A high-throughput assay quantifies thermal scaling of Drosophila development with minute-scale precision

Abstract Precise regulation of developmental timing is essential for coordinated growth and robust development, yet staging remains technically challenging in many model systems. In Drosophila melanogaster, developmental timing has traditionally been assessed using low-throughput or coarse staging methods, limiting insight into how individual larval stages respond to environmental and genetic perturbations. Here, we present a high-throughput, real-time luminometry assay that enables continuous, automated measurement of postembryonic development in individual Drosophila larvae. By monitoring feeding-dependent luciferase activity, this method reliably detects transitions between larval instars and molts of individual larvae with minute-scale temporal resolution. Using this platform, we provide a quantitative, stage-resolved description of larval development across large cohorts, revealing distinct patterns of variability and weak temporal coupling between stages. We next examine how temperature shapes developmental timing across a broad thermal range. Increasing temperature uniformly accelerates larval development while preserving the proportional contribution of each stage, indicating that the temporal architecture of development is maintained as overall pace changes. Developmental rate follows predictable thermodynamic scaling within a defined temperature window, enabling precise estimation of thermal parameters at both whole-organism and stage-specific levels. Finally, we demonstrate that the assay is compatible with genetic perturbations and uncover a previously unrecognized intergenerational effect of parental temperature on offspring developmental timing. This work establishes a scalable, high-precision framework for measuring postembryonic development in Drosophila melanogaster. By revealing the modular and coordinated nature of larval growth, it enables systematic dissection of developmental timing and provides a platform to explore fundamental principles of developmental robustness across species.