The development of genetic transformation systems in insects has revolutionized the field of entomology. Transgenic insects provide tools to identify, isolate and analyze insect genes and to genetically modify insects for the purposes of insect control or disease vector modification. When transformation frequencies are high, multiple transgenic lines can be generated with relative ease. However, in most mosquito species, the results of transformation experiments have been suboptimal. Increased mosquito transformation efficiency is a research priority. Additionally, incorporation of refractory transgenes will not be sufficient to modify natural populations. A gene drive system will be required to allow transgenes to proliferate throughout populations and potentially reach fixation. This study proposes the use of germline-specific regulatory elements to promote confined, regulated transposase expression within the germ tissue. Creation of helper constructs utilizing endogenous promoters will potentially increase genetic transformation frequencies. The generation of lab strains of mosquitoes expressing an endogenous source of transposase within the germline will also serve as a powerful research tool. Endogenous sources of transposase will allow for comparative analysis of integration rates using different donor plasmids. Finally, the generation of autonomous transposable elements will provide a gene drive mechanism to move a tightly-linked refractory gene into a population. Four genes have been identified, cloned, and characterized, revealing expression patterns expected of germline-specific genes. Transcription profiles and in situ hybridization data support these conclusions. Putative cis-acting regulatory elements have been cloned and incorporated into DNA plasmid constructs. These elements are cloned in a manner such that they will regulate fluorescent gene expression. Additionally, similar elements have been cloned upstream of the Mos1 open reading frame, within the inverted terminal repeats of the mariner transposable element, thus creating autonomous elements and a potential gene drive mechanism.

The development of genetic transformation systems in insects has revolutionized the field of entomology. Transgenic insects provide tools to identify, isolate and analyze insect genes and to genetically modify insects for the purposes of insect control or disease vector modification. When transformation frequencies are high, multiple transgenic lines can be generated with relative ease. However, in most mosquito species, the results of transformation experiments have been suboptimal. Increased mosquito transformation efficiency is a research priority. Additionally, incorporation of refractory transgenes will not be sufficient to modify natural populations. A gene drive system will be required to allow transgenes to proliferate throughout populations and potentially reach fixation. This study proposes the use of germline-specific regulatory elements to promote confined, regulated transposase expression within the germ tissue. Creation of helper constructs utilizing endogenous promoters will potentially increase genetic transformation frequencies. The generation of lab strains of mosquitoes expressing an endogenous source of transposase within the germline will also serve as a powerful research tool. Endogenous sources of transposase will allow for comparative analysis of integration rates using different donor plasmids. Finally, the generation of autonomous transposable elements will provide a gene drive mechanism to move a tightly-linked refractory gene into a population. Four genes have been identified, cloned, and characterized, revealing expression patterns expected of germline-specific genes. Transcription profiles and in situ hybridization data support these conclusions. Putative cis-acting regulatory elements have been cloned and incorporated into DNA plasmid constructs. These elements are cloned in a manner such that they will regulate fluorescent gene expression. Additionally, similar elements have been cloned upstream of the Mos1 open reading frame, within the inverted terminal repeats of the mariner transposable element, thus creating autonomous elements and a potential gene drive mechanism.

Female mosquitoes lay eggs after the consuming a blood meal. The iron storage protein ferritin could be involved with iron movement among body tissues in insects. Mosquito ferritin is present in hemolymph (blood) and the messages for the heavy and light chain subunits contain secretion signal sequences. These subunits may be targeted to the secretory pathway. We found that Aedes aegypti larval cells (CCL-125) exposed to iron as ferric ammonium citrate (FAC) increased ferritin secretion in a dose-dependent manner. In order to study the secretory pathway of ferritin, we attempted to disrupt the Golgi by treating CCL-125 cells with brefeldin A (BFA) and monensin. Unexpectedly, neither BFA nor monensin inhibits iron-induced ferritin secretion. These data suggest that either CCL-125 cells are highly resistant to these agents or ferritin is secreted independently of the classical ER-Golgi pathway.

RNA interference (RNAi) is a gene silencing mechanism induced by double-stranded RNA (dsRNA). RNAi has been used extensively to create loss-of-function mutants in many species to identify the functions of genes, but it also has the potential to be used as a species-specific pesticide if the dsRNA can silence essential genes in pests. The mosquito Aedes aegypti is a vector of numerous viruses including Dengue and West Nile virus, and is frequently controlled by chemical insecticides. With growing concerns about the extensive use of broad-spectrum pesticides, new control methods are eagerly sought. In this study, I examined the efficacy of feeding pesticidal dsRNAs to mosquito larvae. A dose-dependent RNAi response and mortality was observed when larvae were fed dsRNA targeting several different genes. Unlike RNAi in the related dipteran Drosophila melanogaster, RNAi in A. aegypti also appeared to be systemic, spreading beyond the gut to other tissues. A degree of species-specificity was also observed, as dsRNA specific to the D. melanogaster β-tubulin gene killed D. melanogaster larvae but did not kill mosquito larvae. RNAi was also used to determine the function of a newly-identified A. aegypti cytochrome P450 (CYP) gene, Aacyp. This gene showed male-biased expression in the mosquitoes, and was expressed primarily in the male abdomen and/or thorax, but unlike some other insect male-biased CYPs, Aacyp was not highly expressed in the reproductive structures. While dsRNA injections successfully knocked down expression of Aacyp, no discernable change in reproductive or male-specific behaviours were noted. Nevertheless, RNAi is still considered a highly versatile tool for both gene function studies and has promising potential to be developed into a novel class of pesticides.

No description available.

No description available.

RNA interference (RNAi) is a gene silencing mechanism induced by double-stranded RNA (dsRNA). RNAi has been used extensively to create loss-of-function mutants in many species to identify the functions of genes, but it also has the potential to be used as a species-specific pesticide if the dsRNA can silence essential genes in pests. The mosquito Aedes aegypti is a vector of numerous viruses including Dengue and West Nile virus, and is frequently controlled by chemical insecticides. With growing concerns about the extensive use of broad-spectrum pesticides, new control methods are eagerly sought. In this study, I examined the efficacy of feeding pesticidal dsRNAs to mosquito larvae. A dose-dependent RNAi response and mortality was observed when larvae were fed dsRNA targeting several different genes. Unlike RNAi in the related dipteran Drosophila melanogaster, RNAi in A. aegypti also appeared to be systemic, spreading beyond the gut to other tissues. A degree of species-specificity was also observed, as dsRNA specific to the D. melanogaster β-tubulin gene killed D. melanogaster larvae but did not kill mosquito larvae. RNAi was also used to determine the function of a newly-identified A. aegypti cytochrome P450 (CYP) gene, Aacyp. This gene showed male-biased expression in the mosquitoes, and was expressed primarily in the male abdomen and/or thorax, but unlike some other insect male-biased CYPs, Aacyp was not highly expressed in the reproductive structures. While dsRNA injections successfully knocked down expression of Aacyp, no discernable change in reproductive or male-specific behaviours were noted. Nevertheless, RNAi is still considered a highly versatile tool for both gene function studies and has promising potential to be developed into a novel class of pesticides.

No description available.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas. Programa de Pós-Graduação em Biotecnologia. / Made available in DSpace on 2012-10-21T12:47:32Z (GMT). No. of bitstreams: 0 / Os mosquitos são insetos dípteros pertencentes a família Culicidae. As fêmeas possuem o hábito hematofágico, sendo responsáveis pela transmissão de várias doenças como: viroses, filarioses e protozoonoses. O aparecimento de resistência aos inseticidas químicos e os danos ambientais causados por eles nos apontam para a necessidade de novas alternativas para o controle de insetos. Neste trabalho, a atividade larvicida e repelente de dezesete extratos vegetais e fúngicos foram testados contra Culex quinquefasciatus. Dos dezesete extratos hidroalcoólicos e hexânicos testados cinco apresentaram atividade larvicida: Polygala sabulosa, Pachyrkizys bolbosa, Schinnus terebinthifolius, Croton celtidifolius e Nerium oleander, sendo que os extratos de P. bolbosa manteve a atividade após dois meses de armazenamento em temperatura ambiente ao abrigo da luz. De posse destes resultados, foi realizada a extração clorofórmica, butanólica, etanólica com acetato de etila e hexânica. Observamos que extratos de C. limon e C. aurantifolia apresentaram atividade larvicida sendo mantida após dois meses a de C. limon. Observamos que os extratos clorofórmico, acetato de etila e hexânico de P. bolbosa e extrato clorofórmico de P. sabulosa mantiveram a atividade larvicida.e que nenhum dos extratos testados apresentou atividade de repelência. Conclui-se que os extratos de P. bolbosa, P. sabulosa e C. limon possuem compostos inseticidas contra larvas de Cx. quinquefasciatus e que essa atividade se encontra nas frações extraídas com os solventes, clorofórmio, acetato de etila e hexano.

Resumo: Os imaturos da família Culicidae (Diptera) desenvolvem-se em coleções hídricas naturais ou artificiais. Os criadouros fitotélmicos são considerados naturais, entre eles as bromélias, com estruturas vegetais que permitem o acúmulo de água e possibilitam o desenvolvimento de fauna associada como larvas de culicídeos. As bromélias, incluídas na família Bromeliaceae distribuídas na região Neotropical apresentam 2900 espécies descritas, contribuem para o aumento da diversidade e quantidade de habitats disponíveis para o estabelecimento das espécies de Culicidae. Os grupos que ocorrem predominantemente em bromélias são Culex (Microculex) spp. e Wyeomyia spp., sendo também encontrados espécies dos gêneros Aedes e Anopheles. A determinação das espécies de Culicidae que ocorrem em bromélias é dificultada pela ausência de informações sobre a biologia e a morfologia dos estágios imaturos e adultos de muitas espécies. Diante dessa dificuldade foi realizado um estudo em uma área dentro da Floresta Estadual do Palmito no bioma Floresta Atlântica (Floresta Ombrófila Densa de Terras Baixas) localizada na planície litorânea do Estado do Paraná, no município de Paranaguá, apresentando 530 hectares de vegetação nativa inseridos sobre a planície costeira. O objetivo foi complementar e aperfeiçoar o quadro de informações sobre a morfologia dos estágios imaturos e adulto de espécies de Culicidae que se desenvolvem em bromélias no bioma Mata Atlântica e confeccionar uma chave de identificação para machos das espécies de Culex (Microculex) que ocorrem em bromélias. Foram realizadas quatro coletas de imaturos em 62 bromélias com o auxílio de uma bomba de sucção cujo conteúdo hídrico de cada bromélia foi transferido para um recipiente e transportado ao laboratório onde as amostras foram triadas. Os imaturos foram individualizados e mantidos em sala de criação sob condições controladas de temperatura e fotoperíodo até a emergência dos adultos. As exúvias das larvas de quarto ínstar, das pupas e as genitálias dos machos foram montadas em lâminas permanentes. Os espécimes adultos foram conservados a seco e montados em alfinetes entomológicos. As bromélias foram identificadas como pertencentes aos gêneros Vriesea e Nidularium e as espécies Vriesea philippocoburgii, Vriesea incurvata e Nidularium innocentii. Foram coletadas 434 larvas, identificadas as espécies Anopheles cruzii, Culex ocellatus, Culex neglectus, Culex fuscatus, Culex imitator, Culex retrosus, Culex daumasturus e, dentre os espécimes identificados, o taxa mais freqüente foi o gênero Culex, sendo a maioria pertencente ao subgênero Microculex. Foram redescritos os adultos macho e fêmea para a espécie Culex neglectus e descritos os adultos macho e fêmea para as espécies Culex daumasturus, Culex fuscatus, Culex retrosus, além de mais dois morfotipos Culex sp.1 e Culex sp.2 e confeccionada uma chave de identificação para os machos das espécies de Culex (Microculex) que ocorrem em bromélias.