Longevity as a quality control metric for male Aedes aegypti (Diptera: Culicidae): does test design matter?

In mosquito Sterile Insect Technique (SIT) programs, adult male longevity is widely used as a quality control (QC) indicator for process monitoring and post-treatment assessment. However, longevity assays are frequently implemented using non-standardized protocols, limiting their comparability and operational interpretation. In this study, we evaluated whether and how key assay design parameters influence measured longevity and the ability of longevity-based tests to detect high dose irradiation related quality effects in male Aedes aegypti (Linnaeus, 1762). Using a factorial cage-based design, we assessed the effects of adult holding density, female presence, and observation period on male survival under standard holding conditions. Increasing density reduced male survival, primarily reflecting an assay-related stress effect. Irradiation consistently reduced longevity across all experimental conditions, while female presence imposed additional survival costs under some conditions. Combined high-density and mixed-sex environments produced the lowest overall survival but did not disproportionately amplify irradiation related effects. We also evaluated a rapid acute-stress assay conducted without access to sugar or water. Treatment-related differences were detectable during the early stages of the assay but diminished as mortality approached saturation, demonstrating that rapid stress assays are highly dependent on the timing of assessment. Together, these findings demonstrate that longevity-based QC indicators can be strongly influenced by experimental design and must be applied under standardized and purpose-specific conditions. Rapid stress assays can support routine SIT QC as targeted screening tools, but they require prior calibration and should not be considered universal proxies of overall male quality.