Identification of growth factors stimulated by Heliothis virescens midgut regenerative response to Bacillus thuringiensis Cry1Ac intoxication

Cry1Ac is the most active Bacillus thuringiensis toxin against the Lepidopteran pest Heliothis virescens. After feeding on diet containing Cry1Ac toxin, the H. virescens midgut epithelium displays a regenerative response controlled by unidentified growth factors. This healing mechanism has been hypothesized to be involved in resistance to Cry1Ac toxin in a strain of H. virescens. The goal of our project is to characterize this regenerative response as a potential resistance mechanism and identify the specific growth factors involved. We prepared primary mature cell cultures from H. virescens larval midguts and treated them with sublethal amounts of Cry1Ac toxin to induce production and secretion of growth factors. Using anion exchange chromatography we fractionated the secreted proteins (secretome), and to test for bioactivity in these fractions we developed a differentiation assay using purified midgut stem cells from H. virescens larvae. As stem cells differentiated, changes in size and complexity were monitored using flow cytometry to establish the presence of growth factors in distinct fractions. Secretomes from Cry1Ac-treated primary mature midgut cell cultures from Cry1Ac susceptible and resistant H. virescens larvae were compared to characterize the role of enhanced regeneration in resistance.