A new tissue technique for evaluating effects of Bacillus thuringiensis toxins on insect midgut epithelium

Epithelial tissue wholemounts were produced after enzymatic removal of basal lamina and connective tissue from midguts of Trichoplusia ni larvae. Wholemounts were nourished in artificial hemolymph and tissue viability was assessed for up to 24 hr using the vital dyes trypan blue, acridine orange (AO), propidium iodide (PI), and 4', 6-diamidino-2-phenylindole (DAPI). Peritrophic membrane synthesis and modification of Bacillus thuringiensis Cry1Ac protoxin to active toxin confirmed some normal epithelial function. Vital staining using the combination of AO and PI, or DAPI revealed altered membrane permeability in columnar epithelial and regenerative cells of tissues treated with activated Cry1Ac toxin while feeding and oral inoculation bioassays verified Cry1Ac toxicity. DAPI was selected to identify target cells in a rapid and highly sensitive assay.