Gut microbiota shape gene regulatory networks to direct intestinal stem cell differentiation trajectories in Apis mellifera.

Honeybees are key pollinators of flowering plants, and their health is closely associated with gut function. The function is regionally specialized, with nutrient absorption distributed across distinct gut compartments and influenced by the gut microbiota. Here, we constructed a single-nucleus transcriptomic atlas of the honeybee gut under microbiota-free and conventional (CV) conditions and uncovered a microbiota-dependent mechanism regulating intestinal homeostasis. We characterized major intestinal cell types in the honeybee, including intestinal stem cell/enteroblast (ISC/EB), enterocyte (EC), enteroendocrine cell (EE), visceral muscle, and enteric neurons. The cellular data uncovered that regional functional specialization of the intestine is associated with the spatial distribution of EC and EE cell subtypes. Intriguingly, we found that microbiota significantly alter cell composition and gene expression in the ileum, particularly within the ISC/EB population. Relish, a conserved nuclear factor-κB transcription factor in the immune deficiency pathway, was upregulated in the ISC/EB within the ileum of CV bees. The gene regulatory network (GRN) revealed that the Relish regulon is explicitly activated in ISC/EB of CV bees, promoting ISC/EB toward ECs by directly regulating differentiation factors.Our study highlights the cellular and regional complexity of the honeybee gut and demonstrates that region-specific microbiota may modulate GRN in ISC/EBs, thereby shaping epithelial cell composition and maintaining intestinal homeostasis. This work provides a valuable resource for understanding host-microbiota interactions in honeybees and serves as a reference to study gut function, immune regulation, and bee health at single-cell resolution.