This study investigates the mechanisms by which rapamycin extends lifespan in Drosophila melanogaster. The research shows that rapamycin increases lifespan in various laboratory strains and a sterile mutant, while reducing fecundity and improving survival after paraquat injection. Rapamycin's effects are independent of Wolbachia status and are observed in both Wolbachia-negative and Wolbachia-positive flies. It also extends the lifespan of long-lived sterile females with the ovoD mutation. Rapamycin reduces egg-laying in different genetic backgrounds, and its effect on lifespan extension is consistent across various food concentrations. The study also demonstrates that rapamycin reduces S6K phosphorylation in IIS mutants, indicating a potential mechanism for its lifespan-extending effects. Additionally, the study includes detailed experimental procedures, including feeding assays, lipid assays, western blots, and mass spectrometry analysis for rapamycin quantification. The results suggest that rapamycin may extend lifespan through multiple pathways, including autophagy induction and reduced S6K activity, and that its effects are not influenced by Wolbachia status. The study provides a comprehensive understanding of the mechanisms by which rapamycin extends lifespan in Drosophila.This study investigates the mechanisms by which rapamycin extends lifespan in Drosophila melanogaster. The research shows that rapamycin increases lifespan in various laboratory strains and a sterile mutant, while reducing fecundity and improving survival after paraquat injection. Rapamycin's effects are independent of Wolbachia status and are observed in both Wolbachia-negative and Wolbachia-positive flies. It also extends the lifespan of long-lived sterile females with the ovoD mutation. Rapamycin reduces egg-laying in different genetic backgrounds, and its effect on lifespan extension is consistent across various food concentrations. The study also demonstrates that rapamycin reduces S6K phosphorylation in IIS mutants, indicating a potential mechanism for its lifespan-extending effects. Additionally, the study includes detailed experimental procedures, including feeding assays, lipid assays, western blots, and mass spectrometry analysis for rapamycin quantification. The results suggest that rapamycin may extend lifespan through multiple pathways, including autophagy induction and reduced S6K activity, and that its effects are not influenced by Wolbachia status. The study provides a comprehensive understanding of the mechanisms by which rapamycin extends lifespan in Drosophila.