Comparing the midgut regenerative responses in <i>Bacillus thuringiensis</i>-susceptible and resistant <i>Heliothis virescens</i> larvae

The crystal (Cry) toxins from Bacillus thuringiensis (Bt) display high specificity and toxicity against relevant insect pests and the use of Bt-based products continues to contribute to insect pest management. To protect this investment, further its potential, and investigate possible unintended effects, various research questions have been proposed. One issue related to Bt usage is the evolution of pest resistance to Bt toxins. The midgut epithelium is targeted by Cry toxins killing enterocytes, facilitating invasion of the hemocoel, leading to septicemia and mortality. While resistance may emerge from alterations to these steps, most research efforts have been focused on reduced toxin binding to midgut receptors as resistance mechanism. Lepidopteran crop pest Heliothis virescens strains have been hypothesized to have enhanced midgut proliferation and differentiation of stem cell populations allowing for regeneration and resistance to diverse Cry toxins. However, the molecular mechanisms involved are not known. We developed a flow cytometry method to monitor stem cell proliferation and differentiation to compare midgut regenerative responses to Cry intoxication in larvae from susceptible and Bt-resistant strains of H. virescens. The structure of the epithelial healing response was studied in vivo using hematoxylin-eosin stained midguts derived from larvae fed Cry1Ac toxin. We detected less regenerative cells in midguts from a Bt-susceptible strain (YDK) compared to midguts from resistant (KCB and CXC) strains, and an overall increase in the total number of cells per unit surface area in KCB midguts. Using primary midgut cell cultures, the midgut regeneration response to Cry1Ac in CXC was an increase in available differentiated cells compared to YDK. In contrast, KCB exhibited an increased abundance of stem cells compared to both YDK and CXC. Using a differential proteomics approach we characterized the proteins secreted by H. virescens midgut cells in response to Cry1Ac and identified a relevant role for arylphorin in promoting midgut regeneration in response to Cry1Ac and DiPel intoxication in both susceptible and resistant H. virescens larvae. The potential fitness costs associated with altered hexamerin transcript expression were monitored using larval bioassays.