*4.2. Polyphenol and Rotenone Treatment*

The treatment plates were prepared with the polyphenol hydroxytyrosol (HT; Sigma-Aldrich, St. Louis, MO, USA or with the aqueous olive pulp extract "HIDROX®" (HD). The HD extract was provided by Oliphenol LLC (Hayward, CA, USA), and total polyphenolic content of the HD extract was declared as 12% [111]. Among the polyphenolics, the major constituent of HD is hydroxytyrosol (40–50%), while other polyphenols present include oleuropein (5–10%), tyrosol (0.3%), and about 20% of other polyphenols including oleuropein aglycone and gallic acid [41]. HD is a freeze-dried powder prepared from the acidic hydrolysis (citric acid 1%) of the aqueous fraction of olives extracted from the defatted olive pulp, a byproduct during the processing of olives (*Olea europaea* L.) for olive oil extraction [41]. Hidrox® is titrated on the total content of olive polyphenols (12%).

HT and HD were dissolved in bidistilled water at 60 mg/mL and the solutions were stored at −20 ◦C. HT or HD, respectively, were added to the bacteria and agar at a final concentration of 100–500 μg/mL.

To trigger the Parkinsonian phenotype, wild type nematodes were exposed to rotenone. A stock solution of 0.5 mg/mL rotenone was prepared in DMSO and added to a final concentration of 10 μM to the control and polyphenol plates. After distribution with a spatula and drying for 24 h in the dark, OP50 (including 10 μM rotenone and the respective polyphenol) was spread on the plates. L4 nematodes were transferred to the rotenone plates until they were used for bioassays.
