Cuticular Chemical Profiling of Bee Workers

We analysed the cuticular chemical profiles of workers because of its key role in intracolonial communication [51 (link)]. Bees from batch B2 (n = 289, 29 colonies) were thawed at room temperature for 4 min and rinsed in 1 ml n-pentane (SupraSolv, 99.9%, Supelco) for 1 min to extract cuticular chemical compounds. The extracts were concentrated to a final volume of 500 µl under a gentle nitrogen stream. We then added 10 µl of an internal standard (dodecane; 99%, Sigma, Germany, 100 µg ml⁻¹ in n-pentane) for quantitative analysis. Samples were stored at −20°C. Chemical analyses were performed by means of a gas chromatograph (Agilent 7890A, Agilent Technologies, Waldbronn, Germany) with a DB-5 capillary column (30 m × 0.25 mm inner diameter, J&W) and a flame ionization detector. Hydrogen was used as a carrier gas at a constant flow of 2.0 ml min⁻¹. One microlitre of extract was injected splitless into the gas chromatograph at an injector port temperature of 310°C. After an initial time of 1 min at 50°C, the splitter was opened and the oven temperature increased continuously by 10°C min⁻¹ to a final temperature of 310°C at which temperature was held for 35 min, resulting in a total run time of 62 min. Compounds were identified based on analyses of reference substances and earlier studies [52 (link),61 (link)].