The growing promise of Toll-deficient Drosophila melanogaster as a model for studying Aspergillus pathogenesis and treatment.

Despite considerable progress over recent years, the prognosis of invasive aspergillosis (IA) remains unfavorable, reflecting an incomplete understanding of Aspergillus pathogenesis and suboptimal antifungal efficacy in vivo. Mammalian host systems including rodents and rabbits are important tools in elucidating antifungal drug activity and the immunopathogenesis of IA. Nonetheless, they are hampered by limitations that impose a "bottleneck" in mass screening of novel antifungal compounds and putative Aspergillus virulence factors including their cost, labor intensity and ethical constraints. Drosophila melanogaster is an invertebrate host with a long tract record of genetic studies and a simple, yet highly conserved innate immune system. Herein, we describe our experience using this fly model as a facile, non-laborious, inexpensive pathosystem for high-throughput screening of novel antifungal compounds and putative Aspergillus mutants, and studying antifungal innate immunity. We present three infection protocols (i.e., injection, rolling, ingestion) that introduce Aspergillus either directly into the hemolymph or at different epithelial surfaces of Toll-deficient Drosophila flies. As a proof of principle, we demonstrate attenuated virulence of known hypovirulent Aspergillus strains and protection of Aspergillus-infected flies given oral Aspergillus-active agents such is voriconazole. These protocols can be adapted for similar studies of other fungal pathogens. Crossing and generation of Toll-deficient Drosophila flies takes 3 weeks; Aspergillus conidial preparation takes 3 days; fly inoculation depending on the infection assay takes 1 to 6-8 hours; and assessment of fly survival, Aspergillus strain virulence, Drosophila innate host parameters and/or drug activity takes 4-8 days.