Salinomycin

All compounds were purchased from Sigma-Aldrich and included: gambogic acid, sodium salinomycin, ethinyl estradiol, fluoxetidine hydrochloride, bepridil, ciclopirox, miconazole nitrate, chlorpromazine hydrochloride, amphotericin b, niclosamide, rescinnamine, flucytosine, vinblastine, carbidopa, praziquantel and auranofin.
Stock solutions of all compounds were made up at 1 mM in appropriate solvents (Dataset S1) and stored at −80°C. All compounds were added to black-sided, flat-bottom (optically clear), 96-well microtiter plates containing schistosomula (1000 parasites/well in triplicate) at 10 µM concentrations. Schistosomula were cultured (as already indicated; 37°C, 5% CO₂) in the presence of each compound for 24 hr before viability levels were assessed.

The concentration of salinomycin in the muscle was assessed through high-performance liquid chromatography HPLC-MS/MS analysis, using Agilent 1260 infinity (Agilent, Santa Clara, CA) and a Thermo LTQ XL (Waltham, MA) linear ion trap mass spectrometer. Nitrogen was used for sheath gas and aux gas. 5 µL of samples were injected into Kinetex^® 2.6 μm Polar C₁₈ 100 Å LC Column (100 × 2.1 mm) from Phenomenex (Torrance, CA) with a C₁₈ resin guard column. The column temperature was set to 30 ℃. Two LC mobile phase solvents (A) 0.1% formic acid solution and (B) 0.1% formic acid acetonitrile were used. The flow rate was 0.3 mL/min, and the total analysis time per sample was 15 min. Solvent composition initially started from (B) 70% and gradually changed to (B) 100% over 8 min. After 3 min of isocratic flow of (B) 100%, solvent composition decreased to (B) 70% in 0.5 min and was eluted with the initial solvent composition for 4.5 min for re-equilibrium. Mass spectrometer acquisition was achieved by selected reaction monitoring (SRM) mode in positive ESI mode with collision energy 58 and 46 (eV). The desolvation gas temperature was adjusted to 320 ℃, and the desolvation gas was Helium.

All experimental protocols were performed following the guidelines of the Institutional Animal Care and Use Committee of Gyeongsang National University (approval numbers: GNU-220,519-E0051). 300 olive flounder (Paralichths olivaceus), comprising both male and female fish, with an average weight of 70 g were obtained from an aquaculture plant, Yeonhwa susan in Tongyeong, Republic of Korea. Each fish was weighed using a balance. After being put in a 1000 L tank, each fish was allowed a week to adjust. The fish were divided into three groups, designated PO-1, PO-2, and PO-3, at random and each group contains 100 fish. While the water temperature for PO-3 group was maintained at 13.3 ℃, it was retained at 22.3 ℃ for the PO-1 and PO-2 groups. For drug administration, fish was individually weighed and the mixture was administered directly into its gastral cavity using the syringe coupled with an oral zonde; oral doses of 5 mg/kg twice daily for groups PO-1 and PO-3, and oral doses of 10 mg/kg twice daily for group PO-2. To treat fish with the exact dose of salinomycin according to the body weight, we opted for oral administration rather than the more widely used commercial form of therapeutic administration (i.e., dietary exposure). Fish were excluded from the experiment if they regurgitated three minutes after being administered.
Similar to this, 300 black rockfish (Sebastes schleglii), male and female combined, with a mean weight of 28.85 g, were bought from an aquaculture plant, Yeonhwa susan in Tongyeong, Republic of Korea. Before administering the medication, all fish spent a week acclimating to their surroundings in a 1500 L tank. Three treatment groups of 100 fish each were randomly assigned the labels SS-1, SS-2, and SS-3. The water temperature for the SS-1 group was fixed at 23 °C, and 5 mg/kg was given orally once per day using zonde. The SS-2 group received 10 mg/kg of oral medication at a water temperature of 23 °C, whereas the SS-3 group received the same treatment at a water temperature of 13 °C. Each group of 15 olive flounder and black rockfish had muscle samples taken on the first, third, seventh, fourteenth, and twenty-eighth days following the last dose. All experimental fish were humanely euthanized using tricaine (TCI, japan) at a concentration of 250 mg/L prior to sampling to ensure a painless procedure. In order to preserve the materials for later analysis, they were frozen and kept at a temperature of -80 °C.