O mosquito Culex quinquefasciatus é considerado praga urbana e tem a capacidade de se desenvolver em águas altamente poluídas atingindo elevada densidade. Mosquitos desta espécie possuem importância vetorial na transmissão de parasitas e arboviroses. Medidas de controle químico têm se mostrado ineficazes, além de serem altamente prejudiciais ao meio ambiente. Desse modo, novas tecnologias de controle foram desenvolvidas, entre elas o SIT (Sterile Insect Technique) que utiliza radiação para esterilizar machos e liberá-los no ambiente para copular com fêmeas selvagens. Posteriormente métodos genéticos baseados nessa técnica têm sido propostos. O sistema RIDL (Liberação de Insetos Carregando Gene Letal Dominante), consiste na integração de um gene letal dominante associado a um promotor específico de fêmea, dispensando a etapa de esterilização por radiação. Nesse processo os insetos recebem dieta suplementada com um repressor químico. A expressão do gene letal dominante é mantida desligada enquanto este repressor é adicionado ao meio das larvas. Para as amostras que estariam sendo preparadas para liberação, o repressor é retirado, e o gene letal dominante é ativado, causando a morte de todas as fêmeas, restando apenas machos para liberação. Os machos homozigotos para o gene letal seriam liberados para copular com fêmeas selvagens. A progênie seria heterozigota para o gene letal, porém somente os machos sobreviveriam. Parte crucial para o sucesso deste projeto foi a adaptação do método de microinjeção de embriões para a espécie Culex quinquefasciatus tornando possível a injeção dos transgenes LA513, LA882 e LA3653 com o objetivo de obtermos linhagens transgênicas. A obtenção de linhagens transgênicas com estas construções se mostraram mais laboriosa do que o previsto, dificultando a transgenia. Porém, as aplicações práticas em controle de vetores utilizando a técnica do RIDL são imensas e pode se tornar uma importante ferramenta do Manejo Integrado de vetores. / The Culex quinquefasciatus mosquitoes are considered urban plague and are able to grown in polluted water achieving high density. This specie are important vectors in the transmission of parasites and arbovirus. Chemical control methods shown to be ineffective and highly hazards to the environment. In this way, new technologies of control have been developed, among them there is SIT (Sterile Insect Technique) that utilizes radiation to sterilize males and then release in the environment to mate with wild females. New genetic methods are been proposed based on the SIT technique, they relies on the integration of a dominant lethal gene associated to a specific promoter of female avoiding the need for radiation (RIDL). In this process the insects receive diet supplemented with a chemical repressor (tetracycline). The expression of the dominant lethal gene is kept off while this repressor is added to the larvae. For the samples that would be being prepared for release, the repressor is removed, and the dominant lethal gene is activated, causing the death of all the females, and consequently only leaving male for release. The homozygous males for the lethal gene would be set free to mate with the wild females. The lineages would be heterozygous for the lethal gene and only males would survive. Since that, the capacity of mating of the transgenic males produced by method RDIL is an important prerequisite for the success of the program. It was crucial to achieve the objectives of this project adapt the microinjection method to Culex quinquefasciatus, so it became possible inject the LA513, LA882 and LA3653 plasmids seeking for the transgenic transformation. The obtaining of the transgenic mosquito Culex quinquefasciatus seems to be more difficult then expected and its development was harmed. The practical applications using RIDL in vector control is huge and can become a useful tool in the Integrated Management of vectors.

O avanço na distribuição geográfica de mosquitos vetores é seguido pela emergência de vírus e doenças em novas áreas para as quais não há disponibilidade de vacinas efetivas e drogas terapêuticas específicas são insuficientes. Métodos de controle de mosquitos tradicionais perderam efetividade, devido principalmente a grande capacidade reprodutiva e flexibilidade genômica dos mosquitos. Controle químico cada vez mais tornase restrito, acarretando na urgente necessidade de novas formas de controle. A liberação de machos carregando um gene letal dominante (RIDL) oferece novas abordagens aplicáveis ao controle de mosquitos e ainda assim ecológicas e espécie específica. Mosquitos Culex quinquefasciatus foram transformados com sucesso apenas uma vez, apesar do esforço de diversos laboratórios em obter uma linhagem transgênica estável. Foi desenvolvido um método de expressão transiente em mosquitos Culex, que insere plasmídeos contendo genes efetores na hemolinfa e tecidos subjacentes do mosquito. Foi observada a expressão da proteína fluorescente DsRed2, em mosquitos Culex quinquefasciatus adultos mediada por plasmídeos. Esta expressão pode ser considerada um importante passo na transformação de mosquitos Culex, além de potencial uso em estratégias de controle genético e interações gênicas. Para que novas formas de controle sejam realmente efetivas é vital que se conheça a estrutura genética da população alvo. Marcadores moleculares têm sido extensivamente utilizados em estudos filogenéticos e taxonômicos de diversas espécies de insetos. Microssatélites são de grande utilidade para observar estruturas populacionais, tanto em âmbito geográfico, quanto na escala evolucionária. Foi possível observar a formação de clusters e de padrões genéticos distintos entre as populações analisadas, criando um panorama genético dos mosquitos Culex quinquefasciatus coletados no Brasil / The increase in the geographic distribution of vectors is accompanied by the emergence of viruses and diseases in new areas. There are insufficient specific therapeutic drugs available and there are no reliable vaccines to malaria or dengue. Most mosquito control measures have failed to achieve their goals, mostly because of the mosquitos great reproductive capacity and genomic flexibility. Chemical control is increasingly restricted therefore other strategies for mosquito control are desperately needed. Releasing of Insects Carrying a Dominant Lethal Gene (RIDL) offers a new approach that can be applied to mosquitoes yet environmentally friendly and species-specific. Culex quinquefasciatus mosquitoes have been successfully genetically modified only once, despite the efforts of several laboratories to transform and establish a stable strain. We have adapted a transient gene expression method, in Culex, that delivers plasmid DNA directly to the mosquito hemolymph and additional tissues. We were able to express DsRed2 fluorescent protein in adult Culex quinquefasciatus mosquitoes by injecting plasmids directly into their thorax. The expression of DsRed2 in adult Culex quinquefasciatus mosquitoes is an important stepping stone to genetic transformation and the potential use of new control strategies and genetic interactions. If new methods of control are to become really effective it is vital to know the genetic structure of the target population. Molecular markers have been widely used in phylogenetic and taxonomic studies of various groups of insects. Microsatellites are very useful for observing the population structure both geographically and evolutionarily. It has been possible to observe the formation of clusters and distinct genetic patterns among the analyzed populations creating a panorama of Culex quinquefasciatus mosquitoes populations present in Brazil

O mosquito Culex quinquefasciatus é considerado praga urbana e tem a capacidade de se desenvolver em águas altamente poluídas atingindo elevada densidade. Mosquitos desta espécie possuem importância vetorial na transmissão de parasitas e arboviroses. Medidas de controle químico têm se mostrado ineficazes, além de serem altamente prejudiciais ao meio ambiente. Desse modo, novas tecnologias de controle foram desenvolvidas, entre elas o SIT (Sterile Insect Technique) que utiliza radiação para esterilizar machos e liberá-los no ambiente para copular com fêmeas selvagens. Posteriormente métodos genéticos baseados nessa técnica têm sido propostos. O sistema RIDL (Liberação de Insetos Carregando Gene Letal Dominante), consiste na integração de um gene letal dominante associado a um promotor específico de fêmea, dispensando a etapa de esterilização por radiação. Nesse processo os insetos recebem dieta suplementada com um repressor químico. A expressão do gene letal dominante é mantida desligada enquanto este repressor é adicionado ao meio das larvas. Para as amostras que estariam sendo preparadas para liberação, o repressor é retirado, e o gene letal dominante é ativado, causando a morte de todas as fêmeas, restando apenas machos para liberação. Os machos homozigotos para o gene letal seriam liberados para copular com fêmeas selvagens. A progênie seria heterozigota para o gene letal, porém somente os machos sobreviveriam. Parte crucial para o sucesso deste projeto foi a adaptação do método de microinjeção de embriões para a espécie Culex quinquefasciatus tornando possível a injeção dos transgenes LA513, LA882 e LA3653 com o objetivo de obtermos linhagens transgênicas. A obtenção de linhagens transgênicas com estas construções se mostraram mais laboriosa do que o previsto, dificultando a transgenia. Porém, as aplicações práticas em controle de vetores utilizando a técnica do RIDL são imensas e pode se tornar uma importante ferramenta do Manejo Integrado de vetores. / The Culex quinquefasciatus mosquitoes are considered urban plague and are able to grown in polluted water achieving high density. This specie are important vectors in the transmission of parasites and arbovirus. Chemical control methods shown to be ineffective and highly hazards to the environment. In this way, new technologies of control have been developed, among them there is SIT (Sterile Insect Technique) that utilizes radiation to sterilize males and then release in the environment to mate with wild females. New genetic methods are been proposed based on the SIT technique, they relies on the integration of a dominant lethal gene associated to a specific promoter of female avoiding the need for radiation (RIDL). In this process the insects receive diet supplemented with a chemical repressor (tetracycline). The expression of the dominant lethal gene is kept off while this repressor is added to the larvae. For the samples that would be being prepared for release, the repressor is removed, and the dominant lethal gene is activated, causing the death of all the females, and consequently only leaving male for release. The homozygous males for the lethal gene would be set free to mate with the wild females. The lineages would be heterozygous for the lethal gene and only males would survive. Since that, the capacity of mating of the transgenic males produced by method RDIL is an important prerequisite for the success of the program. It was crucial to achieve the objectives of this project adapt the microinjection method to Culex quinquefasciatus, so it became possible inject the LA513, LA882 and LA3653 plasmids seeking for the transgenic transformation. The obtaining of the transgenic mosquito Culex quinquefasciatus seems to be more difficult then expected and its development was harmed. The practical applications using RIDL in vector control is huge and can become a useful tool in the Integrated Management of vectors.

O avanço na distribuição geográfica de mosquitos vetores é seguido pela emergência de vírus e doenças em novas áreas para as quais não há disponibilidade de vacinas efetivas e drogas terapêuticas específicas são insuficientes. Métodos de controle de mosquitos tradicionais perderam efetividade, devido principalmente a grande capacidade reprodutiva e flexibilidade genômica dos mosquitos. Controle químico cada vez mais tornase restrito, acarretando na urgente necessidade de novas formas de controle. A liberação de machos carregando um gene letal dominante (RIDL) oferece novas abordagens aplicáveis ao controle de mosquitos e ainda assim ecológicas e espécie específica. Mosquitos Culex quinquefasciatus foram transformados com sucesso apenas uma vez, apesar do esforço de diversos laboratórios em obter uma linhagem transgênica estável. Foi desenvolvido um método de expressão transiente em mosquitos Culex, que insere plasmídeos contendo genes efetores na hemolinfa e tecidos subjacentes do mosquito. Foi observada a expressão da proteína fluorescente DsRed2, em mosquitos Culex quinquefasciatus adultos mediada por plasmídeos. Esta expressão pode ser considerada um importante passo na transformação de mosquitos Culex, além de potencial uso em estratégias de controle genético e interações gênicas. Para que novas formas de controle sejam realmente efetivas é vital que se conheça a estrutura genética da população alvo. Marcadores moleculares têm sido extensivamente utilizados em estudos filogenéticos e taxonômicos de diversas espécies de insetos. Microssatélites são de grande utilidade para observar estruturas populacionais, tanto em âmbito geográfico, quanto na escala evolucionária. Foi possível observar a formação de clusters e de padrões genéticos distintos entre as populações analisadas, criando um panorama genético dos mosquitos Culex quinquefasciatus coletados no Brasil / The increase in the geographic distribution of vectors is accompanied by the emergence of viruses and diseases in new areas. There are insufficient specific therapeutic drugs available and there are no reliable vaccines to malaria or dengue. Most mosquito control measures have failed to achieve their goals, mostly because of the mosquitos great reproductive capacity and genomic flexibility. Chemical control is increasingly restricted therefore other strategies for mosquito control are desperately needed. Releasing of Insects Carrying a Dominant Lethal Gene (RIDL) offers a new approach that can be applied to mosquitoes yet environmentally friendly and species-specific. Culex quinquefasciatus mosquitoes have been successfully genetically modified only once, despite the efforts of several laboratories to transform and establish a stable strain. We have adapted a transient gene expression method, in Culex, that delivers plasmid DNA directly to the mosquito hemolymph and additional tissues. We were able to express DsRed2 fluorescent protein in adult Culex quinquefasciatus mosquitoes by injecting plasmids directly into their thorax. The expression of DsRed2 in adult Culex quinquefasciatus mosquitoes is an important stepping stone to genetic transformation and the potential use of new control strategies and genetic interactions. If new methods of control are to become really effective it is vital to know the genetic structure of the target population. Molecular markers have been widely used in phylogenetic and taxonomic studies of various groups of insects. Microsatellites are very useful for observing the population structure both geographically and evolutionarily. It has been possible to observe the formation of clusters and distinct genetic patterns among the analyzed populations creating a panorama of Culex quinquefasciatus mosquitoes populations present in Brazil

The mosquito Aedes aegypti is the primary worldwide vector of dengue fever, of which there are an estimated 390 million infections annually. Traditional vector control methods have been unsuccessful in preventing Ae. aegypti from posing a serious threat to human health worldwide, and as a result increasing emphasis is being placed on new control technologies. One of these is RIDL® (Release of Insects carrying a Dominant Lethal), a modified form of the sterile insect technique. Released RIDL males of strain OX513A are genetically engineered to possess a repressible lethal construct. Upon mating with a wild female, the construct is inherited by the offspring, causing their death. The RIDL strategy depends on the mass production and release of vigorous male insects, capable of competing with wild males to mate with wild females. <b>Chapter 2</b> investigates how rearing conditions can influence the size, asymmetry, quantity, and timing of OX513A mosquitoes produced. <b>Chapter 3</b> describes the design and testing of devices for the release of these adult males, and their implementation during the world's first release of transgenic mosquitoes. <b>Chapter 4</b> discusses how the modification of male size and nutritional status may affect subsequent performance, and how these changes to male vigour will be assessed in subsequent chapters. <b>Chapter 5</b> measures changes in competitiveness by examining longevity. Larger males lived longer, and longevity was increased by increasing the duration of the initial sugar feed. <b>Chapter 6</b> examines the potential improvements in male performance by measuring the flight capacity of OX513A males, using a purpose-built mosquito flight mill. Male size or nutritional status was not found to affect flight capacity, although light intensity was unexpectedly found to modulate the flight speed of the males. Finally, <b>Chapter 7</b> examines potential improvements in the mating performance of male OX513A Ae. aegypti. When OX513A males and wild-type males were placed in direct competition for virgin females, smaller OX513A males were found to have a significant mating advantage over larger wild-type males. The results of all three of these performance measures are discussed with relevance to field releases. The results presented in this thesis improve current knowledge of how the performance of male mosquitoes can be effectively measured and improved. This is a vital part of ensuring the success of male-release vector control strategies such as RIDL.

Insect pests represent major threats to food production, biodiversity conservation, and human and animal health. Currently, the most widespread strategy to control their populations is through the spraying of synthetic chemical insecticides. However, the overuse of these compounds has had significant negative environmental consequences. Additionally, our reliance on insecticides has resulted in major reductions in their efficacy through pest-evolved resistance. To successfully manage insect pests, while avoiding environmental degradation, thus requires the development of novel, more sustainable, pest management strategies. Recent advances in our understanding of recombinant DNA methods and molecular biology have allowed the application of transgenic tools to pest management. Here, synthetic genes can be engineered, transformed into the genomes of pest species, and transported into wild target populations through the natural mating behaviour of the insect. A strategy in which these transgenes are lethal to those insects inheriting them in the field is known as RIDL – Release of Insects carrying a Dominant Lethal. A variant of RIDL limits this lethality to females – female specific RIDL (fsRIDL) – which explicitly targets the reproductive capacity of a target population. The aim of this thesis is to investigate the application of such an fsRIDL strategy to the diamondback moth (Plutella xylostella L.). This economically important pest of brassica crops is highly adept at developing resistance to insecticides and is considered extremely difficult to manage effectively. I present findings which demonstrate the power of diamondback moth lines transformed with fsRIDL transgenes to eliminate target pest populations, and combine synergistically with other transgenic control strategies such as Bt crops in counteracting the evolution of pesticide resistance. Additionally, an exploration into an alternative gene expression system to that used in current RIDL strategies – the Q system – suggests that not all expression systems will be suitable for transgene control within this highly specific framework. It is hoped that this work will contribute towards the effective control of the diamondback moth, and form a model for the sustainable control of other lepidopteran species through genetic pest management.

Seedlings of tropical forest species show an extraordinary morphological diversity. Tabebuia and Handrohantiis, belong the Bignoniaceae, are classify in Tecomeae tribe. This study aimed to verify the morphological differences between these two genera from the analysis of fruits, seeds and seedlings of Handroanthus ochraceus (Cham.) Mattos ssp. ochraceus, Handroanthus serratifolius (Vahl) S. Grose, Tabebuia insignis (Miq.) Sandwith ssp. insignis e Tabebuia roseo-alba (Ridl.) Sandwith to subsidize taxonomic decisions inherent in these taxa, and indicate relevant characteristics to recognize these species in the field when, still in the seedling stage, during the evaluations of natural regeneration, allowing the practical application in ecological studies, in the management and flora conservation. Handroanthus ochraceus (Cham.) Mattos ssp. ochraceus presented persistent cup fruit, many trichomes surface, velvety looks, seeds with dark brown line, going from the base to the center, cotyledons creams, reticulated nervation and valve prefoliation eophyll; while Handroanthus serratifolius (Vahl) S. Grose don't presented without persistent cup fruit, glabrous surface, almost black dividing line seeds, from the base to the apex, green cotyledons, craspedodromous nervation and conduplicate prefoliation eophyll. Tabebuia insignis (Miq.) Sandwith ssp. insignis presented fruit with the outer surfaces of the pericarp with trichomes, dehiscence with not twisted valves, ovate seeds with hilum, creams cotyledons, seedlings with petioles of eophylls with trichomes; while Tabebuia roseo-alba (Ridl.) Sandwith presented fruit with the outer surfaces of the pericarp glabrous, dehiscence with twisting revolut valve, seeds with hilum in "v" form and browns cotyledons, seedlings petioles of eophylls glabrous. This study has differentiated Handroanthus of Tabebuia through some morphological characters, but there is need to study other species to confirm whether the characters described in this study are still in genera described. / As plântulas de espécies florestais tropicais demonstram uma extraordinária diversidade morfológica. Os gêneros Tabebuia e Handroanthus, pertencentes à Bignoniaceae, estão classificados na tribo Tecomeae. Este trabalho teve como objetivo verificar as diferenças morfológicas existentes entre esses dois gêneros a partir da análise dos frutos, sementes e plântulas de Handroanthus ochraceus (Cham.) Mattos ssp. ochraceus, Handroanthus serratifolius (Vahl) S. Grose, Tabebuia insignis (Miq.) Sandwith ssp. insignis e Tabebuia roseo-alba (Ridl.) Sandwith a fim de subsidiar decisões taxonômicas inerentes a esses táxons, além de indicar características relevantes para reconhecer estas espécies no campo quando, ainda em estágio de plântula, durante as avaliações de regeneração natural, permitindo a aplicação prática em estudos ecológicos, no manejo e conservação da flora. Handroanthus ochraceus (Cham.) Mattos ssp. ochraceus apresentou fruto com cálice persistente, superfície com muitos tricomas, com aparência aveludada, sementes com linha divisória castanho escura, indo da base ao centro, cotilédones cremes, eofilos com nervação reticulada e prefolheação valvar; enquanto que Handroanthus serratifolius (Vahl) S. Grose apresentou fruto sem cálice persistente, superfície glabra, sementes com linha divisória quase negra, indo da base ao ápice, cotilédones verdes, eofilos com nervação craspedódroma e prefolheação conduplicada. Tabebuia insignis (Miq.) Sandwith ssp. insignis apresentou fruto com superfície externa do pericarpo com tricomas, deiscência com valvas não torcidas, sementes com hilo oval, cotilédones cremes, plântulas com pecíolos dos eofilos com tricomas; enquanto que Tabebuia roseo-alba (Ridl.) Sandwith apresentou fruto com superfície externa do pericarpo glabra, deiscência com torção revoluta das valvas, sementes com hilo em forma de "v" , cotilédones castanhos, plântulas com pecíolos dos eofilos glabros. Este estudo possibilitou diferenciar Handroanthus de Tabebuia através de alguns caracteres morfológicos, porém, há a necessidade de se estudar outras espécies a fim de confirmar se os caracteres descritos neste trabalho se mantêm nos gêneros descritos.