Phase III IUCRC at UF: Center for Arthropod Management Technologies - Intestinal Stem Cells: Differentiation and Applications

CAMTech (University of Florida) Dr. Reddy Palli Abstract We recently developed methods to mark different cell types present in the fall armyworm (FAW), Spodoptera frugiperda alimentary canal. Screening eight fluorescent marker genes in FAW identified TdTomato as the best gene in terms of emitting the maximum fluorescence with the lowest toxicity to the animal (Chen and Palli, 2022a). Transgenic FAW strain expressing TdTomato fluorescent protein gene under the control of intestinal stem cell (ISC) and enteroblast (EB)- specific Escargot promoter was produced (Chen and Palli, 2022b). Methods were developed to separate red fluorescent cells using a cell sorter. Single cell sequencing of larval midgut cells identified eight clusters of cells, including ISC and EB clusters (Fig. 1). One-third of cells isolated from larval midgut belong to these two clusters. Single cell sequencing of SfMG-0617 midgut cell line identified eight clusters of cells, including ISC/EB cluster (Fig. 1). Five percent of cells in SfMG-0617 cell line were identified as ISC/EB. Marker genes expressed in the ISC/EB cluster cells were identified. Transfection of constructs expressing tdTomato gene under the control of promoters of ISC/EB-specific genes showed expression of tdTomato in a sub- population of round smaller SfMG-0617 cells, likely ISC/EB. Methods to separate ISC/EB from other cells in SfMG- 0617 cell line were also developed. We propose to use these two sources of ISC/EB in studies aimed at optimizing the differentiation of ISC/EB and the formation of an epithelial layer and to develop applications using these cells in screening for novel insecticides as well as studying their mode of action. The insect alimentary canal is a major organ that encounters ingested materials. Several insecticides, including Bacillus thuringiensis toxins, target the cells in the midgut. The methods developed could be used by industrial partners in isolation of ISC/EB for use in insecticide screens and insecticide mode of action studies.