Insects
Canton-S, Zimbabwe (S-29; Bloomington #60741) and Dalby-HL (Dalby, Sweden) [65] strains of D. melanogaster were used as wild-type flies for behavioural experiments. Canton-S was used for comparison with knockouts of the same background. Further tests were performed with the sister species D. simulans.
We used the Or69a-Gal4/UAS TeTx, tetanus toxin knockout line to verify the role of Or69a in flight attraction to Z4-11Al. Canton-S/UAS TeTx (Bloomington #28838 and 28997) and Canton-S/Or69a-Gal4 (Bloomington #10000) were used as parental controls.
Flies were reared on a standard sugar-yeast-cornmeal diet at room temperature (19–22 °C) under a 16:8-h light:dark photoperiod. Newly emerged flies were anesthetized under CO2 and sexed under a dissecting microscope. Virgin flies were identified by the presence of meconium, and were kept together with flies of the same sex. Flies were kept in 30-mL Plexiglas vials with fresh food. Experiments were performed with 3- to 5-day-old flies.
Chemicals
Z4-11Al and (E)-4-undecenal were synthesized (see below). Commercially available compounds were (R)-carvone (97% chemical purity, CAS #6485-40-1, Firmenich), (S)-carvone (98%, CAS #2244-16-8, Firmenich), (S)-terpineol (97%, CAS #10482-56-1, Aldrich), (S)-linalool (97%, CAS #126–91–0, Firmenich), (R)-linalool (97%, CAS #126-90-9, Firmenich), citronellol (99%, CAS #106-22-9, Aldrich), geraniol (98%, CAS #106-24-1, Aldrich), 3-octanol (99%, CAS #589-98-0, Aldrich), decanol (99%, CAS #112-30-1, Fluka), and undecanal (99%, CAS #112-44-7, Aldrich).
Chemical synthesis
Dry THF and dry Et2O were obtained from a solvent purification system (Activated alumina columns, Pure Solv PS-MD-5, Innovative technology, Newburyport, USA) and used in the reactions when dry conditions were needed. All other chemicals were used without purification. Reactions were performed under an Argon atmosphere unless otherwise stated. Flash chromatography was performed on straight-phase silica gel (Merck 60, 230–400 mesh, 0.040–0.063 mm, 10–50 g/g of product mixture) employing a gradient technique with an increasing concentration (0–100%) of distilled ethyl acetate in distilled cyclohexane. In cases of very polar products, chromatography was continued with ethanol in ethyl acetate (0–20%). Thin-layer chromatography was performed to monitor the progress of the reaction on silica gel plates (Merck 60, pre-coated aluminium foil), using ethyl acetate (40%) in cyclohexane as an eluent, and plates were developed by means of spraying with vanillin in sulfuric acid and heating at 120 °C. The purity of the product was checked with GC analysis on a Varian 3300 GC instrument equipped with a flame ionization detector (FID) using a capillary column Equity-5 (30 m × 0.25 mm id, df = 0.25 μm, with nitrogen (15 psi) as the carrier gas and a split ratio of 1:20). The oven temperature was programmed at 50 °C for 5 min followed by a gradual increase of 10 °C/min to reach a final temperature of 300 °C. An Agilent 7890 GC equipped with a polar capillary column FactorFOUR vf-23 ms (30 m × 0.25 mm i.d., df = 0.25 μm) was coupled to an Agilent 240 ion-trap MS detector for separation of some isomeric intermediates. The injector was operated in split mode (1:20) at 275 °C and a helium flow rate of 1 mL/min and a transfer line temperature of 280 °C. The analyses were performed in the external ionisation configuration. Electron ionisation spectra were recorded with a mass range of m/z 50–300 at fast scan rate. NMR spectra were recorded on a Bruker Avance 500 (500 MHz 1H, 125.8 MHz 13C) spectrometer using CDCl3 as solvent and internal standard.
(Z)-4-11Al
(Z)-4-11Al was synthesized via a modified version of Wube et al. [66] in 80% stereoisomeric purity. Esterification under acidic conditions with sulfuric acid in methanol resulted in 80% Z-isomer and a 93% yield over two steps. Stereoisomeric purity was controlled with NMR and GC-FID by comparing the analysis for acid and ester, the appearance of a small quartet, in the NMR spectra, at 1.96 indicates the presence of E-isomer. Gas chromatographic separation on a polar Varian factorFOUR vf-23ms of Z- and E-ester proved that the stereochemistry was not affected by the acidic conditions during esterification. Methyl (Z)-4-undecenoate was purified on regular silica gel and on silver nitrate impregnated silica gel to obtain a stereoisomeric purity of 98.6%. Methyl (Z)-4-undecenoate was reduced to (Z)-4-undecenol with lithium aluminium hydride in diethylether and oxidized to Z4-11Al with Dess-Martin periodinane in dichloromethane.
NaHMDS (6.78 mmol, 1 M in hexane) was added dropwise, over 30 min, to a suspension of (3-carboxypropyl)triphenylphosphonium bromide (1.45 g, 3.39 mmol) in THF (25 mL). The mixture was stirred for 2 h then cooled to 0 °C on an ice/water bath, and heptanal (0.387 g, 3.39 mmol) in THF (2.5 mL) was added slowly over 15 min. The mixture was stirred for 5 h at 0 °C then allowed to reach room temperature overnight. The reaction was quenched with H2O (20 mL) and the organic solvent was evaporated. The remaining water phase was extracted with Et2O (3 × 20 mL), the obtained organic phases discarded, and the basic aqueous phase was acidified with HCl (2 M) until pH 1 and extracted with Et2O (3 × 20 mL). The combined organic phases were dried over MgSO4 (anhydr.) and the solvent evaporated off. The obtained crude product was dissolved in pentane, cooled at –18 °C and filtered to remove the precipitated OPPh3 followed by evaporation of the solvent to result in 0.547 g of a yellow oil (87.5% yield). 1H-NMR: 5.52–5.30 (m, 2H), 2.35 (m, 4H), 2.04 (q, J = 6.5 Hz, 1.6H, Z-isomer), 1.96 (q, J = 6.5 Hz, 0.4H, E-isomer), 1.37–1.19 (m, 8H) and 0.89 (t, J = 7 Hz, 3H) ppm. The NMR data is in accordance with data previously reported [66, 67]. The relationship by integration between protons at 2.04 and 1.95 indicates approximately a Z:E ratio of 80:20, which is supported by GC-MS analysis on a Varian factorFOUR vf-23ms column. The obtained crude product was used in the next step without further purification.
Methyl (Z)-4-undecenoate
(Z)-4-11Al (0.547 g, 2.97 mmol), as synthesized above, was dissolved in methanol (15 mL) and seven drops of concentrated H2SO4 were added followed by heating at 70 °C overnight. The mixture was allowed to reach room temperature and the methanol was evaporated and the remaining crude product was dissolved in Et2O (15 mL). The organic phase was washed with H2O (3 × 10 mL) and brine (2 × 10 mL), dried over Na2SO4 (anhydr.) and solvent evaporated, resulting in 0.547 g of a yellow oil (92.8% yield). GC-MS (FactorFour vf-23ms) showed a Z:E ratio of 80:20. 1H-NMR(CDCl3): 5.4 (m, 2H), 3.67 (s, 3H), 2.3 (m, 4H), 2.03 (q, J = 6.5 Hz, 1.6H, Z-isomer), 1.96 (q, J = 6.5 Hz, 0.4H, E-isomer), 1.33–1.21 (m, 8H) and 0.89 (t, J = 6.5 Hz, 3H) ppm (no data found in the literature). 13C-NMR(CDCl3): 134.2, 119.9, 32.3, 31.9, 29.5, 29.3, 27.43, 22.7 and 14.1 ppm; 13C-NMR data are in accordance with the literature [68]. Proton NMR showed a 80:20 Z:E ratio between the diastereomers. Enrichment of the Z-isomer on AgNO3 (10%) impregnated silica resulted in 63 mg of a 98.6:1.4 Z:E ratio product according to GC-FID analysis on the vf-5 column as the diastereoisomeric purity was not possible to measure when using 1H-NMR.
(Z)-4-undecenol
Methyl (Z)-4-undecenoate (63 mg, 0.32 mmol) was dissolved in Et2O (5 mL) and LiAlH4 (2 spatula tips) was added followed by stirring at room temperature for 30 min. HCl (2 M, 2 mL) was added to quench the reaction and the mixture was extracted with Et2O (2 × 3 mL), the combined organic layer was dried over MgSO4 (anhydr.) and solvent was evaporated. Purification with flash chromatography on SiO2 resulted in 37 mg. 1H-NMR(CDCl3): 5.43–5.32 (m, 2H), 3.67 (m, 2H), 2.16–2.10 (m, 2H), 2.08–2.02 (m, 2H), 1.69–1.60 (m, 2H), 1.39–1.22 (m, 8H) and 0.89 (t, J = 6.5 Hz, 3H) ppm. NMR data were similar to that of Kim and Hong [69] and Davis and Carlsson [70]. Diastereomeric purity was checked with GC-FID before the next step.
(Z)-4-undecenal
(Z)-4-Undecenol (37 mg, 0.22 mmol) in DCM (3 mL) was added to Dess–Martin periodinane (0.140 g, 0.33 mmol) in DCM (0.5 mL). After 50 min, NaOH (2 M, 10 mL) was added to quench the reaction. The two layers were separated and the aqueous phase was extracted with Et2O (3 × 10 mL), the combined organic layers were washed with NaOH (2 M, 10 mL), dried over MgSO4 (anhydr.) and solvent was evaporated resulting in 30 mg of a yellow oil (81% yield). The crude product was purified with Kugelrohr distillation at boiling point (65–70 °C; 1.6 mbar), resulting in 17 mg. 1H-NMR(CDCl3): 9.77 (s, 1H), 5.48–5.22 (m, 2H), 2.47 (t, J = 7 Hz, 2H), 2.37 (q, J = 7 Hz, 2H), 2.04 (q, J = 7Hz, 2H), 1.37–1.23 (m, 8H) and 0.88 (t, J = 7 Hz, 3H) ppm. 13C-NMR(CDCl3): 202.1, 131.8, 127.0, 43.9, 31.8, 29.5, 29.0, 27.2, 22.6, 20.1 and 14.1 ppm; both 1H- and 13C-NMR data were in accordance with published results [71, 72]. Analysis on GC-MS (FactorFour vf-23ms) resulted in a 98.6:1.4 Z:E ratio, the E-isomer could not be detected by 1H-NMR.
(E)-4-undecenoic acid
A modified version of Virolleaud’s metathesis [73] was used to produce (E)-4-undecenoic acid in a 56% yield (87.5% of the E-isomer). (E)-4-undecenoic acid was esterified under the same conditions as the (Z)-acid, without isomerisation of the double bond (according to GC-FID and 1H-NMR). The methyl-(E)-4-undecenoate was reduced to the alcohol with lithium aluminium hydride in diethyl ether and purified on silver nitrate-impregnated silica gel to obtain a purity of 99.8% of the (E)-isomer, which was oxidized with Dess-Martin periodinane in dichloromethane to obtain (E)-4-undecenal.
4-Pentenoic acid (0.5 g, 5 mmol) and 1-octene (2.8 g, 25 mmol) were dissolved in DCM (50 mL), Grubbs II catalyst (85 mg, 0.1 mmol) was added and the reaction was refluxed. After 7 h, a second portion of Grubbs II catalyst (85 mg, 0.1 mmol) was added and the reaction refluxed for a further 16 h. The reaction was allowed to reach room temperature and the solvent was evaporated. The obtained crude product was dissolved in Et2O (50 mL) and filtered through a short pad of silica gel. The product was purified with flash chromatography by gradient elution (0–100% EtOAc in c-hexane followed by 0–10% EtOH in EtOAC) resulting in 0.52 g of oil (56% yield). 1H-NMR(CDCl3): 5.51–5.33 (m, 2H), 2.41 (q, J = 7 Hz, 2H), 2.32 (q, J = 7 Hz, 2H), 2.04 (q, J = 6.5 Hz, 0.25 H, Z-isomer), 1.97 (q, J = 6.5 Hz, 1.75H, E-isomer), 1.37–1.22 (m, 9H) and 0.88 (t, J = 7.5 Hz, 3H) ppm. The relation between the proton at 2.04 and 1.97 reveals a 87.5:12.5 E:Z ratio. The isolated product was used in the next step without further purification.
Methyl (E)-4-undecenoate
(E)-4-undecenoic acid (0.52 g, 2.82 mmol) was dissolved in methanol (25 mL), a catalytic amount H2SO4 was added and the mixture was refluxed overnight. After evaporation of the solvent, the crude product was dissolved in Et2O (10 mL) and washed with H2O (20 mL). The aqueous phase was extracted with Et2O (2 × 25 mL), the combined organic layer was washed with H2O (20 mL) and brine (20 mL), dried over MgSO4 (anhydr.) and evaporation of solvent resulted in 0.439 g (78% yield). 1H-NMR(CDCl3): 5.51–5.33 (m, 2H), 3.67 (s,3H), 2.40–2.27 (m, 4H), 1.96 (q, J = 6.5 Hz, 2H), 1.38–1.21 (m, 8H) and 0.88 (t, J = 6.5 Hz,3H) ppm. Purification with flash chromatography resulted in 0.401 g (71.7% yield). GC-FID showed the same stereoisomeric ratio as for the acid above.
(E)-4-undecen-1-ol
LiAlH4 (0.055 g, 1.46 mmol) was added to methyl (E)-4-undecenoate (0.145 g, 0.73 mmol) dissolved in Et2O (5 mL). After 30 minutes, HCl (2 M, 5 mL) was added to quench the reaction. The acidic water phase was extracted with Et2O (3 × 10 mL) and the combined organic layers were dried over MgSO4 (anhydr.) and evaporation of solvent resulted in 0.104 g (99% yield). Enrichment of the E-isomer with medium pressure liquid chromatography on AgNO3 (10% impregnated) silica resulted in 30 mg of a clear oil (>99.8% E). 1H-NMR(CDCl3): 5.43 (m, 2H), 3.65 (m, 2H), 2.08 (q, J = 7 Hz, 2H), 1.97 (q, J = 7 Hz, 2H), 1.63 (pent, 2H), 1.35–1.21 (m, 9H) and 0.88 (t, J = 6.5 Hz, 3H) ppm. 13C-NMR(CDCl3): 134.4, 131.3, 129.4, 62.6, 32.6, 32.5, 31.7, 29.6, 29.5, 28.9, 28.8, 22.6 and 14.1 ppm. All NMR data were in accordance with previously published data [72].
(E)-4-undecenal
Dess-Martin periodinane (0.110 g, 0.26 mmol) was added to (E)-4-undecen-1-ol (0.030 g, 0.22 mmol) in DCM (4 mL). NaOH (2 M, 10 mL) was added after 1 h to quench the reaction. The aqueous phase was extracted with Et2O (3 × 10 mL) and the combined organic layers were dried over MgSO4 (anhydr.); evaporation of the solvent resulted in 30 mg (98% yield). Purification of the crude product with Kugelrohr distillation at 65 °C (2 mbar) resulted in 10 mg of product (33% yield, 97% chemical purity, 3% undecenal). 1H-NMR(CDCl3): 9.76 (t, J = 1.5 Hz, 1H), 5.50–5.36 (m, 2H), 2.48 (d of t, J = 7.5, 1.5 Hz, 2H), 2.33 (q, J = 7 Hz, 2H), 1.97 (q, J = 6.5 Hz, 2H), 1.32–1.19 (m, 8H) and 0.87 (t, J = 6.5 Hz, 3H). 13C-NMR (CDCl3): 202.5, 132.2, 127.6, 43.6, 32.5, 31.7, 29.4, 28.8, 25.2, 22.6 and 14.1 ppm. The NMR data were in accordance with previously published data [72, 74].
Odour collection and chemical analysis
Groups of 20 flies, 3- to 5-day-old, D. melanogaster (Dalby), D. melanogaster (Canton-S), or D. simulans, unmated females or unmated males (n = 5 for each) were placed in a glass aeration apparatus designed for collection of airborne pheromones (effluvia) [75]. The flies were held in a glass bulb with a narrow open outlet (ø 1 mm), which prevented them from escaping. A charcoal-filtered air flow (100 mL/min) passed over the flies over 75 min. Fly effluvia were collected on the glass surface, breakthrough was monitored by attaching a 10-cm glass capillary (ø 1 mm) onto the outlet. After 75 min, flies were removed and 100 ng of heptadecyl acetate (internal standard) was deposited in the glass bulb, which was then rinsed with 50 μL hexane, and the solvent was concentrated to 10 μL in Francke vials.
Cuticular extracts (n = 5) were obtained by placing 20 D. melanogaster females for 5 min in 400 μL hexane containing 100 ng heptadecyl acetate. After 5 min, the extracts were transferred to Francke vials and concentrated to 10 μL before analysis. Fly extracts and volatile collections were stored at –20 °C.
Oxidation of 7,11-HD was analysed by dropping 100 ng of synthetic 7,11-HD into a 1.5-mL glass vial at 19 °C. Vials were rinsed with 10 μL of hexane, which contained 100 ng heptadecyl acetate as an internal standard, after 15, 30, 45, 60 and 75 min (n = 3).
Samples were analysed by combined GC-MS (6890 GC and 5975 MS, Agilent technologies Inc., Santa Clara, CA, USA). The samples (2 μL) were injected (injector temperature 225 °C) splitless (30 s) into the fused silica capillary columns (60 m × 0.25 mm) coated with HP-5MS UI (Agilent Technologies Inc., df = 0.25 μm) or DB-wax (J&W Scientific, Folsom, CA, USA, df = 0.25 μm), that were temperature-programmed from 30 °C to 225 °C at 8 °C/min. Helium was used as mobile phase at 35 cm/s. The MS operated in scanning mode over m/z range 29–400. Compounds were tentatively identified based on their mass spectra and Kovats retention indices, using custom and NIST (Agilent) libraries, followed by comparison with authentic standards. Each series of GC-MS runs is preceded by blank runs, including solvent, glassware and air filters.
Behavioural assays
Upwind flight behaviour was observed in a glass wind tunnel (30 × 30 × 100 cm). The flight tunnel was lit diffusely from above, at 13 lux, and the temperature ranged from 22 °C to 24 °C and relative humidity from 38% to 48%, and charcoal filtered air, at a velocity of 0.25 m/s, was produced by a fan (Fischbach GmbH, Neunkirchen, Germany). Compounds were delivered from the centre of the upwind end of the wind tunnel via a piezo-electric micro-sprayer [50, 76]. Forty flies were flown individually to each treatment. ‘Attraction’ was defined as upwind flight, directly from a release tube at the end of the tunnel over 80 cm towards the odour source, followed by landing. Unmated, fed, 4-day-old Dalby wild-type males and females, D. melanogaster Zimbabwe strain males and D. simulans males were flown towards (Z)-4-undecenal (released at 10 ng/min), (R)-linalool (10 ng/min) and the blend of (Z)-4-undecenal and (R)-linalool (10 ng/min, each).
Mated 4-day-old males of the Or69aRNAi/OrcoGal4 line and the respective parental fly lines were used. Since the transgenic fly lines produced fewer offspring, all flies were used, instead of discarding individuals eclosing during the night, which may have mated. Unmated and mated wild-type flies did not show a significant difference in the response rate to 10 ng/min Z4-11Al (50% and 52.5%, n = 40).
Heterologous expression of Or69aA and Or69aB
Or69aA and Or69aB receptors were cloned from antennae of D. melanogaster (Dalby) [77]. Briefly, cDNA was generated from RNA extracts of antennae of 100 males and females using standard procedures. Or69a variants were PCR amplified with the following primers: Or69aA_5’: GTCATAGTTGAAACCAGGATGCAGTTGC, Or69aB_5’: ATAATTCAGGACTAGATGCAGTTGGAGG, Or69aAB_3’: TGCACTTTTGCCCTTTTATTTAAGGGAC.
Or69aA and Or69aB were amplified with unique 5’ primers and a common 3’ primer, reflective of genomic structure at this locus. These primers encompass the entire open reading frame of the receptor variants, and are located partially upstream and downstream of the start and stop codons. PCR amplicons were gel-purified and cloned into the pCR8/GW/Topo-TA Gateway entry vector (Thermo-Fisher Scientific, Waltham, MA, USA) according to standard procedure, with vector inserts sequenced to confirm fidelity of Or sequence. Or inserts were subsequently transferred to pUAS.g-HA.attB [78] with LR Clonase II enzyme (Thermo-Fisher Scientific), according to the manufacturer’s protocol; vector inserts were sequenced to confirm fidelity of Or sequence.
Mini-prep purified pUAS.g-HA.attB plasmids with Or69aA or Or69aB insert were delivered to Best Gene Inc. (Chino Hills, CA, USA) for generation of transgenic D. melanogaster flies. Using the PhiC31 targeted genomic-integration system [78], vectors with Or69aA or Or69aB were injected into the following fly strain, for integration on the third chromosome M{3xP3-RFP.attP}ZH-86Fb (with M{vas-int.Dm}ZH-2A) (Bloomington Drosophila Stock Number: 24749). For expression of single receptor variants in the empty neuron system, Or69a transgenes were crossed into the Δhalo background to give genotype w; Δhalo/Cyo; UAS-DmelOr69a(A or B), and these flies were crossed to flies with genotype w; Δhalo/Cyo; DmelOr22a-Gal4, as described previously [77]. Experimental electrophysiology assays were performed on flies with genotype w; Δhalo; UAS-DmelOr69a(A or B)/DmelOr22a-Gal4.
For co-expression of Or69aA and Or69aB in the same empty neurons, a second fly-line with Or69aB was generated with Or69aB present on the X-chromosome. The same UASg-HA.attB:Or69aB plasmid generated previously was injected into the fly strain y,w, P{CaryIP}su(Hw)attP8 (Bloomington Drosophila Stock Number: 32233). The Or69aB transgene was crossed into the DmelOr22a-Gal4 line in Δhalo background to give genotype UAS-DmelOr69aB; Δhalo/Cyo; DmelOr22a-Gal4; these flies were crossed to flies with genotype w; Δhalo/Cyo; UAS-DmelOr69aA. Experimental electrophysiology assays were performed on flies with genotype UAS-DmelOr69aB/w; Δhalo; UAS-DmelOr69aA/DmelOr22a-Gal4.
Or identity scores were calculated with Clustal Omega Multiple Sequence Alignment webtool, using default parameters [79].
Conformational analysis
MacroModel version 11.0 (Schrodinger LLC, New York, NY, USA) in the Maestro Version 10.4.017 were used to build, minimize and perform conformational analysis of Z4-11Al, (R)-carvone, (S)-terpineol and (R)-linalool, using default settings (OPLS3 as force field, water as the solvent and mixed torsional/low-mode sampling method). The assumed bioactive conformations of the conformationally more flexible compounds, Z4-11Al and (R)-linalool, were based on the position of the shared functional groups in the conformationally more restricted compounds, (R)-carvone and (S)-terpineol. The carbonyl and the double bond atoms were kept fixed during minimisation of the proposed bioactive conformation of Z4-11Al; the alcohol functional group and the double bond were kept fixed in (R)-linalool. Strain energies, the energy cost for adopting proposed bioactive conformations, were then calculated as the difference between the lowest energy conformations and the assumed bioactive conformation.
Electrophysiological recordings
SSR were performed as described earlier [23]. Unmated males were restrained in 100-μL pipette tips, with half of the head protruding, the third antennal segment or palps were placed on a glass microscope slide and held by dental wax. For the initial screening, all basiconic, trichoid, coeloconic and intermediate sensilla [36] were localized in D. melanogaster (Canton-S strain) males, under a binocular at 1000× magnification. Further recordings were made from small basiconic ab9 sensilla, in D. melanogaster (Canton-S and Zimbabwe strains) and in D. simulans males, and from large basiconic ab3 sensilla in mutant D. melanogaster, where Or69aA and Or69aB were heterologously expressed (see above).
Tungsten electrodes (diameter 0.12 mm, Harvard Apparatus Ltd, Edenbridge, United Kingdom) were electrolytically sharpened with a saturated KNO3 solution. The recording electrode was introduced with a DC-3 K micromanipulator equipped with a PM-10 piezo translator (Märzhäuser Wetzler GmbH, Germany) at the base of the sensilla. The reference electrode was inserted into the eye. The signal from OSNs was amplified with a probe (INR-02; Syntech), digitally converted by an IDAC-4-USB (Syntech) interface, and analysed with Autospike software v. 3.4 (Syntech). Neuron activities were recorded during 10 s, starting 2 s before odour stimulation. Neuron responses were calculated from changes in spike frequency, during 500 ms before and after odour stimulation.
Odorants were diluted in redistilled hexane; 10 μg of test compounds in 10 μL hexane were applied to filter paper (1 cm2) and kept in Pasteur pipettes. The test panel contained the most active ligands known for Or69a [39] and several aldehydes. Diagnostic compounds for confirmation of sensillum identity were 2-phenyl ethanol (ab9) and 2-heptanone (ab3). Control pipettes contained solvent only. Puffs (2.5 mL, duration 0.5 s) from these pipettes, produced by a stimulus controller (Syntech GmbH, Kirchzarten, Germany), were injected into a charcoal-filtered and humidified airstream (0.65 m/s), which was delivered through a glass tube to the antenna.
For GC-SSR recordings, GC columns and the temperature programmes were the same as for the GC-MS analysis. At the GC effluent, 4 psi of nitrogen was added and split 1:1 in a 3D/2 low dead volume four-way cross (Gerstel, Mühlheim, Germany) between the flame ionization detector and the antenna. Towards the antenna, the GC effluent capillary passed through a Gerstel ODP-2 transfer line that tracked the GC oven temperature, into a glass tube (30 cm × 8 mm ID), where it was mixed with charcoal-filtered, humidified air (20 °C, 50 cm/s).
Statistical analysis
Generalized linear models with a Bernoulli binomial distribution were used to analyse wind tunnel data. Landing at source and sex were used as the target effects. Post hoc Wald pairwise comparison tests were used to identify differences between treatments. For all the electrophysiological tests, differences in spike activity derived from SSRs were analysed with the Kruskal–Wallis H test followed by pairwise comparisons with the Mann–Whitney U post hoc test. All statistical analyses were carried out using R (R Core Team 2013) and SPSS Version 22 (IBM Corp).