*4.5. Swim Behaviour Assay*

The study of locomotion was realized with a swim assay according to Restif et al. [61] and Ibáñez–Ventoso et al. [124]. Wells with a depth of 0.5 mm and Ø 10 mm were created with two self-adhesive marking films for microscope slides and filled with M9 buffer. Five to 10 nematodes were transferred per well and covered by a cover slip to facilitate visualization and recorded with a connected camera. Nematodes with a ruptured vulva phenotype were excluded from analysis. The analysis was conducted with wild type nematodes at the 3rd, 7th, and 12th day of adulthood. Furthermore, wild type rotenone-treated worms and the strains OW13 and UA44 were analysed at the 3rd and 7th day of adulthood. Each video was converted into single frames and processed with Photoshop (version 19.1.7; Adobe Inc., San José, CA, USA) to meet the required settings. Thereafter, pictures were analysed with the CeleST software (version 3.1; distributed by GitHub Pages, https://github.com/DCS-LCSR/CeleST, accessed on 08 April 2020) as described by Restif et al. [61] and Ibáñez–Ventoso, et al. [124]. The thrashing rate, the body wave number and the activity index were evaluated as representative parameters of motor activity.
