Large-scale gain-of-function analysis reveals distributed genetic contributions to activity persistence under oxidative stress in Drosophila

Organismal responses to stress are often assessed by survival, yet the genetic basis of sustained functional performance under stress remains poorly understood. Here, we performed a large-scale gain-of-function analysis in Drosophila melanogaster using 2,838 Gene Search (GS) insertion lines that enable GAL4-dependent activation of nearby loci. Adult flies were exposed to hydrogen peroxide, and spontaneous locomotor activity was monitored at high temporal resolution to quantify activity persistence, defined as the duration of activity until sustained cessation under oxidative challenge. A primary screen identified candidate lines, which were then subjected to quantitative reassessment. Most perturbations did not improve activity persistence, whereas only a small subset reproducibly enhanced the phenotype. We identified 26 loci that reproducibly extended activity persistence, 23 of which required GAL4, consistent with activation of nearby loci by GS insertions. The associated loci encompass diverse functional categories, including transcriptional and chromatin regulators, signaling components, metabolic and mitochondrial factors, and structural proteins. These findings indicate that stress-dependent activity persistence is governed by distributed genetic contributions spanning diverse biological functions rather than a single dominant pathway.