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Autophagy, an autophagosome and lysosome-based eukaryotic cellular degradation system, has previously been implicated in lifespan regulation in different animal models. In this report, we show that expression of the RNAi transgenes targeting the transcripts of the key autophagy genes Atg1or Atg18in adult fly muscle or glia does not affect the overall levels of autophagosomes in those tissues and does not change the lifespan of the tested flies but the lifespan reduction phenotype has become apparent when Atg1 RNAior Atg18 RNAiis expressed ubiquitously in adult flies or after autophagy is eradicated through the knockdown of Atg1 or Atg18 in adult fly adipocytes. Lifespan reduction was also observed when Atg1 or Atg18 was knocked down in adult fly enteroblasts and midgut stem cells. Overexpression of wild-type Atg1 in adult fly muscle or adipocytes reduces the lifespan and causes accumulation of high levels of ubiquitinated protein aggregates in muscles. Our research data have highlighted the important functions of the key autophagy genes in adult fly adipocytes, enteroblasts, and midgut stem cells and their undetermined roles in adult fly muscle and glia for lifespan regulation.