Made available in DSpace on 2015-03-26T13:30:23Z (GMT). No. of bitstreams: 1 texto completo.pdf: 308576 bytes, checksum: 4517ced7e005a18483bc835f89df8a70 (MD5) Previous issue date: 2009-02-17 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / The Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) is the most important pest of tomato Lycopersicon esculentum Mill in Brazil. Their larvae consume the leaf mesophyll, attack stems, flowers and fruits of tomato, reducing the productivity of this culture and consequently the profitability of farmers. For reducing this insect attack, farmers use insecticides intensively, and often the efficacy control is not reached as desired. One possible reason for the low efficiency is the resistance development to insecticides in populations of this insect. Therefore, tomato producers need search for detecting and monitoring of the T. absolute populations resistance to insecticides. The objective of this study was identified resistance to insecticides in T. absoluta populations from major regions tomato producing of Brazil, and verified the control failures of this pest. The populations were from Cerrado, Southeast and Northeast of Brazil. Ten insecticides were used, six neurotoxic, three insects growth regulators and one which has the bacterium Bacillus thuringiensis as active ingredient. In populations from Cerrado and Southeast the abamectin, spinosad, deltamethrin, triazophos, indoxacarb and bifenthrin were in ascending order, the insecticides that showed the lowest level of resistance among the products tested. In Northeast s population the same resistance was observed, except for bifenthrin, which was replaced by teflubenzurom. The insecticides diflubenzurom, permethrin, triflumurom and the B. Thuringiensis had larger numbers of resistant populations to insecticides in all regions. The T. absoluta population from Viçosa-MG is the most appropriate to be used as susceptibility pattern in resistance study to insecticides of this pest. In recommended doses by industries, the abamectin, spinosad, deltamethrin and triazophos insecticides had not control failures of the T. absolute populations, while the bifenthrin has control failures of populations from Uberlândia, Paulínea and Camocim de São Felix, the indoxacarb had control failures of populations from Uberlândia, São João da Barra and Viçosa, teflubenzurom had control failures of populations from Goianápolis, São João da Barra, Viçosa and Santa Tereza, diflubenzurom, permethrin, triflumurom and B. thuringiensis had control failures in all the T absolute populations which were tested. The correct management of this pest, through the practices which contribute for increasing of control efficacy, can help prevent, delay or reverse the resistance development of this insect pest to insecticides. / A Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) é a praga mais importante do tomateiro Lycopersicon esculentum Mill no Brasil. As larvas desse inseto-praga consomem o mesófilo foliar, bloqueiam os ponteiros, os botões florais, as flores e frutos do tomateiro, reduzindo a produtividade dessa cultura, comprometendo a lucratividade dos tomaticultores. Com o intuito de minimizar o efeito desse inseto, os agricultores fazem o uso intensivo de inseticidas, que muitas vezes não alcançam a eficiência de controle desejada. Uma das possíveis razões para a baixa eficiência esta o desenvolvimento da resistência a inseticida em populações desse inseto. Dessa forma, a tomaticultura carece de trabalhos que visem à detecção e o monitoramento adequado de populações de T. absoluta resistentes a inseticidas. Assim, o objetivo desse trabalho estudar e identificar a ocorrência de resistência em populações de T. absoluta a inseticidas nas principais regiões produtoras de tomate do Brasil, e verificar falhas no controle desta praga. As populações foram provenientes do Cerrado, Sudeste e Nordeste. Foram utilizados dez inseticidas, sendo seis neurotóxicos, três reguladores de crescimento e um que tem como ingrediente ativo a bactéria Bacillus thuringiensis. Para as macrorregiões do Cerrado e Sudeste, abamectina, espinosade, deltametrina mais triazofós, indoxacarbe e bifentrina foram, em ordem crescente, os inseticidas que apresentaram o menor nível de resistência dentre os produtos testados. Para o Nordeste observou-se o mesmo padrão, com exceção da bifentrina que foi substituída pelo teflubenzurom. Os inseticidas diflubenzurom, permetrina, o triflumurom e o B. thuringiensis apresentaram números maiores de populações resistentes a inseticidas em todas as macrorregiões. A população de Tuta absoluta de Viçosa-MG é a mais adequada a ser utilizada como padrão de suscetibilidade em estudos de resistência a inseticidas. Nas doses recomendadas pelos fabricantes, os inseticidas abamectina, espinosade, deltametrina mais triazofós não apresentaram falhas no controle das populações de T. absoluta, já a bifentrina apresentou falhas no controle das populações de Uberlândia, Paulínea e Camocim de São Felix, o indoxacarbe apresentou falhas no controle das populações de Uberlândia, São João da Barra e Viçosa, o teflubenzurom apresentou falhas no controle das populações de Goianápolis, São João da Barra, Viçosa e Santa Tereza, o diflubenzurom, permetrina, triflumurom e o a base de B. thuringiensis apresentaram falhas no controle de todas as populações de T absoluta testadas. O manejo correto dessa praga através de práticas que contribuam para um controle mais eficiente pode ajuda a prevenir, retardar ou reverter à evolução da resistência deste inseto-praga a inseticidas.

Made available in DSpace on 2014-06-11T19:26:04Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-04-27Bitstream added on 2014-06-13T19:12:52Z : No. of bitstreams: 1 ricci_j_me_sjrp.pdf: 1040646 bytes, checksum: cec6211f3f5843239b67ee910f199d37 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A resistência aos inseticidas é um modelo de processo evolutivo onde o inseticida atua como agente seletivo e, como resposta à seleção, ocorre a evolução da resistência nas populações de insetos. As enzimas citocromo P450 monooxigenases (CYP) formam uma família responsável pela resistência aos inseticidas. Tem sido proposto que a inserção de elementos transponíveis (TEs) em regiões reguladoras ou codificadoras dos genes da família Cyp pode alterar a expressão gênica e induzir a resistência aos inseticidas. No presente estudo foram realizadas análises in silico que permitiram identificar a ocorrência de inserções de fragmentos de TEs em 35 genes Cyps com diferentes funções, e em seus genes flanqueadores, em Drosophila melanogaster e D. simulans, além de 13 genes Cyps de seis espécies do grupo melanogaster de Drosophila. As inserções de TEs ocorreram principalmente nas regiões flanqueadoras 5´ dos Cyps associados à resistência aos inseticidas e à função monooxigenase geral. Os resultados não indicaram qualquer relação entre a distância em relação ao gene e o número de inserções. As análises mostraram que a maioria das inserções pertence à classe de transposons de DNA, sendo o transposon DNAREP1_DM o que apresentou o maior número de cópias. O fato de essas seqüências apresentarem putativos sítios de ligação de fatores de transcrição sugere que possam desempenhar algum papel na regulação dos genes Cyps. Também foi analisada a ocorrência de polimorfismos de inserção de TEs em regiões flanqueadoras de genes da família Cyp, em diferentes linhagens geográficas resistentes e suscetíveis, de D. melanogaster e D. simulans. Análises evidenciaram a presença de polimorfismo interpopulacional de tamanho das regiões flanqueadoras dos genes Cyp6w1, Cyp6a2 e Cyp12d1, porém, não indicaram... / Insecticide resistance is a model of evolutionary process where the insecticide acts as the selective agent and resistance in the insect populations evolves as an answer to selection. Cytochrome monooxygenases (CYP) is family of enzymes responsible for the insecticide resistance. It has been proposed that insertion of transposable elements (TEs) in regulatory or coding regions of the Cyp genes can alter gene expression and induce insecticide resistance. In the present study in silico analyses allowed identifying the insertion of TE fragments in 35 Cyp genes with different functions, in Drosophila melanogaster and D. simulans, as well as in 13 Cyps of six species of the melanogaster group of Drosophila. The TE insertions occurred mainly in the 5´ flanking regions of Cyp genes associated to resistance and to those with a general monooxygenase function. The results did not indicate any relationship between the number of insertions and the distance in relation to the gene. The analyses showed that most of the insertions belong to the DNA transposon class, being DNAREP1_DM the most numerous. Since this element carry putative biding sites of transcription factors it can be suggested they play same role in gene regulation. The polymorphism of TE insertions in the flanking regions of Cyp6w1, Cyp6a2 and Cyp12d1, genes associated to resistance, found in resistant and as well as in susceptible geographical strains of D. melanogaster and D. simulans, does not indicate any relationship between the presence of TEs in those regions and the insecticide resistance. The results also showed that the insertions of TEs in the proximities of the Cyps associated to resistance is differential among six species of the melanogaster group, not following the genomic proportion of TEs in each species. These results also suggest that TEs inserted in the Cyp flanking regions can carry out an adaptive... (Complete abstract click electronic access below)

As abelhas africanizadas (Apis mellifera L.) estão desaparecendo e acredita-se que seja devido ao intenso uso de inseticidas, poluição e alterações climáticas, no entanto muitas culturas dependem da polinização realizada por essas abelhas. Desta forma, o objetivo deste trabalho foi avaliar o efeito de três linhagens de Bacillus thuringiensis (Bt) (IPS 82, BR 81 e BR 147), entomopatógeno utilizado para o controle de lagartas desfolhadoras, e de três produtos fitossanitários sintéticos (PFS 1: Acetamiprido + Alfa-cipermetrina; PFS 2: Imidacloporido + Beta-Ciflutina e PFS 3: Fenitrotiona + Esfenvalerato), sobre a longevidade de operárias adultas de A. mellifera africanizada. Foram realizados três bioensaios com as linhagens de Bt e dos PFSs: A) Pulverização sobre A. mellifera; B) Contato de A. mellifera com superfície pulverizada e C) Pasta cândi incorporada com os produtos e análise histológica do intestino médio das abelhas. Nos três bioensaios a testemunha foi composta por água deionizada esterilizada. Cada tratamento foi composto por 60 abelhas, sendo cada abelha considerada uma repetição. Os bioensaios foram mantidos em câmara climatizada tipo BOD (34 ± 2ºC, U.R. de 60 ± 5%) e a longevidade das operárias avaliada a uma; duas; três; quatro; cinco; seis; nove; 12; 15; 18; 21; 24; 30; 36; 42; 48; 60; 72; 96; e 120 horas.Na pulverização de Bt sobre A. mellifera verificou-se que apenas a linhagem Bt 1 (IPS 82) causou redução da longevidade de operárias (88,7 horas), enquanto no bioensaio de contato a linhagem Bt 3 - BR147 provocou redução (101,2 horas). Todas as linhagens de Bt avaliadas, quando incorporadas à alimentação de A. mellifera reduziram a longevidade das operárias (Bt 1 – IPS 82: 64,50 horas; Bt 2 – BR 81: 64,56 horas e Bt 3 – BR 147: 60,00horas) e com a análise histológica pôde-se perceber a desintegração das células epiteliais do mesêntero das abelhas, causada pelas bactérias que elas ingeriram. Os três produtos fitossanitários sintéticos reduziram a longevidade das operárias de A. mellifera, no bioensaio de pulverização direta (PFS 1 e PFS 2 – 1,0 hora; PFS 3 – 2,21 horas). No contato de A. mellifera com superfície pulverizada com PFS, as abelhas ficaram duas horas em contato com os produtos na placa de Petri, e ao final dessas duas horas, 100% abelhas que entraram em contato com os três produtos fitossanitários sintéticos estavam mortas. Quando incorporados os PSFs na alimentação, ambos reduziram a longevidade das operárias (PFS 1 – 22 horas, PFS 2 – 21,72 horas e PFS 3 – 43,93 horas) e na análise histológica foi possível observar diferenças na estruturação do intestino médio das operárias de A. mellifera alimentadas com os três PFSs. Apesar das linhagens de Bt reduzirem a longevidade das operárias de A. mellifera não é possível afirmar que não sejam seletivas, pois a interferência é mínima quando comparada à interferência provocada pelos PFSs, os quais provocaram drástica redução na longevidade de A. mellifera. / Africanized bees (Apis mellifera L.) are disappearing and are believed to be due to the intense use of insecticides, pollution and climate change, however many crops depend on the pollination performed by these bees. The objective of this work was to evaluate the effect of three strains of Bacillus thuringiensis (Bt) (IPS 82, BR 81 and BR 147), entomopathogen used for the control of Acetamiprid + Alpha-cypermethrin, PFS 2: Imidacloporide + Beta-Cyflutin and PFS 3: Fenitrothione + Esfenvalerate), on the longevity of African female A. mellifera workers. Three bioassays were performed with the Bt and PFS lines: A) Spraying on A. mellifera; B) A. mellifera contact with pulverized surface and C) Candi paste incorporated with the products and histological analysis of the medium intestine of the bees. In the three bioassays the control was composed of sterilized deionized water. Each treatment consisted of 60 bees, each bee being considered a repetition. The bioassays were maintained in a BOD type chamber (34 ± 2ºC, 60 ± 5% U.R) and the workers longevity was evaluated at one; two; three; four; five; six; nine; 12; 13; 18; 21; 24; 30; 36; 42; 48; 60; 72; 96; And 120 hours. In Bt spraying on A. mellifera, it was verified that only the Bt 1 strain (IPS 82) caused a reduction in worker longevity (88.7 hours), while in the contact bioassay the Bt 3 - BR147 strain caused a reduction (101, 2 hours). All strains of Bt evaluated when incorporated into A. mellifera feed reduced workers longevity (Bt 1 - IPS 82: 64.50 hours; Bt 2 - BR 81: 64.56 hours and Bt 3 - BR 147: 60). And the histological analysis showed the disintegration of the epithelial cells of the bees mesentery, caused by the bacteria they ingested. The three synthetic phytosanitary products reduced the longevity of A. mellifera workers in the direct spray bioassay (PFS 1 and PFS 2 - 1.0 hour; PFS 3 - 2.21 hours). At the contact of A. mellifera with a PFS-sprayed surface, the bees stayed two hours in contact with the products on the petri dish, and at the end of these two hours, 100% bees that came in contact with the three synthetic phytosanitary products were dead. When the PSFs were incorporated in the diet, they reduced the longevity of the workers (PFS 1 - 22 hours, PFS 2 - 21.72 hours and PFS 3 - 43.93 hours) and in the histological analysis it was possible to observe differences in the workers from A. mellifera fed with the three PFSs. although the Bt lineages reduce the longevity of A. mellifera workers, it is not possible to assert that they are not selective, since the interference is minimal when compared to interference caused by PFSs, which caused a drastic reduction in the longevity of A. mellifera.

As abelhas africanizadas (Apis mellifera L.) estão desaparecendo e acredita-se que seja devido ao intenso uso de inseticidas, poluição e alterações climáticas, no entanto muitas culturas dependem da polinização realizada por essas abelhas. Desta forma, o objetivo deste trabalho foi avaliar o efeito de três linhagens de Bacillus thuringiensis (Bt) (IPS 82, BR 81 e BR 147), entomopatógeno utilizado para o controle de lagartas desfolhadoras, e de três produtos fitossanitários sintéticos (PFS 1: Acetamiprido + Alfa-cipermetrina; PFS 2: Imidacloporido + Beta-Ciflutina e PFS 3: Fenitrotiona + Esfenvalerato), sobre a longevidade de operárias adultas de A. mellifera africanizada. Foram realizados três bioensaios com as linhagens de Bt e dos PFSs: A) Pulverização sobre A. mellifera; B) Contato de A. mellifera com superfície pulverizada e C) Pasta cândi incorporada com os produtos e análise histológica do intestino médio das abelhas. Nos três bioensaios a testemunha foi composta por água deionizada esterilizada. Cada tratamento foi composto por 60 abelhas, sendo cada abelha considerada uma repetição. Os bioensaios foram mantidos em câmara climatizada tipo BOD (34 ± 2ºC, U.R. de 60 ± 5%) e a longevidade das operárias avaliada a uma; duas; três; quatro; cinco; seis; nove; 12; 15; 18; 21; 24; 30; 36; 42; 48; 60; 72; 96; e 120 horas.Na pulverização de Bt sobre A. mellifera verificou-se que apenas a linhagem Bt 1 (IPS 82) causou redução da longevidade de operárias (88,7 horas), enquanto no bioensaio de contato a linhagem Bt 3 - BR147 provocou redução (101,2 horas). Todas as linhagens de Bt avaliadas, quando incorporadas à alimentação de A. mellifera reduziram a longevidade das operárias (Bt 1 – IPS 82: 64,50 horas; Bt 2 – BR 81: 64,56 horas e Bt 3 – BR 147: 60,00horas) e com a análise histológica pôde-se perceber a desintegração das células epiteliais do mesêntero das abelhas, causada pelas bactérias que elas ingeriram. Os três produtos fitossanitários sintéticos reduziram a longevidade das operárias de A. mellifera, no bioensaio de pulverização direta (PFS 1 e PFS 2 – 1,0 hora; PFS 3 – 2,21 horas). No contato de A. mellifera com superfície pulverizada com PFS, as abelhas ficaram duas horas em contato com os produtos na placa de Petri, e ao final dessas duas horas, 100% abelhas que entraram em contato com os três produtos fitossanitários sintéticos estavam mortas. Quando incorporados os PSFs na alimentação, ambos reduziram a longevidade das operárias (PFS 1 – 22 horas, PFS 2 – 21,72 horas e PFS 3 – 43,93 horas) e na análise histológica foi possível observar diferenças na estruturação do intestino médio das operárias de A. mellifera alimentadas com os três PFSs. Apesar das linhagens de Bt reduzirem a longevidade das operárias de A. mellifera não é possível afirmar que não sejam seletivas, pois a interferência é mínima quando comparada à interferência provocada pelos PFSs, os quais provocaram drástica redução na longevidade de A. mellifera. / Africanized bees (Apis mellifera L.) are disappearing and are believed to be due to the intense use of insecticides, pollution and climate change, however many crops depend on the pollination performed by these bees. The objective of this work was to evaluate the effect of three strains of Bacillus thuringiensis (Bt) (IPS 82, BR 81 and BR 147), entomopathogen used for the control of Acetamiprid + Alpha-cypermethrin, PFS 2: Imidacloporide + Beta-Cyflutin and PFS 3: Fenitrothione + Esfenvalerate), on the longevity of African female A. mellifera workers. Three bioassays were performed with the Bt and PFS lines: A) Spraying on A. mellifera; B) A. mellifera contact with pulverized surface and C) Candi paste incorporated with the products and histological analysis of the medium intestine of the bees. In the three bioassays the control was composed of sterilized deionized water. Each treatment consisted of 60 bees, each bee being considered a repetition. The bioassays were maintained in a BOD type chamber (34 ± 2ºC, 60 ± 5% U.R) and the workers longevity was evaluated at one; two; three; four; five; six; nine; 12; 13; 18; 21; 24; 30; 36; 42; 48; 60; 72; 96; And 120 hours. In Bt spraying on A. mellifera, it was verified that only the Bt 1 strain (IPS 82) caused a reduction in worker longevity (88.7 hours), while in the contact bioassay the Bt 3 - BR147 strain caused a reduction (101, 2 hours). All strains of Bt evaluated when incorporated into A. mellifera feed reduced workers longevity (Bt 1 - IPS 82: 64.50 hours; Bt 2 - BR 81: 64.56 hours and Bt 3 - BR 147: 60). And the histological analysis showed the disintegration of the epithelial cells of the bees mesentery, caused by the bacteria they ingested. The three synthetic phytosanitary products reduced the longevity of A. mellifera workers in the direct spray bioassay (PFS 1 and PFS 2 - 1.0 hour; PFS 3 - 2.21 hours). At the contact of A. mellifera with a PFS-sprayed surface, the bees stayed two hours in contact with the products on the petri dish, and at the end of these two hours, 100% bees that came in contact with the three synthetic phytosanitary products were dead. When the PSFs were incorporated in the diet, they reduced the longevity of the workers (PFS 1 - 22 hours, PFS 2 - 21.72 hours and PFS 3 - 43.93 hours) and in the histological analysis it was possible to observe differences in the workers from A. mellifera fed with the three PFSs. although the Bt lineages reduce the longevity of A. mellifera workers, it is not possible to assert that they are not selective, since the interference is minimal when compared to interference caused by PFSs, which caused a drastic reduction in the longevity of A. mellifera.

O inseticida spinosad tem sido um dos mais utilizados para o controle de Spodoptera frugiperda (J. E. Smith) no Brasil, devido à sua eficácia e ao seu mecanismo de ação único (modulador alostérico de receptores nicotínicos da acetilcolina). Para fornecer subsídios a um programa de manejo da resistência, foram realizados estudos para compreender as bases genéticas e moleculares da resistência de S. frugiperda a este inseticida. Inicialmente, foi selecionada uma linhagem de S. frugiperda resistente a spinosad (Spin-res) em laboratório por meio da técnica \"F2 screen\". A razão de resistência, baseada na CL50, foi de aproximadamente 890 vezes. A partir de cruzamentos recíprocos entre a linhagem suscetível (Sus) e Spin-res, constatou-se que o padrão de herança da resistência de S. frugiperda a spinosad é autossômica e incompletamente recessiva. Retrocruzamentos da progênie F1 de cruzamentos recíprocos com a linhagem Spin-res confirmaram a hipótese de herança poligênica da resistência, com número mínimo de segregações independentes variando de 1,86 a 2,45. Além disso, observou-se um elevado custo adaptativo associado à resistência de S. frugiperda a spinosad, baseado nos parâmetros da tabela de vida e fertilidade. A partir dos dados de seqüenciamento de quatro bibliotecas de cDNA de lagartas de quarto ínstar das linhagens Sus e Spin-res (expostas ou não a spinosad), utilizando a plataforma HiScan1000&reg; (Illumina&copy;), foi realizada a comparação do perfil de transcrição e expressão diferencial de genes entre as linhagens Sus e Spin-R. O transcritoma foi montado utilizando a estratégia de novo contendo cerca de 19 milhões de leituras single-end com qualidades de score acima de 30, gerando 42406 transcritos com o N50 de 598 pb. A busca por similaridade no banco de dados não-redundante (nr) do NCBI, possibilitou a anotação funcional de 24980 (59%) transcritos, alinhando-se a Bombyx mori L., Helicoverpa armigera (Hübner) e Spodoptera spp. com 22,5; 3,81 e 3,6% das sequências respectivamente. Foram identificados 2903 transcritos apresentando expressão diferencial (P <= 0,05, t-test; fold-change > 2) entre as linhagens Spin-res e Sus. Dentre os transcritos relacionados a enzimas do complexo metabólico, 23 P450 monooxigenases, 13 glutathiona S-transferases, uma carboxilesterase e uma esterase foram superexpressas na linhagem Spin-res. Além disso, foi observada a superexpressão de 15 genes relacionados à produção energética na linhagem Spin-res, o que pode estar relacionada ao elevado custo adaptativo associado à resistência. Análises de PCR quantitativo em tempo real confirmaram que os padrões de expressão foram consistentes com os resultados de RNA-seq. Bioensaios com os sinergistas PBO e DEM mostraram pouco envolvimento de enzimas P450 e nenhum envolvimento de glutationa S-transferases na resistência de S. frugiperda a spinosad. O sequenciamento da subunidade &alpha;6 do receptor nicotínico de acetilcolina de ambas linhagens demonstrou a existência de uma mutação sinônima entre as duas linhagens (G567A), indicando que a subunidade &alpha;6 não é a única relacionada à resistência de S. frugiperda a spinosad. / Spinosad has been one of the most used insecticides to manage Spodoptera frugiperda (J. E. Smith) in Brazil, due to its efficacy and unique mode of action (nicotinic acetylcholine receptor allosteric modulator). To support an insect resistance management program (IRM), we selected and characterized in laboratory a spinosad-resistant strain (Spin-res) of S. frugiperda using the F2 screen method. The resistance ratio, based on LC50, was &asymp; 890-fold. Based on reciprocal crosses between susceptible (Sus) and Spin-res, the inheritance of spinosad resistance in S. frugiperda was autosomal incompletely recessive. Backcrosses between the F1 from reciprocal crosses and the parental Spin-res revealed a polygenic resistance, with an estimation of at least 1.86 to 2.45 genes related to spinosad resistance. Furthermore, it was observed a strong fitness cost associated to spinosad-resistance in Spin-res strain, based on the life table and fertility parameters. The characterization of the transcriptional profile and the differential gene expression comparison between susceptible and spinosad-resistant strains of Spodoptera frugiperda were obtained from the sequencing of cDNA libraries from fourth instar larvae of Sus and Spin-res strains (exposed or not to spinosad) using a HiScan1000&reg; platform (Illumina&copy;). The transcriptome was de novo assembled using nearly 19 million single-end reads with quality score over 30, yielding 42,406 transcripts with a N50 of 598 bp. Based on similarity search in the non-redundant (nr) nucleotide database, 24,980 (59%) transcripts were annotated. Most of the transcripts aligned to Bombyx mori L., Helicoverpa armigera (Hübner) and Spodoptera spp., with 22.5%, 3.81, and 3.6, respectively. We identified 2,032 differentially expressed transcripts (P <= 0.05, t-test; fold-change > 2) between the susceptible and spinosad-resistant strains. Among metabolic enzyme transcripts, 23 P450 monooxigenases, 13 glutathione S-transferases, one carboxylesterase and one esterase were up-regulated in the spinosad-resistant strain. In addition, it was observed 15 genes superexpressed in spinosad-resistant strain related to energy production, which can be related to the high fitness cost associated with resistance. Quantitative real-time PCR analysis showed that patterns of gene expression were consistent with RNA-seq results. Synergistic bioassays using PBO and DEM showed little involvement of P450s in spinosad-resistance and lack of involvement regarding the glutathione Stransferases. Furthermore, we sequenced and compared the subunit &alpha;6 from the nicotinic acetylcholine receptor of S. frugiperda Spin-res and Sus strains. Only one synonymous mutation within the two strains (G567A) was found, showing that the &alpha;6 is not the only subunit involved in S. frugiperda resistance to spinosad.

Para o sucesso das estratégias de manejo de Rhipicephalus (Boophilus) microplus (carrapato bovino) são necessários testes práticos, econômicos e confiáveis que possam detectar a presença de fenótipos resistentes a drogas em suas populações. O fipronil é um acaricida de uso relativamente recente não havendo testes padronizados para o diagnóstico de resistência do carrapato à molécula. No presente trabalho, foram padronizados bioensaios in vitro para esta finalidade: Teste de Imersão de Adultas, Teste de Imersão de Larvas e Teste de Pacote com Larvas. Os testes foram aplicados e, de forma inédita, populações resistentes foram diagnosticadas tanto no Brasil quanto no Uruguai. Ensaios com inibidores enzimáticos não evidenciaram participação importante de enzimas detoxificadoras no mecanismo de resistência. Foi demonstrada reação cruzada entre fipronil e lindano, não verificada para ivermectina. Em algumas situações, foi observado interferência do controle químico de pragas agrícolas no desenvolvimento de resistência dos carrapatos. / For the success of the strategies for the management of Rhipicephalus (Boophilus) microplus (cattle tick), practical, economical and reliable tests are needed to detect the presence of drug-resistant phenotypes in their populations. Fipronil is a relatively new acaricide with no standardized tests for the diagnosis of tick resistance to this molecule. In this study, were standardized in vitro bioassays for this purpose: Adult Immersion Test, Larval Immersion Test and Larval Packet Test. The tests were applied and for the first time, resistant populations were diagnosed in Brazil and Uruguay. Tests with enzymatic inhibitors showed no significant involvement of detoxification enzymes in the mechanism of resistance. Cross-resistance was demonstrated between lindane and fipronil but not with ivermectin. In some situations, it was observed interference of the chemical control of agricultural pests in the development of resistance in ticks.

L'évolution de la résistance aux insecticides chez les moustiques responsables de la transmission de maladies infectieuses compromet notre capacité à contrôler ces populations de vecteurs et pose de graves problèmes de santé publique. Mais les nombreuses modifications physiologiques associées au phénomène de résistance aux insecticides pourraient altérer l'épidémiologie de ces maladies de manière plus indirecte en modifiant la capacité vectorielle des moustiques. Afin d'étudier cette question nous avons mis en place un nouveau système expérimental composé du parasite aviaire Plasmodium relictum SGS1 et de son vecteur naturel le moustique Culex pipiens. Nous avons étudié l'effet de différents allèles de résistance aux insecticides (représentant deux mécanismes principaux i.e. la résistance métabolique ou la modification de la cible) sur une série de traits d'histoire de vie du parasite et du moustique. L'impact de ces différents allèles a été étudié d'une part, dans les conditions contrôlées de leur expression dans un même fond génétique (en utilisant plusieurs souches de moustiques isogéniques), et d'autre part, dans les conditions plus réalistes de leur expression dans un fond génétique hétérogène (utilisation de moustiques échantillonnés sur le terrain). Nous montrons que la résistance aux insecticides a des effets pleïotropes sur l'immunocompétence et les traits d'histoire de vie des moustiques. Son effet sur le développement de Plasmodium semble en revanche limité. Nous discutons d'une part, de la nécessité de poursuivre une approche multifactorielle (impliquant la physiologie, l'immunité et le comportement des moustiques) afin de mieux comprendre l'impact de la résistance aux insecticides sur la transmission de Plasmodium, et d'autre part des perspectives intéressantes qu'offrent ce nouveau système expérimental pour l'étude de l'écologie évolutive des maladies à vecteurs. / The evolution of insecticide resistance in mosquitoes threatens our ability to control many-vector-transmitted diseases, thereby raising serious public health issues. Insecticide resistance entails numerous physiological changes in mosquitoes. This thesis investigates whether these physiological changes alter the quality of mosquitoes as vectors of malaria. To address this issue, we developed a new experimental system consisting in the avian malaria parasite Plasmodium relictum SGS1 and its natural vector, the mosquito Culex pipiens. We investigated the impact of two insecticide resistance mechanisms (target site resistance and metabolic resistance) on several mosquito and parasite life history traits relevant for malaria transmission. The effect of different insecticide resistant genes was investigated using both isogenic laboratory mosquito strains (i.e. against a homogeneous genetic background) and sympatric field caught mosquitoes (i.e. under the more realistic, albeit noisier, conditions of a heterogeneous genetic background). We show that insecticide resistance has a pleiotropic effect on several mosquito traits (immunocompetence, longevity, fecundity), whereas it has only a limited effect on Plasmodium development. We discuss, on the one hand, the need to pursue such a multi-factorial approach (combining the mosquito physiology, immunity and behavior) to better understand the impact of insecticide resistance on malaria transmission and, on the other hand, the promising perspectives offered by this new experimental system for studying the evolutionary-ecology of infectious diseases.

<p>  </p> <p>Vector-borne diseases (VBDs) represent a significant health burden worldwide, threatening approximately 80% of the global population. Insecticide-based vector control is the most effective method to manage many VBDs, but its efficacy has been declining due to high levels of resistance in vector populations to the main insecticide classes which operate via limited modes of action. Therefore, the discovery of new chemistries from non-conventional chemical classes and with novel modes of action is a priority for the control of vectors and VBDs. Natural products (NPs) are diverse in chemical structures and, potentially, modes of action. They have been used as insecticides for many decades and have inspired the development of multiple synthetic insecticides, suggesting the discovery of novel NPs could lead to the development of highly effective insecticides. </p> <p><br></p> <p>In this thesis, I report two studies with a main goal to identify novel mosquito-active insecticide leads that operate via modes of action distinct from existing insecticides. First, I tested the hypothesis that new mosquito-active insecticide leads with novel chemical structures, possibly operating via novel modes of action, can be identified by high-content larval phenotypic screening against a natural product collection and using novel phenotypic endpoints in addition to mortality endpoints. Here, I performed a high-content larval phenotypic screen using first instar (L1) larvae of <em>Aedes aegypti</em> (Linnaeus, 1762) against 3,680 compounds from the AnalytiCon MEGx Natural Product Libraries and a screening platform developed by Murgia et al., (2022). Compounds were screened in a 384-well plate format using the Perkin Elmer Opera Phenix and larvae were scored for lethal and novel phenotypic endpoints. Screening revealed five chemistries that caused larval mortality, including rotenone and a new NP chemistry, NP-4. The identification of rotenone confirmed the ability of the screen to detect mosquito-active NP chemistries. NP-4 caused high levels of larval mortality in the screen, and toxicity was confirmed in a subsequent concentration-response assay against third instar (L3) larvae of <em>Ae. aegypti</em>. 140 chemistries that caused atypical larval phenotypes, including cuticular pigmentation and morphometric changes relative to negative controls, were also identified by the screen. Some of these chemistries may operate by disruption of pathways regulating melanization, growth and development, and novel targets in the insect nervous systems, thus representing potential leads for further insecticide toxicity and mode of action studies. To facilitate quantitative analyses of atypical phenotypes, an attempt was made to assess the morphometrics of the thorax in larvae exposed to test chemistry, relative to control larvae. However, assessment was limited by the number of larvae images of suitable quality for measurements. </p> <p><br></p> <p>In the second study, I tested the hypothesis that metergoline (Murgia et al., 2022) and NP-4 (this study), two chemistries identified by the HTP phenotypic screen described in this project, operate via disruption of targets in the insect nervous systems that are distinct from the current insecticidal modes of products used in mosquito control programs. Specifically, I explored the hypothesis that metergoline operates via one or more insect orthologs of the mammalian G protein-coupled serotonin and dopamine receptors. An electrophysiology study was performed using the suction electrode technique and ganglia of the German cockroach, <em>Blattella germanica </em>(Linnaeus, 1767). To facilitate the investigation of metergoline agonism/antagonism and disruption of invertebrate GPCR signaling, 5-hydroxytryptamine (5-HT; serotonin) was included as a chemical probe. Electrophysiological recordings showed 5-HT (10µM and 1mM) and metergoline (10µM) caused no significant neurological activity at the tested concentrations in comparison to the saline control. However, a consistent neuro-inhibitory trend was observed, suggesting possible agonism of a 5-HT1-like receptor ortholog and antagonism of a putative 5-HT7-like receptor ortholog in the cockroach, respectively.  NP-4 caused significant neuro-inhibition at the tested concentration of 20µM, in comparison to the negative saline control. Given the demonstration of rapid contact toxicity to <em>Ae. aegypti</em> larvae and neurological inhibition in <em>B. germanica</em>, we propose NP-4 may act at one or more conserved targets in the insect nervous systems, which remain to be elucidated. </p> <p><br></p> <p>The significance of the present study is three-fold. First, this study reports the first high-content phenotypic screen of mosquito larvae against a NP collection and identification of 145 mosquito-active chemistries associated with lethal and phenotypic endpoints. These data confirm that the screening platform provided an innovative and effective system to rapidly identify mosquito-active small molecules with potential novel modes of action. Second, metergoline and NP-4 represent potential novel chemical leads for the development of new insecticides that can be incorporated into vector control programs targeting insecticide-resistant populations. Lastly, the study describes the first electrophysiology study of 5-HT, metergoline, and NP-4 via the suction electrode technique in an insect system and contributes new knowledge to the study of the insect serotonergic system, which represents an expanding area of vector biology research given its roles in feeding regulation.  </p> <p><br></p> <p>Future studies resulting from this thesis might include: (1) development of a set of morphometric criteria for quantitative analyses of atypical larval phenotypes, (2) incorporation of new phenotypic endpoints to expand the capacity of the screen to identify novel mode of action chemistries for insecticide discovery, and (3) identification of chemistry candidates suitable for further development from the 140 chemistries associated with atypical larval phenotypes in the primary screen using chemo-informatic and toxicological studies. In addition, studies using reverse transcription-polymerase chain reaction (RT-PCR), cell-based expression systems, mutant/insecticide resistant strains, and patch clamp electrophysiology could be pursued to further investigate the molecular mode of action of metergoline and NP-4, and potential for vector control.</p>

<p>Bed bugs (<i>Cimex lectularius</i> L.) are globally important human ectoparasites. Their management necessitates the use of multiple control techniques. Plant-derived essential oils are extracts from aromatic plants that represent one of the alternative control measures for bed bug control, in addition to mechanical options and synthetic pesticides. However, there is limited information available on the efficacy and toxicology of plant essential oils against bed bugs. This project was designed with the aim to provide in-depth information on efficacy, toxicology and mode-of-action of essential oils and their insecticidal constituents in bed bugs. Initially, I evaluated topical and fumigant toxicity of fifteen essential oil components against adult male bed bugs of the Harlan strain (an insecticide susceptible strain). Neurological effects of the six most toxicologically active compounds were also determined. In both topical and fumigant bioassays, carvacrol and thymol were the most active compounds. Spontaneous electrical activity measurements of the bed bug nervous system demonstrated neuroinhibitory effects of carvacrol, thymol and eugenol, whereas linalool and bifenthrin (a pyrethroid class insecticide) produced excitatory effects. Further, I evaluated the efficacy and neurological impacts of a mixture of three neuroinhibitory compounds; carvacrol, eugenol and thymol in 1:1:1 ratio against adult male bed bugs of the Harlan strain. This mixture of monoterpenoids as well as the mixture of synthetic insecticides exhibited a synergistic affect in topical bioassays. In electrophysiology experiments, the monoterpenoid mixture led to higher neuroinhibitory effects, whereas a mixture of synthetic insecticides caused higher neuroexcitatory effects in comparison to single compounds. </p> <p>In the next objective of my dissertation, I compared the efficacy of five plant essential oils (thyme, oregano, clove, geranium and coriander), their major components (thymol, carvacrol, eugenol, geraniol and linalool) and EcoRaider^® (commercial product) between pyrethroid susceptible (Harlan) and field collected (Knoxville) bed bug populations. Initially, I found that the Knoxville strain was 72,893 and 291,626 fold resistant to topically applied deltamethrin (a pyrethroid class insecticide) compared to the susceptible Harlan strain at the LD₂₅ and LD₅₀ lethal dose levels, respectively. Synergist bioassays and detoxification enzyme assays showed that the Knoxville strain possesses significantly higher activity of cytochrome P450 and esterase enzymes. Further, Sanger sequencing revealed the presence of the L925I mutation in the voltage gated sodium channel gene. The Knoxville strain, however, did not show any resistance to plant essential oils, their major components or EcoRaider^® in topical bioassays (resistance ratios of ~ 1). In the final objective, I evaluated the efficacy of binary mixtures of above-mentioned essential oils or their major components or EcoRaider^® with deltamethrin in susceptible and resistant bed bugs. In topical application bioassays, binary mixtures of essential oils or their major components or EcoRaider^® and deltamethrin at the LD₂₅ dose caused a synergistic increase in toxicity in resistant bed bugs. Further, I studied the inhibitory effects of major essential oil components on detoxification enzyme activities (cytochrome P450s, esterases and glutathione transferases). Detoxification enzyme assays conducted using protein extracts from bed bugs pre-treated with essential oil constituents showed that these compounds significantly inhibited cytochrome P450 activity in the resistant strain, but esterase and glutathione transferase activity were unaffected. No inhibition of detoxification enzyme activities was observed in the Harlan strain bed bugs pre-treated with essential oil constituents.</p> <p>In conclusion, my dissertation research has created the foundation for utilization of natural products for bed bug management by (i) describing the efficacy of plant essential oils and their components against bed bugs, (ii) discovering synergistic interactions between essential oil components at the nervous system level, (iii) determining susceptibility of deltamethrin-resistant bed bugs to plant essential oils and their constituents and (iv) identifying synergistic effects of essential oils or their components on toxicity of pyrethroid insecticides and underlying mechanisms of this synergistic interaction. </p> <br>

No description available.