1-hexanol

Our work was designed to obtain a generalisation matrix with 16 different odours. Ideally, after conditioning each of the 16 odours as CS, the response to each odour (including the CS) should be measured (i.e., 16 × 16 = 256 cells). However, testing 16 odours implies presenting them without reward, a situation that may result in extinction of the learned response due to the repeated unrewarded odour presentations. Preliminary experiments were performed in which four groups of 180 bees were trained along three trials to 1-hexanol, 2-octanol, linalool, and limonene, respectively. Training was followed by tests with the four different odours, including the conditioned one. These experiments showed that after three conditioning trials, the response of the bees to the CS in the four tests remained at the same level, independently of the order of occurrence of the CS such that it was not influenced by extinction. We thus kept this protocol for the 16 × 16 matrix. Each of the 2,048 bees used in this study was thus subjected to three conditioning trials with their respective CS, and to four test trials, each with a different odour chosen among the 16 possible odours. Intertrial intervals of 10 min were used throughout. A randomisation schedule (detailed below) was developed for the test phase to reduce any possible day- and odour-combination effects.

Aldehydes, acetal, methanol, esters and higher alcohols (also known as fusel alcohols) were quantified by GC-FID. Twenty-one compounds were determined following two different procedures. In both cases, the equipment used was an Agilent 7890B Gas Chromatograph (Agilent Technologies, Santa Clara, CA, USA) coupled with Flame Ionization Detector.
For the analysis of acetaldehyde, acetaldehyde—diethyl acetal, methanol, ethyl acetate, n-propyl alcohol, 2-butyl alcohol, isobutyl alcohol, n-butyl alcohol, 2-methyl-1-butanol and 3-methyl-1-butanol, the samples were injected in a split mode (split 1:46, 250 °C) into a DB-624 (30 m × 250 µm × 1.4 µm, Agilent Technologies, Santa Clara, CA, USA) column. The oven temperature for the analysis was programmed as follows: 30 °C (30 min), then 6 °C/min to 100 °C (0 min). Temperatures of the injector and the detector were 250 °C and 300 °C, respectively. Nitrogen was used as a carrier at flow of 1.0 mL/min. Data acquisition and analyses were performed using OpenLAB CDS Chemstation (Agilent Technologies, Santa Clara, CA, USA) software.
For the analysis of n-hexanol, 2-phenylethyl alcohol, ethyl lactate, ethyl succinate, ethyl caproate, ethyl caprylate, ethyl caprate, ethyl laureate, ethyl myristate and ethyl palmitate, samples were injected in a splitless mode (1 min, 250 °C) onto CP-WAX 57 CB (25 m × 250 µm × 0.2 µm, Agilent Technologies, Santa Clara, CA, USA) column. The oven temperature program during analysis was as follows: 45 °C (20 min), then 3 °C/min to 170 °C (20 min). Temperatures of the injector and the detector were 250 °C and 300 °C respectively. Nitrogen was used as the carrier gas at a flow of 1.3 mL/min. The data acquisition and analyses were performed using OpenLAB CDS Chemstation (Agilent Technologies, Santa Clara, CA, USA) software.
Standards were made in an ethanol/ultrapure water solution at 40%vol. The linear standard curve of 3-methyl-1-butanol ranges from 1 to 250 mg/100 mL of 100% vol. alcohol. The linear standard curve of methanol, ethyl acetate, n-propyl alcohol, isobutyl alcohol and 2-methyl-1-butanol ranges from 1 to 100 mg/100 mL of 100% vol. alcohol. The linear standard curve of acetaldehyde and acetaldehyde—diethyl acetal ranges from 1 to 50 mg/100 mL of 100% vol. alcohol. The linear standard curve of ethyl lactate ranges from 0.5 to 25 mg/100 mL of 100% vol. alcohol. The linear standard curve of 2-butyl alcohol, n-butyl alcohol, n-hexanol, 2-phenylethyl alcohol, ethyl succinate, ethyl caproate, ethyl caprylate, ethyl caprate, ethyl laureate, ethyl myristate, and ethyl palmitate ranges from 0.1 to 5 mg/100 mL of 100% vol. alcohol. The samples were diluted at 40%vol. with ultrapure water and injected in duplicate. The results were expressed in mg of compound per 100 mL of 100% vol. alcohol.