Fipronil

All samples were prepared according to Raju et al. (2016 (link)) and previously described in Contador-Kelsall et al. (2022 (link)). An Agilent 6490 Triple Quad Liquid Chromatography – mass spectrometry (LCMS) was used to analyse all samples. fipronil parent and metabolite compounds (fipronil, fipronil desulfinyl, fipronil sulfide, and fipronil sulfone) standards were purchased from Sigma-Aldrich. The R² for each calibration curve was above 0.996. All samples were run in negative mode and clearly defined, confirmed by their most abundant product ions at optimised collision energies. To ensure there was no contamination throughout, a positive control (0.01 μg/mL fipronil) and a negative control (acetonitrile) were run every five samples.

Fipronil (5-amino-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-[(trifluoromethyl)sulfinyl]- 1H-pyrazole-3-carbonitrile) (FPN) was provided by Sigma Aldrich, Dusseldorf Germany. All other reagents used were from available commercial sources.

Picrotoxinin (PTX), fipronil, bicuculline, propofol, salicylidene salicylhydrazide, zinc chloride, GABA, dexamethasone, zeocin, and geneticin were purchased from Sigma Aldrich (St. Louis, MO, United States). Diazepam, allopregnanolone, and DS2 (4-chloro-N-[2-(2-thienyl)imidazo[1,2-a]pyridin-3-yl]benzamide) were purchased from Tocris Bioscience (Bristol, United Kingdom). TETS was synthesized in the laboratory of Dr. Bruce Hammock, University of California, Davis, CA (Zhao et al. 2014 (link)). 10 mM stocks of GABA were made fresh daily using Ringer solution (see below for composition). 10 mM stocks of PTX and TETS were prepared in DMSO and diluted down into Ringer solution immediately before application onto the cell. Both TETS and PTX waste were treated with nitric acid and disposed of using the waste accumulation program at UC Davis.

The IRE/CTVM19 cell line is derived from the embryonic stage of I. ricinus [22 (link)]. Cells were seeded in flat-sided culture tubes (Nunc™, Thermo Scientific, Milan, Italy) and maintained at 28 °C in Leibovitz’s L-15 medium (Life Technologies, Milan, Italy) supplemented with 10% tryptose phosphate broth, 20% fetal bovine serum, 2mM L glutamine, penicillin (100 U/ml) and streptomycin (100 μg/ml) (Life Technologies) as described previously [9 ]. The medium was replaced weekly and cells were passaged at least every 15 days. Cells derived from cultures of the same passage level were centrifuged, re-suspended in fresh complete medium to a concentration of 1 × 10⁶ cells/ml and seeded into new tubes (2 ml cell suspension per tube). Cultures were treated with a range of concentrations of analytical standard amitraz (Sigma-Aldrich, Milan, Italy), fipronil (Sigma-Aldrich) or permethrin (Sigma- Aldrich). Drugs were dissolved in dimethyl sulfoxide (DMSO) and then diluted in complete culture medium to final concentrations of 25, 50, 100 and 150 μM, maintaining the DMSO concentration at 0.5%. Control samples were treated with 0.5% DMSO only. Cultures were maintained for 10 days and medium was changed once on day 7. Experiments were carried out with four replicates per treatment protocol.

Glycine, GABA, strychnine, picrotoxin, fipronil, lindane, zinc chloride and bicuculline were obtained from Sigma. Stock solutions of Glycine (1 M), GABA (1 M) and zinc chloride (1 M) were prepared in water, strychnine (10 mM), picrotoxin (100 mM), fipronil (30 mM), lindane (30 mM) and bicuculline (100 mM) were dissolved in dimethylsulphoxide (DMSO). All stocks were frozen at −20°C. From these stocks, solutions for experiments were prepared on the day of recording.