Floral odour collections

Floral odour collections were made in cleaned 1 litre amber borosilicate glass bottles (VWR). For the diesel exhaust experiment, bottles were filled with either ambient air or diesel exhaust, collected at 1 l min−1 for 3 min from a diesel generator's exhaust (Suntom SDE 6500 E; Fuzhou). The diesel generator was run using a standard operating protocol of warm-up, engine load and time to first collection; the fuel and the engine oil were consistently purchased from the same supplier. The generator was maintained according to the manufacturers instructions. The concentrations of nitrogen dioxide (NO 2 ), nitric oxide (NO), carbon monoxide (CO) and sulphur dioxide (SO 2 ) produced at the generator's exhaust were measured using a toxic gas probe (TG501+; Graywolf Sensing Solutions). For the NO x experiments, NO x for the 10:1 NO:NO 2 ratio were produced from a commercially purchased gas cylinder (BOC Group) and for the 1:1 NO:NO 2 ratio NO x were produced by reducing nitric acid with elemental copper. Concentrations of 10 ppm, 1 ppm, 0.1 ppm per bottle were achieved by using gas tight syringes and volumetric calculations. Bottles were sealed with 2 layers of Parafilm (Pechiney Plastic Packaging Company) and a GL45 cap (VWR). One microliter of the synthetic odour blend (Supplementary Table 1), applied to a 2.1 cm diameter filter paper (Grade 3 M), was placed into the glass bottle along with a stir bar (operated at 300 rpm to mix air). After 1, 30, 60 and 120 min (only after 30 min for NO x experiments) of mixing a solid-phase microextraction fibre (SPME, blue fibre 65 μm PDMS-DVB; Supelco) was inserted into the bottle through a 1 mm bore hole in the cap, for a 5 min exposure/adsorption period. For the diesel experiment, the process was repeated 5 times for both ambient air and diesel exhaust. For the NO x experiments, the process was repeated 4 times for ambient air and 4 times for each NO:NO 2 ratio and concentration.

Floral odour analysis

Chemicals were thermally desorped from the SPME fibres in the injector (250°C) of a Hewlett-Packard 6890 gas chromatograph, coupled to a 5972 mass spectrometer. The carrier gas was helium (1 ml min−1) and the injector was operated in a split mode (10:1). The capillary column was an HP-INNOWAX (30 m, 0.25 mm i.d., 0.25 mm film; Agilent Technologies). The oven temperature was held at 50°C for 2 min and then increased at 5°C min−1 to 70°C and then at 10°C min−1 to 240°C. The mass spectrometer (250°C) scanned from mass 350 to 40 at a rate of 2.43 times s−1 and data were captured and analysed by Enhanced Chemstation software (v. B.01.00; Agilent Technologies). The data for each chemical at each time point (or each NO x concentration and ratio) were examined for the normality of their distributions using a series of Shapiro-Wilk tests and normal Q-Q plots. For those time points that were normally distributed, a series of unpaired two-tailed t-tests (SPSS v.19; IBM) were used to compare the mean abundances of each floral chemical between ambient air and diesel exhaust treatments. For each time point equal variances were assumed, unless Levene's tests demonstrated that variances were not equal. For those time points that were not normally distributed two-tailed Mann-Whitney U tests were performed (Supplementary Table 3).

Proboscis extension reflex (PER)

Honeybees, Apis mellifera, were from colonies kept at the University apiary (50° 56′ 10″N, 1° 23′ 39″W). For each assay, 30 returning forager honeybees (identified by full pollen baskets) were collected in individual plastic tubes between 14.00–16.00 BST. Honeybees were immobilized at 4°C, harnessed in 1 ml pipette tips30, fed to satiation with 30% sucrose solution and kept at 20°C. The morning after collection, honeybees were randomly assigned into groups of 7–10 individuals. Each honeybee was trained to associatively learn the synthetic odour blend. A harnessed honeybee was placed in a well-ventilated chamber in front of a flow of ambient air. After 10 s the honeybee was exposed to odours from a glass tube containing a 2.1 cm diameter filter paper impregnated with 8 μl of the synthetic blend, after a further 10 s the air flow was switched back to ambient. Five seconds into the odour stimulus the honeybees' antennae were touched with 30% sucrose solution and honeybees were allowed to feed for 10 s. Each honeybee underwent 6 exposures with 10 min intervals between each exposure. Honeybees which extended their proboscis (Fig. 2c) in response to the odour stimuli on the 6th exposure were considered to have learnt the blend and were used in recognition trials. In the recognition trials the groups of honeybees were tested to one of four odours, either the synthetic blend or a blend where α-farnesene, α-terpinene or both chemicals were omitted. Recognition mirrored the conditioning trials, with the omission of sucrose. Extension of the proboscis within 10 s in response to the onset of the odour stimulus was classified as a positive recognition. Responses to each of the three manipulated blends are expressed as the per cent PER recognition of each blend relative to the per cent PER recognition of the full synthetic blend. A X2 test was used to compare the total numbers of honeybees recognizing each odour between all four odour groups, combined with a z-test to perform pairwise comparisons between odour groups (SPSS v.19; IBM).