Identification of key midgut cell clusters Co-responding fungal and viral infections in silkworms

• The analyses identified and annotated 20 distinct cell clusters (C0-C19) into 5 cell types in the silkworm midgut using snRNA-seq and established a comprehensive single-cell atlas. • New marker gene Cel specific to goblet cells (GCs) was discovered and validated. • We identified C4 and C13, which are in the state of differentiation from ISCs to ECs, as susceptible cell clusters for both N.b and BmNPV. • We found that the low-infection cell clusters C2 and C16 in ECs exhibit low expression of immune genes and high immune response characteristics after infection with pathogens. • A type of EC in the midgut of silkworms which is considered as EC-like (C0 and C12 in this study) highly expresses innate immune genes. The midgut of insects is a crucial organ that enables them to obtain nutrients and resist pathogen invasion. In this study, the midgut cell clusters of the silkworm ( Bombyx mori ), a model species of lepidopteran insects, were analyzed using single-nucleus RNA sequencing technology (snRNA-seq) (PRJNA1280782) to understand the functional diversity of midgut tissues, identify the key cell clusters involved in the response to pathogen infection, and screen molecular targets for disease resistance. Twenty cell clusters (C0-C19) were obtained and annotated. The known marker genes, KEGG and GO pathway enrichment analysis were used to identify five types of cells: enterocyte cells (ECs, 54.4%), goblet cells (GCs, 25.9%), enteroendocrine cells (EEs, 10.6%), intestinal stem cells (ISCs, 2.17%), and muscle cells (MCs, 7.0%). Additional representative cell marker gene Cel of GCs was identified. We conducted snRNA-seq on the midgut tissues of B. mori infected with the fungal and viral pathogens Nosema bombycis (N.b) and Bombyx mori nucleopolyhedrovirus (BmNPV) at 24 h (early stage of infection) and 96 h (later stage of infection) to determine the immune response patterns of different cell clusters. The immune-related genes of ECs C2 and C16 were significantly altered after pathogen infection, making them important cell clusters for screening disease resistance target genes. The common susceptible cell clusters C4 and C13 between different pathogens were successfully identified, and related genes that could simultaneously affect N.b and BmNPV proliferation were screened in C2, C13 and C16. The results provide a basis for exploring potential common molecular target genes of various pathogens.