Interação da toxina Cry1ac de Bacillus thuringiensis às microvilosidades apicais das células colunares do intestino médio de Helicoverpa armigera Hübner, 1805 (Lepidoptera: Noctuidae) em diferentes ínstares larvais
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Data
2017-07-26
Autores
Silva, Igor Henrique Sena da [UNESP]
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Universidade Estadual Paulista (Unesp)
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Helicoverpa armigera é uma praga altamente polífaga e ataca culturas de grande importância agrícola em diversos países do mundo. O controle desta praga é realizado primariamente por inseticidas químicos. Porém, o uso indiscriminado do controle químico levou a resistência de populações desta praga a maioria dos inseticidas químicos usados para seu controle, dificultando o seu manejo no campo. Além do controle químico, o controle de H. armigera tem sido realizado com uso de plantas transgênicas que expressam a proteína Cry1Ac de Bacillus thuringiensis (Bt) ou por bioinseticidas que contem esta e outras proteínas. No entanto, estudos têm demonstrado uma diminuição significativa na susceptibilidade de H. armigera às proteínas Cry com o aumento de seu desenvolvimento larval. O mecanismo de resistência mais comum dos insetos às proteínas Cry é a redução de ligação da proteína aos receptores presentes na membrana, levando a uma menor afinidade de ligação da proteína aos receptores intestinais. Desta forma, o objetivo deste trabalho foi avaliar a susceptibilidade de lagartas de diferentes ínstares de H. armigera à Cry1Ac e correlacionar com a capacidade de ligação da proteína Cry1Ac às microvilosidades apicais das células colunares do intestino médio (BBMVs) isoladas de todos ínstares larvais. Além disso, por meio de ensaios de imunoprecipitação e análise por cromatografia líquida acoplada a espectrofotometria de massa, identificar as proteínas envolvidas na interação com a proteína Cry1Ac no segundo e quinto ínstares de H. armigera. Foi observada uma redução significativa na susceptibilidade dos últimos ínstares larvais de H. armigera à proteína Cry1Ac comparado aos ínstares iniciais. Os valores estimados de CL50 variaram de 31,1 a 2525,7 ng de proteína/cm² de dieta, em lagartas de primeiro e sexto ínstar, respectivamente. Estes resultados evidenciam uma diferença de 80 vezes na susceptibilidade à proteína Cry1Ac do último para o primeiro ínstar. Nos testes de ligação de ELISA da proteína Cry1Ac às BBMVs dos diferentes ínstares, foi constatada uma diminuição total na capacidade de ligação da proteína Cry1Ac as BBMVs dos estádios mais tardios comparados aos iniciais, com afinidade de ligação aparente de 3,88 vezes menor no último ínstar comparado ao primeiro. Assim, uma clara correlação direta entre toxicidade de Cry1Ac e a afinidade de ligação da proteína às BBMVs de H. armigera foi demonstrada. Os resultados dos ensaios de imunoprecipitação demonstraram um padrão diferenciado de interação com a proteína Cry1Ac no segundo e quinto ínstar. A proteína fosfatase alcalina (ALP) foi identificada apenas no segundo ínstar, bem como, outras proteínas de membrana, como proibitina e uma proteína de canal iônico, que podem estar envolvidas para a alta toxicidade de Cry1Ac em ínstares iniciais de H. armigera. A identificação e o papel funcional das proteínas de ligação nos diferentes estádios de desenvolvimento de H. armigera, facilitará a elucidação do mecanismo de ação da proteína Cry1Ac e poderá ajudar a propor estratégias que retardem a evolução da resistência dos insetos às cultivares transgênicas que expressam esta proteína.
Helicoverpa armigera is a highly polyphagous pest and attacks important crops worldwide. The control of this pest is carried out primarily by chemical insecticides. However, the indiscriminate use of chemical control, led to pest populations to develop resistance to most of the chemical insecticides used for their control, making it difficult to management in the field. In addition to chemical control, H. armigera has been done by transgenic crops expressing Cry1Ac toxin from Bacillus thuringiensis (Bt) or by biopesticides that contains Cry1Ac or other toxins. However, studies have demonstrated a susceptibility decrease of H. armigera to Cry toxins with their larval development increase. The most common mechanism of resistance used by insects against Cry toxins is the reduced toxin binding to receptors present on the membrane, leading to a lower binding affinity of the toxin to intestinal receptors. Thus, the objective of this work was to evaluate the susceptibility of different instar larvae of H. armigera to Cry1Ac toxin and to correlate with the Cry1Ac toxin binding capacity to BBMV isolated from all larval instar. Furthermore, by pull-down techniques and liquid chromatography coupled to mass spectrometry analysis, to identify the proteins involved in the Cry1Ac toxin interaction in the second and fifth instars of H. armigera. A significant reduction in the susceptibility of the late instars of H. armigera to Cry1Ac toxin was observed compared to early instars. LC50 estimated values ranged from 31.1 to 2525.7 ng of toxin/cm2 of diet in first and sixth instar larvae, respectively. These results point a difference of 80-fold in the susceptibility to Cry1Ac toxin from late to first larval instar. In the ELISA binding assays results to BBMV of the different instars was found a total decrease in the binding capacity of Cry1Ac toxin to BBMVs from late instars compared to BBMV from early instars, presenting an apparent binding affinity of 3.88 times lower in the last instars than the first. Thus, a clearly correlation between Cry1Ac toxicity and binding toxin affinity to H. armigera BBMV has been demonstrated. The pull-down assays demonstrated a different pattern of the interaction between Cry1Ac toxin with the second and fifth instars. The protein phosphatase alkaline (ALP) was identified only in the second instar, as well as, other membrane proteins, as prohibitin and an ion channel protein, which may be involved for higher toxicity of Cry1Ac in early instars of H. armigera. The identification and functional role of binding proteins in the different stages of development of H. armigera will facilitate the elucidation of the Cry1Ac toxin mechanism of action and will may help to propose strategies that delay the insect resistance evolution to transgenic crops that express this protein.
Helicoverpa armigera is a highly polyphagous pest and attacks important crops worldwide. The control of this pest is carried out primarily by chemical insecticides. However, the indiscriminate use of chemical control, led to pest populations to develop resistance to most of the chemical insecticides used for their control, making it difficult to management in the field. In addition to chemical control, H. armigera has been done by transgenic crops expressing Cry1Ac toxin from Bacillus thuringiensis (Bt) or by biopesticides that contains Cry1Ac or other toxins. However, studies have demonstrated a susceptibility decrease of H. armigera to Cry toxins with their larval development increase. The most common mechanism of resistance used by insects against Cry toxins is the reduced toxin binding to receptors present on the membrane, leading to a lower binding affinity of the toxin to intestinal receptors. Thus, the objective of this work was to evaluate the susceptibility of different instar larvae of H. armigera to Cry1Ac toxin and to correlate with the Cry1Ac toxin binding capacity to BBMV isolated from all larval instar. Furthermore, by pull-down techniques and liquid chromatography coupled to mass spectrometry analysis, to identify the proteins involved in the Cry1Ac toxin interaction in the second and fifth instars of H. armigera. A significant reduction in the susceptibility of the late instars of H. armigera to Cry1Ac toxin was observed compared to early instars. LC50 estimated values ranged from 31.1 to 2525.7 ng of toxin/cm2 of diet in first and sixth instar larvae, respectively. These results point a difference of 80-fold in the susceptibility to Cry1Ac toxin from late to first larval instar. In the ELISA binding assays results to BBMV of the different instars was found a total decrease in the binding capacity of Cry1Ac toxin to BBMVs from late instars compared to BBMV from early instars, presenting an apparent binding affinity of 3.88 times lower in the last instars than the first. Thus, a clearly correlation between Cry1Ac toxicity and binding toxin affinity to H. armigera BBMV has been demonstrated. The pull-down assays demonstrated a different pattern of the interaction between Cry1Ac toxin with the second and fifth instars. The protein phosphatase alkaline (ALP) was identified only in the second instar, as well as, other membrane proteins, as prohibitin and an ion channel protein, which may be involved for higher toxicity of Cry1Ac in early instars of H. armigera. The identification and functional role of binding proteins in the different stages of development of H. armigera will facilitate the elucidation of the Cry1Ac toxin mechanism of action and will may help to propose strategies that delay the insect resistance evolution to transgenic crops that express this protein.
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Palavras-chave
Aminopeptidase N, Fosfatase alcalina, Controle biológico, Ensaios de ligação, Espectrofotometria de massa, Cromatografia líquida, Imunoprecipitação, Biological control, Binding assay, Alkaline phosphatase, Mass spectrometry analysis, Liquid chromatography, Pull-down