Brilliant blue fcf

The binary food choice assay was performed as described (30 (link)), with slight modifications. Briefly, 40 to 60 flies (5 to 7 d old) were starved for 24 to 26 h in vials containing two Kimwipes soaked in 6 mL water. The assays were conducted in 72-well microwell mini trays (Thermo Fisher Scientific, 15461158) with the two food alternatives in alternate wells. Both foods contained 0.75% agarose, either red (sulforhodamine B, 0.1 mg/mL; Sigma S9012) or blue (Brilliant Blue FCF, 0.1875 mg/mL; Wako, 027-12842) food dyes and either 2 mM sucrose alone or 2 mM sucrose mixed with a range of HCl concentrations. The foods were loaded into the wells using a repeating pipettor. The flies were anesthetized using CO₂ and introduced into the plates. The assays were conducted in a dark humid chamber at 25 °C for 90 min. Subsequently, the plates were transferred to −20 °C for ≥1 h to kill the flies. The flies were then viewed under a stereomicroscope to determine their food preferences based on the color of their abdomens: red, blue, or purple. Flies that ate both foods would have purple abdomens. We observed robust feeding with 80 to 100% of the flies having colored abdomen across all experiments. Their PIs were calculated using the following formula: $$\text{P}.\text{I}.=\left({\text{N}}_{\text{sugar}+\text{acid}}-{ \text{N}}_{\text{sugar}}\right)/\left({\text{N}}_{\text{sugar}+\text{acid}}+{\text{N}}_{\text{sugar}}+{\text{N}}_{\text{both} \text{foods}}\right).$$

Sucrose (CAS No. 57-50-1, Cat No. S9378), sulforhodamine B (CAS No. 3520-42-1, Cat No. 230162), and cucurbitacin B (CAS No. 6199-67-3, Cat No. PHL82226) were purchased from Sigma-Aldrich (USA). Brilliant blue FCF (CAS No. 3844-45-9, Cat No. 027-12842) was purchased from Wako Pure Chemical Industries (Japan).