Bifidobacterium adolescentis SBT2786-induced early-night sleep requires CCHamide-1 signaling in Drosophila ellipsoid body ring neuron circuits

We previously identified the sleep-promoting bacterium Bifidobacterium adolescentis SBT2786; however, its molecular mechanism remains unknown. In Drosophila melanogaster, tissue-/region-specific RNA interference (RNAi) targeted the gut-brain neuropeptide CCHamide-1 (CCHa1) and the CCHa1 receptor (CCHa1R). The increase in SBT2786-induced early-night sleep was abolished by systemic CCHa1 and pan-neuronal CCHa1R knockdown. This phenotype persisted after enteroendocrine cell-specific CCHa1 knockdown, suggesting that it is not fully explained by gut enteroendocrine cell-derived CCHa1 alone. Region-specific RNAi revealed that CCHa1R is required in the ellipsoid body (EB), not the fan-shaped body and tested dopaminergic populations. Ring-driver mapping implicated EB ring neuron circuits, including R3-enriched populations, in the SBT2786 effect: CCHa1R knockdown with the 189 years driver abolished the effect, whereas knockdown with drivers labeling R2/R4m populations did not. These findings show that SBT2786-induced early-night sleep requires CCHa1-CCHa1R signaling and EB ring neuron circuits, and is not fully explained by enteroendocrine cell-derived CCHa1.