Quinine

Perceived food value in experiments 1 and 2 may have been so high as to be close to maximum. This may have resulted in a ceiling effect, preventing additional increases in perceived value due to pheromone trails. To counter this, we reduced food acceptability here by adding small amounts of quinine. We used the same setup and procedure as in experiments 1 and 2. However, instead of presenting a 0.2 M sucrose syrup, we instead used 0.5 M sucrose and decreased its attractiveness by adding quinine (Merck KGaA, Darmstadt, Germany). We piloted the ants’ food acceptance on a serial dilution starting with a 10 mM quinine in 0.5 M sucrose solution and halving the quinine content in each step until we reached a food acceptance of around 50%, meaning that half of the ants interrupted drinking within the first 3 seconds. 50% acceptance was reached in step 8, which corresponded to a 78.1 μM quinine solution. Furthermore, we also added an 8.5 dilution to get closer to a food acceptance of ∼50%, which corresponded to a 58.6 μM quinine solution. We only tested ants on DCM-only and 4 gl/ml pheromone solutions.

DB, SB, PTC, PHE, FFA, quinine, aloin, TA, PGG, BERB, ARG, CuE, C8-AHL, and C12-O-AHL were purchased from MilliporeSigma.

Pharmacological reagents were obtained from Fujifilm Wako Pure Chemical, except for EDTA, HEPES (Dojindo Laboratories), and quinine (MilliporeSigma). quinine and tetrapentylammonium (TPA) were dissolved in DMSO at concentrations of 150 and 100 mM, respectively, as a stock solution.

Hemin chloride, chloroquine, quinacrine, amodiaquine, mefloquine, 8-hydroxyquinoline, quinine, quinidine and the β-carbolines norharman, tryptoline, harman, and harmine were obtained from Sigma-Aldrich. Indazole compounds 1,1´-[2,2´-biphenyldiyl)bismethylene]bis(5-nitro-1H-indazol-3-ol) (DIM-32) and 1,1´-(o-xylylene)bis(5-nitro-1H-indazol-3-ol) (DIM-5) were previously synthetized^{45 (link)}. 3,3′,5,5′-Tetramethylbenzidine (TMB), 2,2′-azinobis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) and tween 20 were obtained from Sigma and cysteine from Merck. DMSO and hydrogen peroxide (H₂O₂) were from Scharlau and Panreac, respectively. Globin was obtained from bovine hemoglobin (Sigma) by precipitation in acetone-0.1% HCl at low temperature^{89 (link),90 (link)}, and lab-stored crystallized and lyophilized bovine serum albumin (BSA) was from Sigma. cysteine proteases: papain from Carica papaya, ficin from fig tree latex and cathepsin B from bovine spleen were obtained from Sigma. The peptide Z-Phe-Arg-AMC was purchased from Bachem, and 7-amino-4-methylcoumarin (AMC) from Sigma.

To assess voluntary drinking, mice were singly housed with access to two 50 mL conical tubes fitted with sipper tops following established procedures^{40 (link),57 (link)}. Briefly, every morning fluid consumption was measured, and bottle placement was switched to account for individual side bias. Mice that displayed a > 30% side/bottle preference during morphine preference were excluded from analysis. Mice were allowed to habituate to both bottles containing only water for 4 days, then water bottles were replaced for sucrose or morphine preference assessment for an additional 4 days. For sucrose preference, water bottles were replaced with bottles containing either 1% sucrose or water. For morphine preference, water bottles were replaced with bottles containing a 0.2% sucrose solution with either 0.05 mg/mL morphine sulfate or 0.01 mg/mL quinine (Sigma, 22640, bitter taste control)^{60 (link),61 (link)}. Drinking was measured to determine solution preference over time. Results are reported as percent preference (solution consumed/total fluid consumed × 100), average preference (4-day average of percent preference), and fluid intake (volume normalized to mouse body weight, 4-day average).

Ninety Six percent ethanol (Synth, Diadema, Brazil) was diluted in filtered water at 20% (w/v); Quinine (Sigma-Aldrich, St. Louis, MO, United States) was dissolved in a 20% ethanol solution in increasing concentrations (0.005, 0.01, 0.025, and 0.05g/L).