Isolation of third, fourth, and fifth instar larval midgut epithelia of the moth, Trichoplusia ni

A new tissue isolation technique was used to create intact midgut epithelial wholemounts from three Trichoplusia ni (Lepidoptera: Noctuidae) larval instars. The protease, dispase, removed the basal lamina and associated connective tissue and allowed for high resolution light microscopy of entire epithelia. Columnar, goblet, differentiating, and stem cells were characterized by double fluorescent labelling of f-actin and nuclei. A comparison of cell populations by digital image analysis revealed significant regional and temporal changes in the density and number of differentiating and stem cells. Growth of the midgut epithelium from third to fourth instar, and from fourth to fifth instar, was accomplished by both cell differentiation and cell division. Cell division however, was greatly reduced from fourth to fifth instar with a concomitant sharp decrease in the stem cell population.