I8898

Haemonchus contortus eggs were incubated for 24 h to obtain L1. Solutions consisting of 1 mL of L1 (~250 larvae) and 1 mL of treatment solution diluted with 3% Tween 80 and distilled water were incubated with GIN-free sheep feces for 6 days at room temperature (27 °C) [21 (link)]. LEV concentrations (T1512—Sigma-Aldrich^®) ranged from 0.08 to 0.005 mg/mL, IVM concentrations (I8898—Sigma-Aldrich^®) ranged from 0.05 to 0.0003 mg/mL, and CA from 2 at 0.125 mg/mL. The combination CA/LEV was evaluated at concentrations of 0.014 mg/mL LEV + 0.383 mg/mL AC to 8 × 10⁻⁴ mg/mL LEV + 0.023 mg/mL AC; for the combination CA/IVM, the concentrations were from 8.4 × 10⁻⁴ mg/mL IVM + 0.383 mg/mL CA to 5 × 10⁻⁵ mg/mL IVM + 0.023 mg/mL CA. In the negative control, 3% Tween 80 was used. L3 were retrieved according to the technique described by [22 (link)] and counted under the microscope. Three replicates were performed with five repetitions for each treatment and the control.

All EPG recordings were made in M9 buffer (Stiernagle, 2006 ), to which drugs, solvents or bacteria were added. Stocks of 5-Hydroxytryptamine creatinine sulfate complex (5HT, Sigma-Aldrich H7752; St. Louis, MO) were prepared in M9 at 40 mM, stored at −20 °C and diluted to desired concentrations in M9. Chip perfusion was initiated within 70 min of preparing a 5HT solution. Ivermectin (IVM; Sigma-Aldrich I8898) stocks (5 mM) were prepared in 100% DMSO and stored at −20 °C. The highest concentrations of DMSO that did not perturb EPG activity in control experiments were 0.2% (Fisher D-136) or 0.5% (Sigma-Aldrich Hybri-Max D2650) (data not shown) so these were the highest concentrations used in working solutions. Levamisole hydrochloride (LEV; Sigma 31,742) stocks (100 mM) were prepared in dH₂0 and stored at 4 °C until dilution on the day of use. Piperazine hexahydrate (PPZ; Sigma P7003) stocks (1M) were prepared in dH₂0 and stored at 4 °C until use. The pH of PPZ solutions was adjusted to 7.0 using HEPES buffer in M9 and titrating with 12 N HCl. Working solutions of all anthelmintic drugs were prepared from stocks and used on the same day. In some initial experiments, 0.005% Fast Green (Fisher F-99) was added to solutions to confirm uninterrupted perfusate flow (Lockery et al., 2012 (link)).