Interaction of Cry1ac toxin from Bacillus thuringiensis to brush border membrane of Helicoverpa armigera Hübner, 1805 (Lepidoptera: Noctuidae) midgut in different larval instars

Author(s)

Igor Henrique Sena da Silva

• Document type: Master's Dissertation
• Institution: Universidade Estadual Paulista (Unesp)
• Defense date: 2017-07-26
• Advisor: Ricardo Antonio Polanczyk; Fernando Hercos Valicente

Abstract

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. (AU)

• FAPESP's process: 15/24330-5 - Interação da toxina Cry1Ac de Bacillus thurigiensis às microvilosidades apicais das células colunares (BBMVs) do intestino médio de diferentes ínstares larvais de Helicoverpa armigera (Lepidoptera: Noctuidae)
• Grantee: Igor Henrique Sena da Silva
• Support Opportunities: Scholarships in Brazil - Master