<i>dilp2</i>-Mediated Insulin SignalingPathway Was Involved in O₃‑Induced MultigenerationalEffects of Shortened Lifespan in <i>Drosophila melanogasters</i>

As a long-standing atmospheric pollutant, ozone (O₃) exerts enduring effects on biological health. However, experimental research on its impact on organism lifespan and generational effects is limited. This study exposed three generations of fruit flies (Drosophila melanogaster) to O₃, revealing a shortened lifespan across generations. Specifically, after O₃ exposure, the lifespan of the F2 generation was significantly reduced compared with F0 and F1 generations, indicating a cumulative multigenerational effect. Transcriptome analysis unveiled significant disruptions in metabolic pathways, notably the insulin signaling pathway. Subsequent qRT-PCR analysis showed elevated mRNA levels of insulin pathway-related genes (<i>dilp2</i>, <i>dilp3</i>, <i>dilp5</i>, <i>InR</i>, and <i>TOR</i>), alongside decreased expression levels of <i>FOXO</i>, <i>4E-BP</i>, and <i>Atg5</i> in flies exposed to O₃. Notably, knocking down <i>dilp2</i>, rather than <i>dilp3</i>, <i>dilp5,</i> and <i>InR</i>, rescued the O₃-induced lifespan shortening. Overall, O₃ exposure triggered activation of the dilp2-mediated InR-FOXO/TOR-4E-BP-Atg5 signaling pathway, potentially contributing to shortened lifespan with cumulative effects. This study highlights the viability of employing fruit flies as a model to evaluate the multigenerational toxicity of environmental pollutants, particularly atmospheric pollutants.