Design and implementation of an automated Drosophila locomotor assay using computer vision tracking

Drosophila has long served as a powerful model for investigating locomotor behavior, using geotaxis assays to generate valuable insights into genetics, aging, and neurobiology. Nonetheless, its use has been constrained by subjective scoring, modest throughput, and challenges in reproducibility. We developed and validated an integrated hardware–software platform that enables automated, high-resolution locomotor analysis across 12 vials in parallel.

The system integrates 3D-printed mechanical components, Raspberry Pi–based video acquisition, and programmable environmental controls to ensure standardized conditions. A deep learning pipeline segments vials with near-perfect accuracy (IoU > 0.95), while computer vision algorithms quantify climbing trajectories, velocity, and positional zone occupancy at 60 frames per second. The end-to-end workflow converts raw video into time-resolved metrics, supports sex-specific aggregation, and incorporates advanced statistical analyses, including Linear Mixed Effects regression, harmonic mean p-values, and Mann–Whitney U tests.

Relative to manual scoring, this automated pipeline yields 2.8-fold faster processing and about 800-fold higher data density. Application of the platform uncovered reproducible phenotypes of multiple genotypes. For example, in circadian Clockout mutation, males displayed progressive climbing deficits with age, whereas females maintained age-resilient trajectories.

Moreover, male Clockout exhibited a reduced locomotor performance compared to age-matched control (w1118) males; however, female Clockout showed subtle reduction in locomotor performance. Additionally, glial-specific knockdown of PolG, encoding the DNA polymerase gamma catalytic subunit, revealed striking sex-dimorphic aging patterns: females outperformed controls at older age in Glaz-driven line and at younger age in Elav-driven line, while males exhibited a marked decline. To promote broad adoption, a user-friendly Python interface (Tkinter GUI) enables accessibility independent of computational expertise.

Collectively, this standardized, high-throughput framework advances the resolution of genotype-, age, and sex-dependent locomotor dynamics, offering new opportunities in aging, circadian biology, and neurodegeneration research. We are grateful for access to the University of Alabama at Birmingham’s High-Performance Computing (Cheaha) resources, ensuring efficient processing of large-scale video datasets. Research reported in this publication was supported by the National Institutes of Health (NIH) grants AG065992 and RF1NS133378 to G.C.M.

Department of Pathology, Division of Molecular and Cellular Pathology, Heersink School of Medicine, University of Alabama at Birmingham, Birmingham, AL, 35294, USA Dave Melkani, Neelaksh Harnwal, Shubhankar Desai, Dev Patel & Girish C. Melkani UAB Nathan Shock Center, Birmingham, AL, 35294, USA The authors declare no competing interests. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.