Quinine hydrochloride dihydrate

High molecular weight (MW) PVA 197,000 with a degree of hydrolysis of 85–89% and ethanol were obtained from Merck-Sigma Aldrich Company Ltd. (Darmstadt, Germany). PSP was purchased from Cambridge Bioscience Ltd., (Cambridge, UK). The chemical structure of PSP and PVA are presented in Figure 1. Quinine hydrochloride dihydrate, potassium chloride, tartaric acid, absolute ethanol, and hydrochloric acid (32%) were bought from Sigma-Aldrich (Dorset, UK), while distilled water was generated by an ELGA Option 4 Water Purifier (Veolia Water Technologies, High Wycombe, UK).

Quinine hydrochloride dihydrate, chloroquine diphosphate and artesunate (from Artemisia annua) were obtained from Sigma Aldrich (St. Louis, MO, USA). All anti-malaria drugs were diluted in sterile PBS to a concentration of 50 mg/kg in a 200 µL volume and administered intraperitoneally.

All drugs were dissolved in standard fly food at the following concentrations: 400 μM rapamycin (LC Laboratories, Woburn, MA, USA), 10 mM LiCl (Sigma-Aldrich, Burlington, MA, USA), 100 μM mifepristone (Sigma-Aldrich), 10, 30, and 50 nM SB269970 hydrochloride (Cayman Chemical, Ann Arbor, MI, USA), and 100 μM fluoxetine hydrochloride (Prozac, Sigma-Aldrich). For behavioural experiments, we used whatman paper embaptised in 0.1 M quinine hydrochloride dihydrate (Sigma-Aldrich).

The TAS2R agonists chloroquine diphosphate, quinine hydrochloride dihydrate, saccharin sodium hydrate, denatonium benzoate, 1,10-phenanthroline hydrochloride monohydrate, ofloxacin, strychnine hemisulphate, erythromycin, dapsone, carisoprodol, and sodium cromoglycate were obtained from Sigma-Aldrich (Saint-Quentin Fallavier, France) and diphenidol hydrochloride was provided by TCI Europe (Zwijndrecht, Belgium). All products were solubilized and diluted in sterile water, with the exception of erythromycin, dapsone, and carisoprodol, which were solubilized in DMSO and then diluted in water. Antibiotics, DMSO, L-glutamine, trypan blue dye, heat-inactivated fetal calf serum and LPS (from E. coli serotype 0111:B4) were purchased from Sigma (St. Louis, MO, United States). RPMI medium was from Eurobio Biotechnology (Les Ulis, France).

Animals were filled with DiI as described previously (Perkins et al. 1986 (link); Herman and Hedgecock 1990 (link)) and examined under a compound microscope. Five pairs of sensory neurons in the head consistently and robustly dye-fill in wild-type animals; the ASI neurons dye-fill more variably and were excluded from this analysis. Mutants exhibiting weak or no dye-filling in at least one of these five head neuron pairs in ≥50% of animals were considered partially dye-fill defective (pDyf). Animals in which all five head sensory neuron pairs exhibited weak or no dye-filling in 50–80% of animals were considered strongly dye-fill defective (sDyf). Mutants that failed to dye-fill all five pairs of head neurons in 80–100% of animals were considered fully dye-fill defective (Dyf).
Dauer formation assays using ascarosides were performed as previously described (Neal et al. 2013 (link)). Animals were maintained under standard culture conditions at either 20° or 25° for at least three generations prior to being tested for dauer formation at 25°. For dauer formation experiments in the presence of quinine, 3 µL of a 100 mM stock solution of quinine hydrochloride dihydrate (Sigma) was added to the molten agar during the preparation of assay plates.