Study On Physiological And Biochemical Mechanism Of Vip3Aa Against Cotton Bollworm Helicoverpa Armigera (Hübner)

Large-scale cultivation of transgenic Bt cotton has played an important role against cottonbollworm Helicoverpa armigera (Hübner). However, as the selection of Bt toxin in long time, theresistance problem of cotton bollworm to Bt has become the key factor in continuable effective using ofBt cotton. Vip3A protein, which has different mode of action and different mechanism of actioncompared with Bt insecticide crystal proteins, is one kind of important protein in modern insectmanagement strategy, but its insecticidal mechanism is unclear. In order to clarify the action mode ofVip3Aa and provide the theoretical bases for applying Vip3Aa as an important protein in New toxinstrategy. The effects of Vip3Aa and Cry1Ac on protease, detoxification enzymes and aminopeptidaseN (APN) activities in larvae of cotton bollworm Helicoverpa armigera (Hübner) were compared, thepathological changes in midgut tissues of cotton bollworm Helicoverpa armigera (Hübner) larvae fedartifical diet containing Vip3Aa protein were observed using transmission electron microscope, thedifferences of binding proteins on BBMVs between the tolerant and susceptible strain were preliminarycompared. The results were as follows:1. Bioassay results suggested that the lethal effect of Vip3Aa protein against H. armigera was lowerthan Cry1Ac protein, but the Vip3Aa had significantly inhibition to the growth of H. armigera. TheVip3Aa and Cry1Ac could be digested to the effective core fragment by trypsin, but the enzymatichydrolysis rate of Vip3Aa was faster than Cry1Ac.2. The activities of mid-gut protease in the susceptible and resistant strains after they ingestedVip3Aa or Cry1Ac toxin differently were compared, including main protease、detoxifying enzymes andAPN activity, and the activity of the several enzymes effected by Vip3Aa and Cry1Ac together werestudied. The results showed that the activities of total protease and tryptase were quickly increased afterH. armigera fed on artifical diet containing Cry1Ac or Vip3Aa or Cry1Ac+Vip3Aa respectively. Butcompared with control, these two enzyme activities had no significant difference or lower than controlafter treated by Cry1Ac12h. While the activities increased times of these two enzymies in larvaetreated by Vip3Aa was clearly prolonged, and the activity of chymotrypsin-like enzyme in this treatmentwas also higher than control. The combined action of Cry1Ac and Vip3Aa can extend the time ofenzymatic hydrolysis. Glutathione-S-transferase enzymes and-acetic acid naphthyl esterase activityincreased after cotton bollworm fed on feed containing Vip3Aa or Cry1Ac or Cry1Ac+Vip3Aa protein.However, the Vip3Aa and Cry1Ac had little effect on aminopeptidase N activities, maybe their toxicityaction process had no relationship with enzyme activity changes of APN.3. The pathological changes in midgut tissues of cotton bollworm Helicoverpa armigera (Hübner)larvae fed artifical diet containing Vip3Aa protein were observed using transmission electronmicroscope, the differences between its changes and the changes induced by Cry1Ac were compared.The results showed that the midgut tissues gradually appeared pathological changes after H. armigerafed artifical diet containing Vip3Aa protein, microvilli, nucleus, chromatin, mitochondria, endoplasmic network changed. Compared with the larvae which fed on artificial diet containing Cry1Ac, thepathological changes of H. armigera induced by Vip3Aa were slowly.4. The differences of binding proteins on BBMVs between the tolerant (selected by Vip3Aa,96-3A)and susceptible (96S) H.armigera were preliminary compared using the two-dimensionalelectrophoresis. The variety of binding proteins in96-3A was obviously reduced than96S H. armigera,there had some proteins bands missed in96-3A strain. However, at the same time, some specific bandswere found in96-3A which hadn’t appeared in96S strain.