The investigation of factors potentially involved in resistance to Bacillus thuringiensis in native Plutella xylostella (l.) (Lepidoptera: Plutellidae) populations
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Data
2018-02-16
Autores
De Bortoli, Caroline Placidi
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
Plutella xylostella é uma praga de grande importância para as crucíferas em todo o mundo. Embora seja controlada com inseticidas sintéticos e biológicos, ela pode desenvolver resistência rapidamente a uma variedade de inseticidas. Os biopesticidas mais comuns utilizados para controlar P. xylostella são baseados na bactéria entomopatogênica Bacillus thuringiensis. Embora muitos estudos tenham sido realizados com Bt, o modo de ação ainda não é totalmente compreendido. Uma grande diversidade de genes é diferencialmente expressa no intestino médio de insetos resistentes, o que sugere que vários processos celulares podem estar envolvidos na resistência. Descobertas recentes mostraram que as mutações no gene que codifica o transportador ABCC2 são responsáveis pela resistência às toxinas Bt em diferentes espécies de insetos. O objetivo desta pesquisa foi testar a hipótese de que a susceptibilidade de P. xylostella a Bt se correlaciona com o nível de expressão dos componentes desse regulador de estresse. Os níveis de expressão dos genes ALP, APN, CDKAL 1, MAP4K4 e ABCC2 foi comparado utilizando qPCR, entre populações suscetíveis e resistentes de P. xylostella. A investigação da sequência de DNA do cDNA do ABCC2 foi realizada, por PCR e sequenciamento, para testar a hipótese de que a susceptibilidade de P. xylostella a Bt se correlaciona com mutações no gene ABCC2. Foram realizados retrocruzamentos entre populações suscetíveis e resistentes e cruzamentos de complementação entre populações resistentes. Nossa pesquisa demonstrou que não há padrões na expressão dos genes testados demonstrando nenhuma associação entre expressão e resistência/susceptibilidade. No entanto, ao investigar a sequência do gene ABCC2, encontrou-se uma mutação no gene da população brasileira resistente, que poderia ser responsável pela causa da resistência da população estudada neste trabalho. Os ensaios de retrocruzamentos não confirmaram que a resistência foi devida à supressão de 1 pb encontrada, no entanto, os ensaios complementares indicaram que a população brasileira compartilha um alelo de resistência com a população havaiana resistente.
Plutella xylostella is a major insect pest of cruciferous crops worldwide. Although controlled with both synthetic and biological insecticides it can rapidly evolve resistance to a variety of insecticides. The most common biopesticides used to control P. xylostella are based on the entomopathogenic bacterium Bacillus thuringiensis. Although many studies have been performed on Bt, the mode of action is still not fully understood. A wide diversity of genes are differentially expressed in the midgut of resistant insects, this suggests that a variety of cell processes may be involved in the preservation of resistance. Recent discoveries have shown that mutations in the gene encoding an ABCC2 transporter are responsible for resistance to Bt toxins in various different insect species. The aim of this research was to test the hypothesis that the susceptibility of P. xylostella to Bt correlates with the level of expression of components of this putative stress-response regulon. The level of expression of ALP, APN, CDKAL 1, MAP4K4 and ABCC2 genes were compared using qPCR, between susceptible and resistant P. xylostella populations. The investigation of the DNA sequence of ABCC2 cDNA was performed, through PCR and sequencing, to test the hypothesis that the susceptibility of P. xylostella to Bt correlates with mutations on the ABCC2 gene. Backcrosses between susceptible and resistant populations and complementation cross between resistant populations were performed. Our research demonstrated that there were no patterns in the expression of the genes tested demonstrating no association between expression and resistance/susceptibility. However, when investigating the ABCC2 gene sequence, a mutation in the gene of the Brazilian resistant population was found, which could be responsible for the resistance of the Brazilian population studied in this research. Backcrossing assays didn’t confirm that the resistance was due to the 1bp deletion found, however complementation assays indicated that the resistant Brazilian population shares a resistance allele with the resistant Hawaiian population.
Plutella xylostella is a major insect pest of cruciferous crops worldwide. Although controlled with both synthetic and biological insecticides it can rapidly evolve resistance to a variety of insecticides. The most common biopesticides used to control P. xylostella are based on the entomopathogenic bacterium Bacillus thuringiensis. Although many studies have been performed on Bt, the mode of action is still not fully understood. A wide diversity of genes are differentially expressed in the midgut of resistant insects, this suggests that a variety of cell processes may be involved in the preservation of resistance. Recent discoveries have shown that mutations in the gene encoding an ABCC2 transporter are responsible for resistance to Bt toxins in various different insect species. The aim of this research was to test the hypothesis that the susceptibility of P. xylostella to Bt correlates with the level of expression of components of this putative stress-response regulon. The level of expression of ALP, APN, CDKAL 1, MAP4K4 and ABCC2 genes were compared using qPCR, between susceptible and resistant P. xylostella populations. The investigation of the DNA sequence of ABCC2 cDNA was performed, through PCR and sequencing, to test the hypothesis that the susceptibility of P. xylostella to Bt correlates with mutations on the ABCC2 gene. Backcrosses between susceptible and resistant populations and complementation cross between resistant populations were performed. Our research demonstrated that there were no patterns in the expression of the genes tested demonstrating no association between expression and resistance/susceptibility. However, when investigating the ABCC2 gene sequence, a mutation in the gene of the Brazilian resistant population was found, which could be responsible for the resistance of the Brazilian population studied in this research. Backcrossing assays didn’t confirm that the resistance was due to the 1bp deletion found, however complementation assays indicated that the resistant Brazilian population shares a resistance allele with the resistant Hawaiian population.
Descrição
Palavras-chave
ABCC2, mecanismo de ação, resistência, suscetibilidade, traça-das-crucíferas, virulência, ABCC2, mode of action, resistance, susceptibility, diamondback moth, virulence