Deciphering cellular heterogeneity in Spodoptera frugiperda midgut cell line through single cell RNA sequencing

Using the 10x Genomics Chromium single-cell RNA sequencing (scRNA-seq) platform, we discovered unexpected heterogeneity in an established cell line developed from the midgut of the Fall armyworm, Spodoptera frugiperda, a major global pest. We analyzed the sequences of 18,794 cells and identified ten unique cellular clusters, including stem cells, enteroblasts, enterocytes and enteroendocrine cells, characterized by the expression of specific marker genes. Additionally, these studies addressed an important knowledge gap by investigating the expression of genes coding for respiratory and midgut membrane insecticide targets classified by the Insecticide Resistance Action Committee. Dual-fluorescence tagging method, fluorescence microscopy and fluorescence-activated cell sorting confirmed the expression of midgut cell type-specific genes. Stem cells were isolated from the heterogeneous population of SfMG-0617 cells. Our results, validated by KEGG and Gene Ontology analyses and supported by Monocle 3.0, advance the fields of midgut cellular biology and establish standards for scRNA-seq studies in non-model organisms.