Characterization of Digestive Enzymes of Bruchid Parasitoids–Initial Steps for Environmental Risk Assessment of Genetically Modified Legumes

Genetically modified (GM) legumes expressing the α-amylase inhibitor 1 (αAI-1) from Phaseolus vulgaris L. or cysteine protease inhibitors are resistant to several bruchid pests (Coleoptera: Chrysomelidae). In addition, the combination of plant resistance factors together with hymenopteran parasitoids can substantially increase the bruchid control provided by the resistance alone. If the strategy of combining a bruchid-resistant GM legume and biological control is to be effective, the insecticidal trait must not adversely affect bruchid antagonists. The environmental risk assessment of such GM legumes includes the characterization of the targeted enzymes in the beneficial species and the assessment of the in vitro susceptibility to the resistance factor. The digestive physiology of bruchid parasitoids remain relatively unknown, and their susceptibility to αAI-1 has never been investigated. We have detected α-amylase and serine protease activities in all five bruchid parasitoid species tested. Thus, the deployment of GM legumes expressing cysteine protease inhibitors to control bruchids should be compatible with the use of parasitoids. In vitro inhibition studies showed that sensitivity of α-amylase activity to αAI-1 in the parasitoids was comparable to that in the target species. Direct feeding assays revealed that harmful effects of α-amylase inhibitors on bruchid parasitoids cannot be discounted and need further evaluation

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ix.; 84.; bibl.; 21 c

Interactions of anti -insect dietary proteins in the cowpea bruchid, Callosobruchus maculatus

Breeding resistant plant varieties with multiple resistance factors against insects rather than a single resistance factor may result in a broader spectrum of insecticidal activity, a higher level of protection, and an increase in the useful life of the resistant varieties. Given that genetic engineering may be used to introduce multiple resistance genes into a crop plant, the possible interactions of two or more anti-insect dietary proteins fed in the artificial diet of cowpea bruchid (Callosobruchus maculatus) were investigated. The anti-insect dietary protein that was tested in combination with other anti-insect dietary proteins was a soyacystatin N (scN). scN is a cysteine proteinase inhibitor, an inhibitor of one of major enzymes in the midgut of cowpea bruchid larvae. Combinations of scN and E-64 (another cysteine proteinase inhibitor with a known toxic effect on C. maculatus) were synergistic when fed to cowpea bruchids. Similar results were observed using combinations of scN together with inhibitors of different mechanistic classes of proteinases, e.g., Kunitz inhibitor, a serine proteinase inhibitor, and pepstatin A, an aspartyl proteinase inhibitor. Mixtures of scN and wheat alpha-amylase inhibitor were synergistic, while scN mixed with GS II lectin was not. The results of these experiments indicated that not all of combinations of resistance factors will have additive or synergistic effects on suppressing the development of the cowpea bruchid. The mechanism of the synergism was investigated using SDS-PAGE gel electrophoresis. The synergistic effect appears to be related to protection of the protein toxin against degradation by digestive cysteine proteinases and/or aspartyl proteinases in the bruchid midguts. These results confirmed another function of major proteinases in the gut of cowpea bruchid larvae. They serve not only to release free amino acids from dietary proteins, but also have a defensive role in helping reduce the concentration of a toxic dietary proteins

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x.;82 Hal.; ill.;19 c