Safety Evaluation Of Cry1Ac On Macrocentrus Cingulum And Physiological Regulation Of Parasitization And Cry1Ac On Ostrinia Furnacalis

It is necessary for the safety of genetically modified crops to be strictly assessed before commercial cultivation, our research systematically evaluated the effect on transgenic corn expressing Cry 1Ac toxin to Macrocentrus cingulum which is the parasitic wasp of target pest, the Asian corn borer, Ostrinia furnacalis. Thus far, no similar research about the relationship among Bt crops or Bt protein, Asian corn borer and Macrocentrus cingulum has been reported. Firstly, an approach of evaluation risk of genetically modified crops or insecticidal protein on parasitic wasp was set up. Then, this research used the methods of physiology, biochemistry, molecular biology and proteomics to study the parasitism of Macrocentrus cingulum and the effect on Cry 1 Ac protein to nutrition metabolism, enzyme activity in metabolism and immune responses of sensitive and resistant Asian corn borer, in order to further reveal the mutual regulatory mechanism between parasitoids and host sand determine the inner differences caused by parasitism between sensitive and resistant Asian corn borer, providing theoretical basis and methodological guidance to the function of parasitic enemy in biological control, the development of delaying resistance and new ways of resistance management. The major findings are as follows:1. This research developed a set of experiment system about evaluating the direct and indirect effect on transgenic insecticidal protein to Macrocentrus cingulum, and we used this evaluation system to assess the effect on Cry 1 Ac protein to M. cingulum, the results revealed that the mortality rate of the host increased significantly after feeding Cry 1 Ac protein when used sensitive Asian corn borer as host, and the parasitism rate and the weight of cocoon decreased significantly. However, feeding Cry 1 Ac protein had no adverse effect on the life table parameters of M. cingulum when used resistant Asian corn borer as host. It demonstrated that the effect on Cry 1Ac protein to Macrocentrus cingulum caused by the decreased quality of hosts, while Cry 1 Ac protein had no adverse effect on M. cingulum. And adults of M cingulum were fed directly with Cry 1 Ac protein while PA as positive control, the results shows no effect on adults of M. cingulum when they were fed different concentration of Cry 1 Ac protein, but the longevity and the number of M cingulum progeny per host decreased significantly when they were fed PA. The results of this research revealed that Cryl Ac protein have no adverse effect on M. cingulum.2. We studied the relationship among M. cingulum, Bt protein and hosts by proteomics method. This study was mainly focused on the effects of M, cingulum and Cry1 Ac protein on hemolymph proteomics of susceptible and resistant ACB (Asia corn borer) larvae. iTRAQ technology was used to analyze the expression profiles of hemolymph protein and 286 proteins with quantitative information were identified. Go functional annotation and KEGG pathway were used to analyze the function of differential expression proteins among different treatments. Then three important differential expression proteins (storage protein, phenol oxidase and lysozyme) were selected for MRM verification, and the storage protein with significant various expression levels among different treatments was cloned and analyzed by qPCR. The results showed that parasitoids and Cry1 Ac protein alone or combined effects will have differentially expressed proteins between of susceptive and resistant Asian corn borer.3. Our research studied the nutrition regulation of hemolymph in susceptible and resistant ACB larvae, which were treated by M. cingulum and CrylAc protein. We measured the change of proteins, amino acids, carbohydrates and fats within the host larvae hemolymph after different treatments. It turned out that protein content, lipid droplet concentration, trehalose content and total reducing sugars changed significantly within the hemolymph of susceptible and resistant larvae while the total amino acid content and trehalase activity didn’t change obviously; the protein content, lipid droplet concentration and total reducing sugars suffered significant changes in susceptible larvae treated by CrylAc protein and no obvious change was detected for other substances. However, there were no significant changes in the content of all detected substances in resistant larvae feeding on CrylAc no matter the larvae were parasitic or not. The changes of these nutrients may be the internal cause of the decline of host quality.4. We measured the effects of M. cingulum and CrylAc protein on detoxifying enzymes, protective enzyme and midgut protease in hemolymph of susceptible and resistant ACB larvae. The results showed that:after being parasitic, both susceptible and resistant larvae exhibited decreased ACHE activity, significantly increased GST activity and SOD enzyme activity increased at first and decreased subsequently while no obvious change happened to other enzymes. For CrylAc protein, ACHE activity increased significantly in both susceptible and resistant larvae, GST activity reduced in susceptible larvae and rised in resistant larvae, SOD enzyme activity increased at first and then decreased in susceptible larvae but unchanged in resistant larvae, tryptase activity rised apparently only in susceptible larvae and also no difference in resistant larvae. No significant activity change had been detected for other enzyme. These results suggest that the changes of the three kinds of enzyme activities may also be related to the decline in the quality of the host.5. Our research studied the impact of M. cingulum and CrylAc protein on cellular immunity and humoral immunity within hemolymph of susceptible and resistant ACB larvae. According to the results, the hemocytes in hemolymph of susceptible and resistant larvae exhibited a series of pathological changes after 2 days treated by M. cingulum and CrylAc protein. Total number of hemocytes changed variously when susceptible larvae were fed on CrylAc before or after parasitism. Hemocytes of susceptible larvae feeding on CrylAc had significantly declined cysts capacity to Sephadex A-25 microsphere and apoptosis rate was obviously higher than other treatments. Parasitism of M. cingulum inhibited the PPO activity and melanism reaction in Hemocytes of both susceptible and resistant larvae, however, lysozyme activity rised after parasitism. The differences in these immune responses may be another intrinsic mechanism for the decline of host quality.