<i>dilp2</i>-Mediated Insulin Signaling Pathway Was Involved in O₃-Induced Multigenerational Effects 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 (<i>Drosophila melanogaster</i>) 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.