Behavioral toxicity of polyhydroxyalkanoate microplastics in Drosophila melanogaster larvae revealed by deep-learning pose estimation

Polyhydroxyalkanoates (PHAs) are microbially synthesized polyesters developed as biodegradable alternatives to conventional plastics, but incomplete degradation can leave PHA-derived particles in the environment, and their biological effects remain poorly characterized. This study investigated whether 96-h dietary exposure to 5-µm PHA particles altered locomotor organization in third-instar Drosophila melanogaster larvae. Larvae were exposed to 0.25, 2.5, or 7.5 mg L⁻¹ PHA, and movement in the standardized 5-65 s window was quantified using SLEAP tracking, speed and angular-speed interval distributions, and nine spatial trajectory features. PHA exposure altered larval locomotion, with the broadest response at 7.5 mg L⁻¹. Relative to the control, this treatment reduced mean crawling speed by 37.8% and distance travelled by 40.7%, increased low-speed occupancy, decreased medium- and high-speed occupancy, and reduced displacement, straightness, convex-hull perimeter and area, and radius of gyration. Angular-speed occupancy shifted from the low category toward the medium and high categories, whereas mean angular speed did not differ significantly among groups. Changes in the low-speed and low-angular-speed proportions were also detected at 0.25 mg L⁻¹. These coordinated changes identify a sublethal alteration of locomotor organization under the tested conditions. Locomotion supports resource acquisition, habitat use, dispersal, and escape; the observed response constitutes an ecologically relevant effect in a terrestrial insect. The findings show that biodegradable-polymer particles can elicit sublethal effects in terrestrial insects and warrant inclusion in ecological hazard assessment.