase de pré-supressão com a liberação de 540.000 machos ao longo de seis semanas e obtenção de 17% de larvas transgênicas oriundas do cruzamento desses machos com fêmeas do campo. Baseado nesses dados iniciou-se a fase de supressão com a liberação alvo de 400.000 por semana, aproximadamente 05 vezes mais esperando alcançar o estágio de supressão. / New alternatives are needed to control mosquitoes that transmit Dengue. Based on insect technique Sterile (SIT), which comprises sterilizing, mass rearing and release a large number of sterile males in an area target, RIDL technology based on SIT, but using transgenesis instead of radiation. Doing the same process for mass rearing and release of male mosquitoes carrying lethal gene to their offspring, in this case the strain OX513A of Aedes aegypti is under test in this project, through the evaluation of laboratory and field trials with mark-release-recapture (MRR), comparing transgenic with wild-type. We measured competitiveness, longevity and dispersal. The tests were performed in Itaberaba neighborhood (Juazeiro / BA) and at University of São Paulo. The evaluation was carried out with monitoring through ovitraps and adult mosquitoes collection with aspiration in houses. It was also developed a communication plan to society/community in general. The results indicate that the compatibility between the lines (transgenic and wild-type) was positive, and competitiveness showed no trend among females to choose one lineage or another. Statistically there were no difference between the number of eggs and larvae (resulting fertility) between the transgenic line and wild-type. The study area monitoring confirmed the presence of Ae. aegypti, and no Aedes albopictus was captured. To evaluate dispersion, transgenic males were released into the environment and they showed a field survival of 2.3 days and a flight range of 80 meters from the release point. The relative sterility index was determined based on the competitiveness and fluorescent proportion of eggs. Mass production was established to perform the pre-suppression phase releasing 540,000 males over six weeks and obtaining 17% of transgenic larvae in response of transgenic males mating field females. Based on these data suppression process have started with a release target of 400,000 per week, this is about 05 times more to reach the suppression stage briefly.

O mosquito Aedes aegypti é vetor de doenças como a febre amarela, dengue, chinkungunya e zika. O sistema digestório é responsável pela digestão e absorção de nutrientes, é também uma interface com o ambiente externo sendo a porta de entrada de organismos infecciosos. A presença de duas lipases digestivas foi confirmada por qPCR, uma na fase larval (L-Aa7051) e outra na fase adulta (L-Aa7055). Estas enzimas foram agrupadas na família das lipases neutras e apresentam alterações em resíduos envolvidos na especificidade, domínio tampa e alça β9. A L-Aa7055 recombinante foi expressa heterologamente em Escherichia coli na porção insolúvel, com atividade após a renaturação. Observamos que a expressão da lipase L-Aa7055 sofre uma redução de 30% na infecção Plasmodium gallinaceum, não sendo afetada pelo vírus dengue sorotipo 2 (DENV2). A digestão de lipídeos é importante na fase larval, com altos níveis de transcrito. Um estudo mais aprofundado ainda será necessário para compreender completamente o papel das lipases no processo infeccioso. / The Aedes aegypti borne diseases yellow fever, dengue fever, chinkungunya and zica are important public healthy problems. The digestion and absorption of nutrients are performed in the digestive system, which is also an external environment interface that allows the infection by pathogenic microorganisms. The presence of two digestive lipases were identified by qPCR, L-Aa7051 in the larval phase and L-Aa7055 in the adult female. The lipase sequences were grouped in the neutral family, and exhibit alterations in residues involved in specificity, lid domain and β9 loop. The recombinant L-Aa7055 was expressed in the insoluble fraction, and show activity after a renaturation process. We notice that the expression levels of L-Aa7055 are reduced by 30% in the Plasmodium gallinaceum infection and were not affected by serotype 2 dengue virus (DENV2). The lipid digestion is important in the larval phase, with higher transcript levels. New studies will be necessary to the complete understanding of lipase contribution in the infectious process.

O mosquito Aedes aegypti é vetor de doenças como a febre amarela, dengue, chinkungunya e zika. O sistema digestório é responsável pela digestão e absorção de nutrientes, é também uma interface com o ambiente externo sendo a porta de entrada de organismos infecciosos. A presença de duas lipases digestivas foi confirmada por qPCR, uma na fase larval (L-Aa7051) e outra na fase adulta (L-Aa7055). Estas enzimas foram agrupadas na família das lipases neutras e apresentam alterações em resíduos envolvidos na especificidade, domínio tampa e alça β9. A L-Aa7055 recombinante foi expressa heterologamente em Escherichia coli na porção insolúvel, com atividade após a renaturação. Observamos que a expressão da lipase L-Aa7055 sofre uma redução de 30% na infecção Plasmodium gallinaceum, não sendo afetada pelo vírus dengue sorotipo 2 (DENV2). A digestão de lipídeos é importante na fase larval, com altos níveis de transcrito. Um estudo mais aprofundado ainda será necessário para compreender completamente o papel das lipases no processo infeccioso. / The Aedes aegypti borne diseases yellow fever, dengue fever, chinkungunya and zica are important public healthy problems. The digestion and absorption of nutrients are performed in the digestive system, which is also an external environment interface that allows the infection by pathogenic microorganisms. The presence of two digestive lipases were identified by qPCR, L-Aa7051 in the larval phase and L-Aa7055 in the adult female. The lipase sequences were grouped in the neutral family, and exhibit alterations in residues involved in specificity, lid domain and β9 loop. The recombinant L-Aa7055 was expressed in the insoluble fraction, and show activity after a renaturation process. We notice that the expression levels of L-Aa7055 are reduced by 30% in the Plasmodium gallinaceum infection and were not affected by serotype 2 dengue virus (DENV2). The lipid digestion is important in the larval phase, with higher transcript levels. New studies will be necessary to the complete understanding of lipase contribution in the infectious process.

Les arbovirus (virus transmis par des arthropodes) sont à l'origine de maladies humaines telles que la dengue, le chikungunya ou encore le Zika. Le moustique Aedes aegypti, est le vecteur majeur de ces trois arbovirus. La faible efficacité des méthodes de contrôle des populations de moustiques, principalement réalisées au moyen d'insecticides chimiques ouvre un champ de développement de nouvelles approches en lutte antivectorielle. Le moustique, hôte vecteur, contrôle la réplication virale en limitant les réponses immunitaires antivirales. La machinerie RNA interférence (RNAi) est la voie jouant un rôle majeur dans l'immunité antivirale chez le moustique. Alors que le rôle des deux voies, siRNA (" small interfering RNA ") et piRNA (" piwi-interfering RNA "), est de mieux en mieux compris dans les réactions antivirales du vecteur, peu de connaissances sont disponibles à ce jour en ce qui concernent les interactions entre la voie miRNA (" micro RNA ") et les arbovirus. Ainsi, nous proposons une analyse détaillée des mécanismes par lesquels les miARN tentent de réguler la réplication virale chez le moustique. Dans la première partie de la thèse, nous avons effectué une analyse génomique pour identifier les miRNAs pouvant interagir chez Ae. aegypti avec divers lignées/génotypes des virus chikungunya (CHIKV), de dengue (DENV) et de Zika. Avec l'aide d'outils de prédiction faisant appel à divers algorithmes, plusieurs sites de liaison de miARN avec différents lignées/génotypes de chaque arbovirus ont été identifiés. Nous avons ensuite sélectionné les miARN pouvant cibler plus d'un arbovirus et nécessitant un faible seuil d'énergie lors de la formation des complexes entre l'ARNm. / Mosquito-borne arboviruses cause some of the world’s most devastating diseases and are responsible for recent dengue, chikungunya and Zika pandemics. The yellow-fever mosquito. Aedes aegypti, plays an important role in the transmission of all three viruses. The ineffectiveness of chemical control methods targeting Ae. aegypti makes urgent the need for novel vector-based approaches for controlling these diseases. Mosquitoes control arbovirus replication by triggering immune responses. RNAi machinery is the most significant pathway playing a role on antiviral immunity. Although the role of exogenous siRNA and piRNA pathways in mosquito antiviral immunity is increasingly better understood, there is still little knowledge regarding interactions between the mosquito cellular miRNA pathway and arboviruses. Thus further analysis of mechanisms by which miRNAs may regulate arbovirus replication in mosquitoes is pivotal. In the first part of the thesis, we carried out genomic analysis to identify Ae. aegypti miRNAs that potentially interact with various lineages and genotypes of chikungunya (CHIKV), dengue (DENV) and Zika viruses. By using prediction tools with distinct algorithms, several miRNA binding sites were commonly found within different genotypes/and or lineages of each arbovirus. We further analyzed the miRNAs that could target more than one arbovirus and required a low energy threshold to form miRNA-vRNA (viral RNA) complexes and predicted potential RNA structures using RNAhybrid software. Thus, we predicted miRNA candidates that might participate in regulating arboviral replication in Ae. aegypti. In the second part of the thesis, we developed a miRNA-based approach that results in a dual resistance phenotype in mosquitoes to dengue serotype 3 (DENV-3) and chikungunya (CHIKV) viruses for stopping arboviruses spreading within urban cycles. The target viruses are from two distinct arboviral families and the antiviral mechanism is designed to function through the endogenous miRNA pathway in infected mosquitoes. Ten artificial antiviral 4 miRNAs capable of targeting ~97% of all published strains were designed based on derived consensus sequences of CHIKV and DENV-3. The antiviral miRNA constructs were placed under control of either an Aedes PolyUbiquitin (PUb) or Carboxypeptidase A (AeCPA) gene promoter triggering respectively expression ubiquitously in the transgenic mosquitoes or more locally in the midgut epithelial cells following a blood meal. Challenge experiments using viruses added in blood meals showed subsequent reductions in viral transmission efficiency in the saliva of transgenic mosquitoes as a result of lowered infection rate and dissemination efficiency. Several components of mosquito fitness, including larval development time, larval/pupal mortality, adult lifespan, sex ratio, and male mating competitiveness, were examined: transgenic mosquitoes with the PUb promoter showed minor fitness costs at all developing stages whereas those based on AeCPA exhibited a high fitness cost. Further development of these strains with gene editing tools could make them candidates for releases in population replacement strategies for sustainable control of multiple arbovirus diseases.

<p>Meteorological&nbsp; conditions&nbsp; have&nbsp; a&nbsp; significant influence on the time of occurrence, abundance and activity of the mosquito vector. In the current context of climate change, it is of great importance to assess the&nbsp; impact&nbsp; of&nbsp; shifts&nbsp; in&nbsp; climatic&nbsp; conditions&nbsp; on&nbsp; the suitability for the establishment and annual activity of&nbsp; the&nbsp; vector&nbsp; species.&nbsp; Moreover,&nbsp; changes&nbsp; in&nbsp; the variability&nbsp; of&nbsp; meteorological&nbsp; elements&nbsp; and&nbsp; their extremes&nbsp; can&nbsp; generate&nbsp; unexpected&nbsp; changes&nbsp; in&nbsp; the mosquito&nbsp; vector&nbsp; population&nbsp; which&nbsp; in&nbsp; turn&nbsp; have&nbsp; an&nbsp; important effect on human health. One of the ways to put&nbsp; these&nbsp; causes&nbsp; and&nbsp; effects&nbsp; into&nbsp; perspective&nbsp; is&nbsp; to simulate the activity of the vector within a processbased framework which allows for the analysis of the contribution of individual factors on the different life stages of the vector. Such analysis is presented by use of&nbsp; sophisticated&nbsp; dynamical&nbsp; models&nbsp; simulating&nbsp; the characteristics of the biological population, forced by observed&nbsp; meteorological&nbsp; data,&nbsp; capturing&nbsp; the&nbsp; localmicro-environment&nbsp; of&nbsp; the&nbsp; vector&nbsp; habitat,&nbsp; and validated by the observed entomology.Numerical&nbsp; models&nbsp; are&nbsp; being&nbsp; developed&nbsp; to&nbsp; model vector&nbsp; population&nbsp; dynamics&nbsp; and&nbsp; the&nbsp; expected circulation of the virus within a closed system. Two modelling&nbsp; approaches&nbsp; are&nbsp; standardly&nbsp; applied&nbsp; to modelling vector population dynamics: Mechanistic and&nbsp; Stochastic.&nbsp; The&nbsp; advantage&nbsp; of&nbsp; mechanistic&nbsp; over<br />statistical&nbsp; models&nbsp; is&nbsp; that&nbsp; they&nbsp; can&nbsp; provide&nbsp; a deterministic&nbsp; framework&nbsp; allowing&nbsp; for&nbsp; the&nbsp; isolated evaluation of each input parameter and their effect on the modelled system. Mechanistic dynamical models are used to describe the biophysical processes or part<br />of&nbsp; the&nbsp; process&nbsp; as&nbsp; a&nbsp; response&nbsp; to&nbsp; changes&nbsp; in&nbsp; the meteorological conditions.<br />The&nbsp; work&nbsp; carried&nbsp; out&nbsp; in&nbsp; this&nbsp; thesis&nbsp; can&nbsp; be summarized as follows: (i) Analysis of the association between&nbsp; the&nbsp; most&nbsp; important&nbsp; abiotic&nbsp; drivers influencing the population dynamics, annual activity and&nbsp; time&nbsp; of&nbsp; occurrence&nbsp; of Culex&nbsp; pipiens and Aedes aegypti;&nbsp; (ii)&nbsp; Identifying&nbsp; the&nbsp; most&nbsp; important&nbsp; climatic factors and model settings as a function of climatic characteristics of the study region; (iii) Modelling the vector population dynamics and stability analysis of the&nbsp; dynamical&nbsp; system&nbsp; (iv)&nbsp; Analysis&nbsp; of&nbsp; different verification techniques and implications in terms of model&nbsp; application;&nbsp; (v)&nbsp; Feasibility&nbsp; analysis&nbsp; of<br />improving&nbsp; the&nbsp; model&nbsp; with&nbsp; a&nbsp; Land-Surface Parametrization scheme and short-range forecasting of pest population dynamics.</p> / <p>Meteorolo&scaron;ki&nbsp; uslovi&nbsp; bitno&nbsp; utiču&nbsp; na&nbsp; vreme&nbsp; pojave, brojnost&nbsp; vektora&nbsp; i&nbsp; njihovu&nbsp; aktivnost.&nbsp; U&nbsp; uslovima evidentnih promene klime, od ogromne je važnosti sagledati&nbsp; uticaj&nbsp; očekivanih&nbsp; promena&nbsp; klime&nbsp; na pogodnost&nbsp; uslova&nbsp; na&nbsp; pojavu&nbsp; izabranih&nbsp; vektora.<br />Takodje, značajne promene kolebanja meteorolo&scaron;kih elemenata&nbsp; u&nbsp; odnosu&nbsp; na&nbsp; vi&scaron;egodi&scaron;nji&nbsp; prosek&nbsp; i&nbsp; sve če&scaron;će pojave nepovoljnih vremenskih prilika dovode do neočekivanog pona&scaron;anja populacije komarca &scaron;to značajno&nbsp; utiče&nbsp; na&nbsp; kvalitet&nbsp; života&nbsp; i&nbsp; zdravlje&nbsp; ljudi. Jedini&nbsp; način&nbsp; da&nbsp; se&nbsp; sagledaju&nbsp; uzroci&nbsp; i&nbsp; posledice navedenih pojava zasniva se na simulaciji aktivnosti i&nbsp; brojnosti&nbsp; vektora&nbsp; uz&nbsp; mogućnost&nbsp; testiranja&nbsp; uticaja svakog&nbsp; pojedinačnog&nbsp; faktora.&nbsp; Ovu&nbsp; mogućnost pružaju samo visoko sofistikovani dinamički modeli koju su pro&scaron;li proces kalibracije i validacije zasnovan<br />na izmerenim vrednostima meteorolo&scaron;kih elemenata i karakteristika biolo&scaron;ke populacije.<br />Sofistikovani&nbsp; modeli&nbsp; za&nbsp; simulaciju&nbsp; dinamike populacije vektora i očekivane cirkulacije vektorskih transmisivnih bolesti se koriste sa ciljem modeliranja potencijalnog rizika od zaraze i epidemije. Modeli zasimulaciju dinamike vektora mogu da se podele na dve&nbsp; glavne&nbsp; grupe:&nbsp; Mehanističke&nbsp; i&nbsp; Statističke. Prednost&nbsp; mehanističkih&nbsp; modela&nbsp; nad&nbsp; statističkim&nbsp; je &scaron;to&nbsp; mogu&nbsp; da&nbsp; se&nbsp; koriste&nbsp; za&nbsp; evaluaciju&nbsp; uticaja izolovanog&nbsp; faktora&nbsp; na&nbsp; dinamički&nbsp; sistem&nbsp; i odgovarajuće promene brojnosti unutar svake faze u razvoju&nbsp; vektora.&nbsp; Mehanistički&nbsp; dinamički&nbsp; sistemi&nbsp; se koriste&nbsp; kako&nbsp; bi&nbsp; se&nbsp; opisao&nbsp; mehanizam&nbsp; biofizičkog procesa&nbsp; ili&nbsp; dela&nbsp; procesa&nbsp; u&nbsp; zavisnosti&nbsp; od&nbsp; forsirajuće veličine.<br />Predmet&nbsp; istraživanja&nbsp; u&nbsp; ovom&nbsp; radu&nbsp; jeste identifikovanje&nbsp; najznačajnijih&nbsp; biolo&scaron;kih&nbsp; i&nbsp; fizičkih procesa&nbsp; kao&nbsp; i&nbsp; odgovarajućih&nbsp; faktora&nbsp; koji&nbsp; utiču&nbsp; na brojnost i aktivnost vektora roda Aedes i Culex. Ciljevi istraživanja mogu da se sumiraju na sledeći način: (i)<br />analiza najznačajnijih meteorolo&scaron;kih parametara koji utiču na vreme pojave, brojnost i aktivnost vektora Aedes&nbsp; i&nbsp; Culex&nbsp; roda;&nbsp; (ii)&nbsp; definisanje&nbsp; najznačajnijih klimatskih&nbsp; faktora&nbsp; i&nbsp; stepena&nbsp; osetljivosti&nbsp; procesa&nbsp; na njih; (iii) modeliranje dinamike populacije vektora i analiza&nbsp; stabilnosti&nbsp; dinamičkog&nbsp; sistema;&nbsp;&nbsp; (iv) verifikacija&nbsp; i&nbsp; analiza&nbsp; metoda&nbsp; verifikacije&nbsp; i&nbsp; validacije dinamičkog&nbsp; modela;&nbsp; (v)&nbsp; kratkoročna&nbsp; prognoza dinamike&nbsp; populacije&nbsp; komarca&nbsp; i&nbsp; formulacija hidrolo&scaron;kog modula upotrebom SURFEX povr&scaron;inske<br />&scaron;eme sa ECOCLIMAP fiziogeografskim podacima.</p>

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Stephen Higgs / The genetic composition of arboviruses is a critical determinant of viral infectivity and the capacity for virus dissemination in arthropod vectors. Due to concerns related to a hypothetical potential for loss of attenuation, the supression of vector infection and dissemination is a critical component for the rationale-based design of live-attenuated flavivirus vaccine candidates. The yellow fever virus (YFV) 17D vaccine virus is not only attenuated in vertebrates, but also has low infectivity for Aedes agypti mosquitoes and since it does not disseminate, it is not transmissible. Using a reverse genetics system, the mutations present in the envelope protein YFV 17D virus were characterized in Ae. aegypti to determine the role of mutations in limiting the viral infectivity and dissemination capacity. This knowledge would contribute to the rational design of live attenuated vaccines with the desirable phenotype of being nontransmissible by arthropod vectors. The upper lateral portion of the YFV 17D envelope (E) protein domain III (EDIII) habors the T380R mutation in the FG loop. Experiments demonstrated that the T380R mutation was associated with the viral infectivity phenotype for mosquitoes, but did not influence dissemination into the secondary tissues. The G52R mutation in the molecular hinge region that is located between E protein domains I (EDI) and II, significantly reduced viral infectivity for mosquitoes. In contrast, when cloned into the Asibi wildtype virus genetic backbone, the T173I mutation in the loop structure between the G0 and H0 β- strands did not attenuate viral infection and dissemination. The double mutant virus containing both the G52R and T173I mutations in the E protein, showed a similar attenuated reduced infectivity to the single G52R mutant. The M299I mutation in the linker region between EDI and EDIII resulted in a significantly lower viral infectivity at the initial phase of viral infection at 7 days post-infection in Ae. aegypti. In conclusion, the characterization on four mutations in the YFV 17D vaccine E protein have demonstrated three genetic loci, that can influence the process of YFV infection in Ae. aegypti. These results provide new knowledge and understanding which may have broad applications for the rationale design of safe flavivirus vaccines, via targeting genetic loci and introducing specific mutations that preclude infection of, and transmission by arthropod vectors.

Female mosquitoes of the species, Aedes aegypti (yellow fever mosquito, Diptera), blood feed for oogenesis. Therefore, mosquitoes are exposed to high iron loads and possibly blood-borne pathogens. We are interested in studying iron metabolism in A. aegypti to find methods for controlling mosquito populations, and thereby reduce human exposure to these pathogens. First, we found that the expression of the Aedes ferritin light chain homologue (LCH) is up-regulated by blood feeding. Ferritin LCH and heavy chain homologue (HCH) genes are closely clustered together and both mRNA transcripts increase with iron and oxidative stress (H2O2 and hemin). Second, we show A. aegypti larval cells synthesize and secrete ferritin in response to iron. Cytoplasmic ferritin is maximal at low levels of iron, consists of a specific subunit composition and reflects cytoplasmic iron levels. Secreted ferritin increases in linear relationship to increasing iron dose and is composed of different subunits than cytoplasmic ferritin. HCH and LCH transcripts increase with increasing cytoplasmic iron suggesting transcriptional control of ferritin synthesis. We previously reported that the mosquito HCH mRNA has an iron responsive element (IRE), but LCH mRNA does not have a canonical IRE. We show that iron regulatory protein 1 (IRP1)/IRE binding activity declines in response to increasing cytoplasmic iron levels. These data would indicate that HCH synthesis is controlled at transcription and translation. Third, we report that A. aegypti larval cell cytoplasmic iron concentration does not change temporally with iron treatment. However, membrane iron levels increase with iron over time. Iron temporally up-regulates both HCH and LCH mRNA. Ferritin secretion increases with time in response to iron and reflects that most of the intracellular ferritin is found in the membrane fraction. Membrane ferritin has the same subunit composition as cytoplasmic ferritin. Finally, membrane ferritin is found in both non-iron and iron-treated cells. This suggests a mechanism to store iron from a blood meal in membrane ferritin. These results indicate Aedes ferritin could act as an antioxidant and holoferritin secretion is likely the mechanism whereby mosquito cells protect against iron overload and, thus reduce the intracellular potential for iron-mediated oxidative stress during blood feeding.

Aedes aegypti is one of the most important mosquito vectors of human disease, transmitting dengue, Chikungunya and yellow fever viruses. New control methods are much needed for this species and an ability to skew sex ratios toward males would be very useful for a number of strategies. In this study, male-specific chromosomal regions were searched for in Ae. aegypti using novel approaches such as microdissection of chromosomes. The sex determination pathway of Ae. aegypti was also explored, using Drosophila melanogaster as a model, to find candidate genes that could be used to induce male biased sex ratios. The transformer-2 (tra-2) gene is necessary for sexual differentiation of females in D. melanogaster, but its role remains unknown in mosquitoes. A homolog of tra-2 was identified in Ae. aegypti. Rapid Amplification of cDNA ends (RACE) experiments were conducted to characterize this gene in Ae. aegypti. The Ae. aegypti tra-2 gene, as for D. melanogaster, was highly variable in transcription due to alternative splicing and alternative polyadenylation, with 9 different variants identified. RNA interference (RNAi) was then used to determine if knockdown of all variants of Ae. aegypti tra-2 can be achieved and if it would cause gender switching in individuals, initially by means of direct injection of double stranded RNA, and then progressing to germline transformation. A construct designed to produce tra-2 dsRNA was injected into Ae. aegypti embryos and integrated into the Ae. aegypti genome. The transgenic population showed up to 100% male bias in single pair crosses. The male bias effect could still be seen in the heterozygous population. The results suggest that knockdown of tra-2 could provide a useful tool for sex ratio distortion as part of the development of novel control methods for Ae. aegypti.

Submitted by Dominick Jesus (dominickdejesus@hotmail.com) on 2015-11-06T18:12:42Z No. of bitstreams: 2 Dissertação_Joelma Soares da Silva.pdf: 3973131 bytes, checksum: e2d308bc76b0d2e137227d73bfb0719f (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-11-06T18:12:43Z (GMT). No. of bitstreams: 2 Dissertação_Joelma Soares da Silva.pdf: 3973131 bytes, checksum: e2d308bc76b0d2e137227d73bfb0719f (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2011-02-23 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / In Amazonas state have been isolated 57 strains of Bacillus thuringiensis, these five have shown toxicity to Aedes aegypty. Isolates did not show the genes of Cry toxins 4AA; 4ba; 10Aa; 11Aa and 11Ba, except BtAM- 27 lineage that showed all genes analyzed. Data from the larvicidal activity of Amazonian isolates were not superior to those obtained with the standard strain Bti IPS-82, entrtanto BtAM 27 showed similar toxicity to this, the isolated enters obtained in this work, IBt-07 and IBt-28 were the most toxic . / No estado do Amazonas foram isolados 57 estirpes de Bacillus Thuringiensis, destas, cinco mostraram toxicidade às larvas de Aedes aegypty. Os isolados não apresentaram os genes das toxinas Cry 4Aa; 4Ba; 10Aa; 11Aa e 11Ba, com exceção da linhagem BtAM- 27 que apresentou todos os genes analisados. Os dados da atividade larvicida dos isolados do Amazonas não foram superiores aos obtidos com a estirpe padrão Bti IPS-82, entrtanto BtAM 27 mostrou toxicidade similar á esta, entra os isolados obtidos neste trabalho, IBt-07 e IBt-28 foram os mais tóxicos.

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