VIP (Vegetative Insecticidal Protein) proteins are a recently discovered family of Bacillus thuringiensis (Bt) insecticidal proteins, which are secreted by the bacteria during the vegetative growth, with no structural relationship to the best known family of Bt toxins, the Cry proteins, although it has been shown that, similarly to them, the target of VIP proteins is the insect midgut. Despite the interest in this new family of insecticidal proteins, there is very little information on their mode of action and especially on the events that take place once the VIP proteins are ingested. In order to use in the near future VIP proteins as part of the relatively scarce arsenal of environmentally-friendly insecticidal products, a deeper understanding of their mode of action is pivotal. The proposed project aims to study in detail the mode of action of different Vip3 toxins, active against larvae of two lepidopteran species, Helicoverpa armigera and Spodoptera exigua, which are world-wide pests. H. armigera is relatively tolerant to Vip3 proteins while S. exigua is very sensitive. The first part of the working program will consist on searching for differences in the activation of the Vip3 proteins by midgut juice from the two species. The activity of the resulting products will be tested in bioassays with larvae from the two species. In a second phase of the project we will determine the binding properties of labelled activated Vip3 proteins with brush border membrane vesicles (BBMV) prepared from larval midguts. In a third phase, the permeabilisation capacity of different Vip3 proteins on BBMV will be studied by means of a spectrofluorimetric technique using a membrane-voltage-sensitive dye. Finally, primary cultures of isolated midgut cells will be established and tested for their susceptibility to Vip3 toxins, in order to shed light on in vivo mechanisms of cell death which might be independent of membrane permeabilisation induced by this family of proteins.