AD 20

To synthesize cDNA, we transcribed a standardized quantity of 1 µg RNA per sample using a random hexamer primer (0.1 nmol, 1 µl, SO142, Life Technologies GmbH, Darmstadt, Germany), an oligo-dT18 primer (0.1 nmol, 1 µl, SO131, Life Technologies), 5 × M-MLV-buffer (4 µl, M1705, Promega, Fitchburg, WI, USA) and dNTP mix (40 nmol, 1 µl–10 nmol/dNTP, Roti^®-Mix PCR3, L785.2, Carl-Roth GmbH) ad 20 µl nuclease-free H₂O (T143, Carl-Roth GmbH). After incubation (3 min, 70 °C) the mixture was quickly cooled on ice. We then added reverse transcriptase (200 U, 1 µl, M1705, Promega) and an RNase inhibitor (40 U, 1 µl, EO0381, Life Technologies), continued incubation (37 °C, 60 min) and heat-inactivated the reverse transcriptase (95 °C, 2 min). To minimize experimental variations, synthesis of cDNA, which was stored at −20 °C until use, was performed concurrently for all samples.

Colonies of bumblebees (Bombus terrestris audax) were obtained from Agralan, (Swindon, United Kingdom) or Koppert Biological Systems Nederland (Berkel en Rodenrijs, the Netherlands). Bees were housed in 30.0 × 14.0 × 16.0 cm bipartite wooden nest boxes, and all individuals were marked with numbered Opalith tags for individual identification during transfer to these nest boxes. This involved trapping each bee in a small cage, gently pressing it against the mesh with a sponge, and affixing the tag to the dorsal thorax with a small amount of glue. The nest boxes were connected to flight arenas (66.0 × 60.0 × 30.0 cm or 132.0 × 60.0 × 30.0 cm for single- and multiple-demonstrator experiments, respectively) via 26.0 × 3.5 × 3.5 cm clear acrylic tunnels, which could be blocked to limit access to the flight arena. Bees were allowed to forage ad libitum on 20% w/w sucrose solution provided in mass feeders in these arenas overnight, and pollen was provided every 2 days. Colonies were maintained at standardised room temperature throughout the study, and experiments were conducted under standardised artificial lights (12:12, high-frequency fluorescent lighting; TMS 24F lamps with HF-B 236 TLD [4.3 kHz] ballasts [Koninklijke Phillips NV, Amsterdam, the Netherlands], fitted with Activa daylight fluorescent tubes [OSRAM Licht AG, Munich, Germany]).

The concentration of adenine nucleotides (ATP, ADP, AMP) and adenosine (Ado) released into the culture medium were determined using HPLC. The medium (400 µl) was transferred into a cold Eppendorf tube containing 50 µl of 0.1 m perchloric acid and 10 μm theophylline (as an internal standard). The medium was centrifuged (at 3510 × g for 10 min at 0°C-4°C), and the supernatant was kept at −20°C until analysis. Online solid phase extraction coupled to the column-switching technique was applied for quantification of nucleotide content in the samples. HPLC separation was performed by a Shimadzu LC-20 AD Analytical System using UV (Agilent 1100 VW, set at 253 nm) detection. A phenyl-hexyl packed (7.5 × 2.1 mm) column was used for online sample enrichment, and separation was completed by coupling to an analytical C-18 (150 × 2.1 mm) column. The flow rates of the mobile phases [Phase A: 10 mm potassium phosphate buffer with 0.25 mm EDTA; Phase B: additional components, such as 0.45 mm octane sulfonyl acid sodium salt, 8% acetonitrile (v/v), 2% methanol (v/v), pH 5.55] were 350 and 450 µl/min, and they were applied in a step gradient (Baranyi et al., 2006 (link)). The sample enrichment flow rate of buffer A was 300 µl/min for 4 min, and the total run time was 55 min. Concentrations were calculated by an internal standard 2 point calibration curve method [(A_i × f × B)/(C × D_i), where A_i is the area of nucleotide components, B is the sample volume, C is the injection volume, D_i is the response factor for 1 pmol nucleotide standard, and f is the international standard factor (IS area in the calibration/IS area in the actual sample)]. The data are expressed as pmol per ml.

Chemicals such as sodium hydroxide (NaOH), 3-aminopropyl-trimethoxysilane (APTMS), and dimethyl sulfoxide (DMSO) were obtained from the Chengdu Kelong chemical reagent factory (Chengdu, China). Monoamine oxidase B (MAO-B, 100.23 U/mL) was prepared in-house [12 (link)]. Kynuramine dihydrobromide was purchased from Sigma-Aldrich (St Louis, MO, USA). Safinamide mesylate and rasagiline were purchased from Meilunbio (Dalian, China). The MAO-B inhibition assay was carried out using Thermo Scientific Varioskan Flash equipped with a 96-well microplate (Thermo, Waltham, MA, USA). FT-IR spectra were recorded in KBr with a PerkinElmer FT-IR spectroscope (PerkinElmer, Waltham, MA, USA). Ultrapure water produced with a UP water purification (18.25 MΩ) system (Ultrapure, Chengdu, China) was used for HPLC. HPLC-grade methanol was obtained from JT Baker (Phillipsburg, NJ, USA). The HPLC system consisted of a Shimadzu LC-20 AD series equipped with a thermostatic column compartment, an SPD20A UV-vis detector (Shimadzu, Kyoto, Japan), and an Agilent ZORBAX SB-C18 column (4.6 × 250 mm, 5 μm). The mobile phase was composed of solvent A (0.1%, v/v, formic acid/H₂O) and solvent B (100% MeOH) at a flow rate of 0.8 mL/min, and an injection volume of 20 µL. The eluting gradient was set to 30–100% MeOH for 0–30 min. The MS detection was in the positive ion mode using a capillary voltage of 3.0 kV; a source temperature of 180 °C; a desolvation temperature of 350 °C; and a desolvation gas flow of 800 L/h, while the nebulizer was set at 0.8 Bar and the sample flow rate was set at 0.3 mL/min for the ESI-MS (Bruker Compass Data Analysis 4.0; microTOF-Q11-10203). For the FT-IR, the samples were pretreated with a tablet press using potassium bromide (KBr) in a dry environment, and the wavenumbers of the FT-IR measurement were set in the range of 450 to 4500 cm⁻¹.