Methods for Studying Infection Biology in <i>Drosophila</i>

Drosophila melanogaster has been an indispensable model for dissecting out innate immune pathways in a controlled and genetically tractable system. Two modes of infection, oral and systemic, in flies reveal distinct facets of host defense. Oral infection captures how the gut senses and responds to ingested microbes, whereas systemic challenges expose the mechanisms that act once pathogens breach external barriers and multiply systemically. Here, we provide a set of protocols that together offer a framework for studying infection biology in flies. They comprise a step-by-step guide to studying infection biology in adult flies using the two routes of infection-systemic infection via needle pricking/injection into the hemolymph and oral infection via pathogen-contaminated food or filter disks. We also outline assays to track infection outcomes, both oral and systemic, including survival rates, bacterial load assessments, gut function evaluations, and wound-repair responses. All experiments in this article are described for wild-type flies, but the workflow can be readily applied to different genetic backgrounds or mutant strains as a means to compare post-infection phenotypes and probe shifts in underlying immune signaling. © 2026 Wiley Periodicals LLC. Basic Protocol 1: Preparation of fly food vials Basic Protocol 2: Starvation of adult Drosophila melanogaster flies Basic Protocol 3: Preparation of pathogenic pellets Basic Protocol 4: Systemic infection of adult Drosophila melanogaster flies Basic Protocol 5: Oral infection of adult Drosophila melanogaster flies Basic Protocol 6: Assessment of survival in infected adult Drosophila melanogaster flies Basic Protocol 7: Scoring of melanization in adult Drosophila melanogaster flies Basic Protocol 8: Tracking bacterial load and pathogen clearance in adult Drosophila melanogaster flies Basic Protocol 9: Monitoring defecation patterns in adult Drosophila melanogaster flies.