HsOr cloning and UAS fly generation

RNA was extracted from resected and homogenized H. saltator worker antennae using TRIzol reagent (Life Technologies, Carlsbad, CA, USA). Total antennal RNA was then oligo(dT)-primed for cDNA synthesis with Superscript II Reverse Transcriptase (Life Technologies). Predicted full-length HsOr coding sequences were PCR amplified from antennal cDNA templates and cloned into either pENTR/D-TOPO (Life Technologies) or pATTL entry vectors. HsOrs in entry vectors were then sub-cloned into the pUASg.attB (a gift from Konrad Basler, University of Zurich) using LR Clonase II (Life Technologies). These constructs were then coinjected into y, w; attP40 D. melanogaster embryos with a phiC31 integrase plasmid (Genetic Services, Inc., Sudbury, MA, USA).

Harpegnathos saltator extract preparation

Whole bodies of freeze-killed gamergates and non-reproductive workers were individually extracted in 500 µl hexane for 5 min and the extracts were analysed by GC/MS using a DB1 column to check for possible contamination or inadequate extraction. Sufficiently clean extracts from 23 gamergates from 19 colonies and 26 non-reproductive workers from 15 colonies were consolidated, and the two consolidated extracts were again analysed by GC/MS. Whole bodies of 50 males originating from 14 colonies were extracted in two batches of 25 individuals in 2 ml hexane for 5 min each before consolidation into one batch. The gamergate extract consisted of 99.1%, non-reproductive worker of 96.7%, and male of 99.9% CHCs, respectively. Other compounds present in the extract were largely short-chain hydrocarbons smaller than pentacosane that do not regularly appear on the worker cuticle. Total cuticular hydrocarbon yield was determined by calibration using tetracosane as an internal standard. Consolidated CHC extracts were then diluted to 0.6 µg/µl in pentane for use in electrophysiology assays.

Syntheses of CHCs

The syntheses of several unsaturated and branched CHCs in the panel have been previously described33,34,35.

General. All reactions were performed under an argon atmosphere with oven-dried glassware. Crude products were purified via flash or vacuum flash column chromatography unless otherwise stated. Yields refer to isolated yields of purified products. Tetrahydrofuran (THF) was distilled from sodium/benzophenone ketyl under an argon atmosphere, and all solvents utilized were Optima grade (Fisher Scientific, Pittsburg, PA, USA). Mass spectra were obtained with a Hewlett-Packard (HP) 6890 GC (Hewlett-Packard, Avondale, PA, USA) interfaced to an HP 5973 mass selective detector, in EI mode (70 eV) with helium as carrier gas. The GC was equipped with a DB17-MS column (25 m × 0.20 mm i.d., 0.33 μm film). 1H and 13C NMR spectra were recorded with a Varian INOVA-400 (400 and 100.5 MHz, respectively) spectrometer (Palo Alto, CA, USA), as CDCl 3 solutions. 1H NMR chemical shifts are expressed in ppm relative to residual CHCl 3 (7.27 ppm) and 13C NMR chemical shifts are reported relative to CDCl 3 (77.16 ppm).

Synthesis of 13,23-dimethylheptatriacontane 10. Figure 5 Triphenylphosphine (1.09 g, 4.15 mmol) and ethyl 9-bromononanoate (1.1. g, 4.15 mmol; TCI Americas, Portland, OR, USA) were refluxed in 10 ml acetonitrile for 36 h. Most of the acetonitrile was removed by rotary evaporation, and the resulting gum was triturated 4 times with ethyl ether. The remaining gum would not crystallize, and so it was taken up in 5 ml CH 2 Cl 2 , and ~2/3 of the solution was transferred to a tared vial. Removal of the solvent gave 1.23 g (2.3 mmol) of the phosphonium salt 2, which was used without further purification.

Fig. 5 Synthesis of 13,23-dimethylheptatriacontane. a Ph 3 P, acetonitrile, reflux; b NaHMDS, then 2-tetradecanone 3, CH 2 Cl 2 ; c LiAlH 4 , THF; d Ph 3 P, CBr 4 , CH 2 Cl 2 ; e Ph 3 P, 110 °C; f NaHMDS, then 2-hexadecanone, CH 2 Cl 2 ; g 5% Pd/C, H 2 , hexane Full size image

The phosphonium salt 2 was taken up in 10 ml CH 2 Cl 2 , cooled to −78 °C under argon, and NaHMDS (2 M in THF) was added dropwise until the solution turned orange, then a further 1.2 ml (2.4 mmol) of NaHMDS solution was added. The resulting solution was stirred 30 min, then 2-tetradecanone 3 (0.3 g, 1.4 mmol) in 5 ml CH 2 Cl 2 was added dropwise. The resulting solution was warmed to room temp over 4 h, then quenched with 1 M HCl, and the mixture was extracted with hexane. The hexane layer was washed with saturated aqueous NaHCO 3 solution and brine, then dried and concentrated. The residue was purified by vacuum flash chromatography, eluting with 3% EtOAc in hexane, yielding ethyl 10-methyldocos-9-enoate 4 (0.42 g, 79%) as a 4:3 mixture of E-isomers and Z-isomers. Isomer 1 MS (m/z, abundance) 380 (22), 335 (33), 227 (11), 211 (29), 210 (27), 180 (44), 171 (94), 165 (35), 155 (13), 147 (17), 137 (19), 125 (69), 111 (37), 97 (81), 83 (76), 69 (91), 55 (100), 43 (52), 41 (48). Isomer 2: MS (m/z, abundance) 380 (23), 335 (35), 227 (12), 211 (27), 210 (27), 180 (46), 171 (100), 165 (34), 155 (13), 147 (16), 137 (21), 125 (65), 111 (38), 97 (72), 83 (73), 69 (89), 55 (97), 43 (45), 41 (41).

The mixture of esters 4 in ~2 ml THF was then added to a slurry of LiAlH 4 (84 mg, 2.2 mmol) in 5 ml THF cooled to –78 °C under argon. The resulting mixture was warmed to room temperature and stirred 2 h, then quenched by sequential dropwise addition of water (88 µl), 20% aqueous NaOH (66 µl), and water (310 µl). After stirring 20 min, the mixture was filtered through a plug of Celite, rinsing well with ether. Concentration yielded 10-methyldocos-9-en-1-ols 5 (0.21 g, 65%, 96% pure by GC) as a 4:3 mixture of isomers, which were used immediately in the next step.

Carbon tetrabromide (232 mg, 0.7 mmol) and triphenylphospine (187 mg, 0.7 mmol) were added to an ice-cooled solution of alcohol 5 (0.21 g, 0.62 mmol) in 5 ml CH 2 Cl 2 , the mixture was stirred 30 min, then the cooling bath was removed and the mixture was stirred a further 3 h. The mixture was then concentrated, and the residue was purified by vacuum flash chromatography, eluting with hexane, yielding 10-methyldocos-9-en-1-yl bromide 6 (237 mg, 0.57 mmol, 92%) as a colourless oil, as a 4:3 misture of isomers. Isomer 1, MS (m/z, abundance) 402 (10), 400(11), 248 (17), 246 (20), 233 (13), 231 (15), 220 (10), 218 (8), 210 (12), 195 (12), 151 (14), 125 (15), 123 (11), 111 (42), 97 (72), 83 (73), 69 (94), 57 (79), 56 (87), 55 (100), 43 (45), 41 (45). Isomer 2, MS (m/z, abundance) 402 (9), 400(10), 248 (14), 246 (14), 233 (13), 231 (11), 220 (10), 218 (8), 210 (12), 195 (8), 151 (10), 125 (16), 123 (8), 111 (38), 97 (64), 83 (76), 69 (89), 57 (77), 56 (89), 55 (100), 43 (41), 41 (465).

The mixture of bromides 6 was mixed with triphenylphosphine (152 mg, 0.57 mmol) and heated at 110 °C overnight under Ar. Toluene (5 ml) was then added to the resulting viscous oil, and the mixture was refluxed for 1.5 days, yielding a pale brown solution. The solvent was removed by rotary evaporation, and 10 ml diethyl ether was added to the residue, giving a viscous oil and a cloudy solution, which was removed. A further 10 ml ether was added and the mixture was stirred 1 h, resulting in a very fine pale brown suspension. The suspension was centrifuged, the liquid phase was removed, and the remaining solids were taken up in ~3 ml CH 2 Cl 2 . An aliquot of the solution was transferred to a clean, dry flask, and concentrated to give 160 mg (~0.25 mmol) of the phosphonium salt 7 as a gum.

The gum was taken up in 5 ml CH 2 Cl 2 , the solution was cooled to −78 °C under argon, and NaHMDS (2 M in THF) was added dropwise until the solution turned yellow. An additional 0.13 ml of NaHMDS solution (0.26 mmol) was added, the mixture was stirred 30 min, and then a solution of 2-hexadecanone 8 (60 mg, 0.25 mmol) in 2 ml CH 2 Cl 2 was added dropwise. The mixture was warmed to room temperature and stirred overnight, then quenched with 1 M HCl, and extracted with hexane. The hexane extract was washed with saturated aqueous NaHCO 3 solution and brine, then dried and concentrated. The residue was purified by vacuum flash chromatography, eluting with hexane. The resulting clear oil was taken up in 5 ml hexane, 5% Pd on carbon (10 mg) was added, and after flushing with hydrogen, a balloon of hydrogen was attached to the septum-sealed flask. The mixture was stirred 3 h, then filtered through a plug of Celite, rinsing well with hexane. The resulting white semisolid (~80% pure by GC) was recrystallized from a mixture of 5 ml each of hexane and acetone at −20 °C, yielding 25 mg of the desired 13,23-dimethylheptatriacontane as a white solid, >96% pure by GC. The remaining liquor containing the bulk of the product, contaminated with impurities, was not purified further. MS (m/z, abundance): 533 (4, M+-15) 519 (2), 379 (13), 351 (14), 224 (23), 196 (34), 183 (4), 181 (6), 169 (6), 155 (), 141 (11), 127 (15), 113 (19), 99 (27), 85 (65), 71 (82), 57 (100), 43 (44).

Synthesis of (±)-5-methylhentriacontane 8. (±)-2-Methylhexanol (2). Figure 6 LiAlH 4 (2.87 g, 75.6 mmol) was suspended in dry Et 2 O (50 ml), the mixture was cooled to 0 °C, and 2-methylhexanoic acid 1 (5.0 g, 37.8 mmol, Sigma-Aldrich, Milwaukee, WI, USA) was added dropwise over 10 min. The resulting mixture was stirred 30 min at 0 °C, then warmed to room temperature and stirred 3 h. The mixture was then cooled to 0 °C and worked up by sequential dropwise addition of 2.9 ml of H 2 O, 2.9 ml of 15% (wt/wt) aqueous NaOH, and 9 ml H 2 O. The resulting suspension was stirred 20 min, then filtered with suction, and the solids were washed with Et 2 O (3 × 75 ml). The combined Et 2 O washes were concentrated and purified by vacuum flash chromatography (EtOAc/hexanes, 1:4) to afford (±)-2-methylhexanol (4.15 g, 95 % yield, 98.7% pure by GC) as a colourless oil. 1H NMR (CDCl 3 ): ∂ 0.87 (3H, t, 5.7 Hz), 0.89 (3H, d, 6.3 Hz), 1.05 (1H, m), 1.24–1.35 (5H, broad m), 1.62 (1H, m), 1.82 (OH, broad s), 3.35 (1H, dd, J = 11.8, 5.8 Hz), 3.48 (1H, dd, J = 12.3, 6.8 Hz). 13C NMR (CDCl 3 ): ∂ 14.1, 17.0, 23.5, 29.8, 33.0, 38.5, 68.5 ppm. EIMS (m/z, abundance): 115 (5, M+-1), 98 (12), 84 (28), 70 (45), 56 (100), 42 (57).

Fig. 6 Synthesis of ±-5-methylhentriacontane. a LiAlH 4, Et 2 O (95%); b Tf 2 O, pyridine, CH 2 Cl 2 (quantitative); c tert-Butyldimethyl(tridec-12-ynyloxy)silane, n-BuLi, THF (74%); d AcCl, dry MeOH (96%); e Tf 2 O, pyridine, CH 2 Cl 2 (quantitative); f hexadecynyllithium, THF (49%); g 10% Pd/C, H 2 , hexane (~quantitative) Full size image

(±)-2-Methylhexan-1-yl triflate (3). Pyridine (277 μl, 3.44 mmol) and triflic anhydride (704 μl, 4.1 mmol) were added sequentially to a cold (−10 °C) solution of (±)-2-methylhexan-1-ol 2 (400 mg, 3.44 mmol) in CH 2 Cl 2 (15 ml). The mixture was stirred for 1 h at −10 °C and then diluted with pentanes (50 ml), warmed to room temperature, and filtered through a plug of silica gel. The filter cake was rinsed with 3:1 hexane:CH 2 Cl 2 (3 × 50 ml) to ensure that all of the alkyl triflate intermediate was recovered. The filtrates were then combined and concentrated to give triflate (±)-3 (854 mg, quantitative) as a colourless oil, which was used immediately in the next step.

(±)-tert-Butyldimethyl(15-methylnonadec-12-ynyloxy)silane (4). tert-Butyldimethyl(tridec-12-ynyloxy)silane (1.013 g, 3.27 mmol) was dissolved in dry THF (15 ml), the solution was cooled to −78 °C, and n-BuLi (2.2 M, 1.48 ml) was added dropwise. The reaction was stirred at −78 °C for 1 h, then (±)-2-methylhexan-1-yl triflate 3 (854 mg, 3.44 mmol) in 3 ml THF was added by syringe pump over 30 min, and the reaction was warmed −40 °C and stirred for 5 h. The mixture was then quenched with water (20 ml) and extracted with hexane. The hexane extract was washed with brine, dried, and concentrated, and the residue was purified by vacuum flash chromatography (EtOAc/hexanes, 1:5) to afford (±)-tert-butyldimethyl(15-methylnonadec-12-ynyloxy)silane (988 mg, 74% yield, 98.3% pure by GC) as a colorless oil. 1H NMR (CDCl 3 ): ∂ 0.21 (6H, s), 0.89 (3H, t, 6.3 Hz), 0.93 (3H, d, 6.5 Hz), 0.98 (9H, s), 1.2–1.5 (25H, br m), 2.08 (4H, m), 3.38 (2H, t, J = 6.3 Hz). 13C NMR (CDCl 3 ): ∂ -1.9, 14.0, 18.2, 19.5, 26.3, 28.7, 29.5, 30.2, 30.5, 31.3, 33.2, 36.5, 38.0, 64.1, 78.3, 80.1 ppm. EIMS (m/z, abundance): 351 (35, M+-57), 277 (3), 265 (5), 249 (1), 207 (1), 193 (1), 179 (1), 165 (1), 151 (1), 137 (5), 99 (11), 85 (75), 71 (61), 57 (100), 43 (67).

(±)-15-Methylnonadec-12-yn-1-ol (5). Acetyl chloride (30 µl, 0.32 mmol) was added to a cooled (0 °C) solution of (±)-tert-butyldimethyl(15-methylnonadec-12-ynyloxy)silane 4 (900 mg, 2.15 mmol) in dry MeOH (10 ml). The reaction was stirred for 10 min at 0 °C, then warmed to room temperature for another 20 min. The reaction was then diluted with Et 2 O (25 ml) and quenched with 10% aqueous NaHCO 3 . The layers were separated and the aqueous layer was washed with Et 2 O (2 × 50 ml). The combined organic layers were washed with water (2 × 50 ml) and brine (2 × 50 ml), dried, and concentrated. The residue was purified by vacuum flash chromatography (EtOAc/hexanes, 1:5) to afford (±)-15-methylnonadec-12-yn-1-ol (602 mg, 96% yield, 98.3% pure by GC) as a colourless oil. 1H NMR (CDCl 3 ): ∂0.88 (3H, t, 6.1 Hz), 0.90 (3H, d, 6.5 Hz), 1.2–1.5 (25H, br m), 1.83 (OH, broad s), 2.08 (4H, m), 3.51 (2H, t, J = 6.0 Hz). 13C NMR (CDCl 3 ): ∂ 14.0, 17.3, 19.2, 26.5, 28.7, 29.7, 30.2, 30.8, 31.3, 33.2, 36.5, 38.0, 62.3, 78.5, 81.1. EIMS (m/z, abundance): 293 (5, M+-1), 276 (3), 263 (1), 249 (1), 235 (1), 209 (5), 195 (2), 171 (2), 151 (1), 137 (5), 123 (11), 112 (14), 99 (23), 85 (76), 71 (68), 57 (100), 43 (77).

(±)-15-methylnonadec-12-yn-1-yl triflate (6). Pyridine (41 μl, 0.51 mmol) and triflic anhydride (104 μl, 0.61 mmol) were added sequentially to a cold (−10 °C) solution of (±)-15-methylnonadec-12-yn-1-ol 5 (150 mg, 0.51 mmol) in CH 2 Cl 2 (5 ml). The mixture was stirred for 1 h at −10 °C, then diluted with hexanes (50 ml), warmed to room temperature, and filtered through a plug of silica gel, rinsing with 3:1 hexane:CH 2 Cl 2 (3 × 50 ml) to ensure that all of alkyl triflate 6 was recovered. The filtrate was concentrated to give 15-methylnonadec-12-yn-1-yl triflate (±)-6 (219 mg, quantitative) as a colourless oil, which was used immediately without further purification or characterization.

(±)-5-methylhentriacontane (8). n-BuLi (2.1 M, 0.42 ml, 0.88 mmol) was added dropwise to a cooled solution (−78 °C) of 1-dodecyne (0.21 ml, 0.97 mmol) in 5 ml dry THF. The mixture was stirred at −78 °C for 1 h, followed by dropwise addition of triflate 6 (0.35 g, 0.81 mmol) in 2 ml THF. The mixture was slowly warmed to room temperature, then quenched with water and extracted with hexane. The hexane extract was washed with brine, dried, and concentrated, and the residue was purified by vacuum flash chromatography (hexane) giving (±)-5-methylhentriaconta-7,20-diyne (7) as a colourless oil (170 mg, 49%). The oil was taken up in 2 ml hexane and added to a slurry of 10% palladium on carbon (20 mg, 10 % wt, Signa Aldrich) and K 2 CO 3 (250 mg, 1.81 mmol) in 5 ml hexane. The heterogenous mixture was stirred for 8 h under a slight positive pressure of H 2, then filtered through a plug of silica gel and concentrated. Recrystallization of the residue from acetone (5 ml) at −20 °C gave 170 mg of (±)-5-methylhentriacontane (99% pure by GC), mp 36 °C. 1H NMR (CDCl 3 ): ∂ 0.87 (3H, d, J = 6 Hz), 0.89 (3H, t, J = 6.1 Hz), 0.91 (3H, t, J = 6.2 Hz), 1.16–1.4 (56H, broad m), 1.53 (1H, m). 13C NMR (CDCl 3 ): ∂ c 11.6, 15.3, 19.4, 21.8, 23.5, 24.7, 25.2 27.6, 29.3, 29.7, 30.0, 30.3, 31.8, 32.6, 34.2, 36.8 ppm. EIMS (m/z, abundance): 435 (1, M+-15), 421 (1), 407 (1), 393 (35), 365 (4), 351 (1), 337 (1), 323 (1), 309 (2), 295 (2), 281 (2), 267 (2), 253 (2), 239 (3), 225 (3), 211 (2), 197 (3), 183 (3), 169 (3), 155 (5), 141 (5), 127 (9), 113 (11), 99 (18), 85 (100), 71 (70), 57 (81), 43 (69).

Synthesis of 13- and 15-methylbranched hydrocarbons. Hexadecyltriphenylphosphonium bromide was purchased from Alfa-Aesar (Ward Hill MA) and tetradecyltriphenylphosphonium bromide was obtained from Lancaster Synthesis (Windham NH). Octadecyltriphenylphosphonium bromide was prepared by refluxing a mixture of triphenylphosphine (26.2 g, 100 mmol) and octadecyl bromide (33.3 g, 100 mmol) in 50 ml toluene for 5 days. The resulting viscous oil was poured into a 1:1 mixture of toluene and hexane, and after stirring 30 min, the resulting solids were collected by vacuum filtration, then pumped under high vacuum (0.05 mm Hg) for 8 h, yielding the desired product as an amorphous white powder (47.5 g, 80%).

A slurry of hexadecyltriphenylphosphonium bromide (1.703 g, 3 mmol) in dry THF (6 ml) was cooled to −10 °C under argon, and lithium diisopropylamide (1.5 M in cyclohexane, 2.1 ml, 3.15 mol) was added over ~10 min. The mixture was stirred 3 h, then cooled to −30 °C, and 2-tetradecanone (0.64 g, 3 mmol) in 1.5 ml THF was added dropwise. The resulting mixture was warmed to room temperature and stirred overnight, then quenched with saturated aqueous NH 4 Cl solution. The organic layer was separated, and the aqueous phase was extracted with ethyl ether (3 × 20 ml). The combined organic layers were washed with water and brine, dried, and concentrated. The residue was purified by vacuum flash chromatography, eluting with hexane, yielding 13-methyl-13E/Z-nonacosene (0.55 g, 44% yield), in a 55/45 ratio of geometric isomers. MS (m/z, abundance): 420 (M+, 8), 266 (7), 251 (7), 210 (9), 195 (11), 181 (3), 167 (5), 153 (5), 139 (10), 125 (19), 111 (38), 97 (59), 83 (53), 69 (68), 57 (100), 55 (86), 43 (84), 41 (55).

The mixture of alkene isomers (5.44 g, 1.29 mmol) was dissolved in 10 ml hexane, 5% Pd on carbon catalyst was added (80 mg), and the septum sealed flask was fitted with a balloon of hydrogen. After flushing with hydrogen, the mixture was stirred overnight, then filtered through a plug of Celite, rinsing with hexane. Removal of the solvent by rotary evaporation yielded (±)-13-methylnonacosane (0.53 g, 96%) as a low-melting white solid. 1H NMR: δ 0.83 (d, 3H, J = 6.4 Hz), 0.88 (t, 6H, J = 6.8 Hz), 1.00–1.14 (m, 2H), 1.18–1.40 (m, 51H). 13C NMR: δ 14.35, 19.96, 22.93, 27.32, 29.60, 29.89, 29.94, 29.97, 30.27, 32.16, 32.98, 37.33. MS (m/z, abundance): 407 (M-15) (2), 393 (1), 253 (10), 252 (14), 224 (4), 197 (12), 196 (23), 183 (2), 168 (5), 155 (4), 141 (7), 127 (10), 113 (13), 99 (20), 85 (58), 71 (77), 57 (100), 43 (60).

In similar fashion, reaction between 2-tetradecanone and octadecyltriphenylphosphonium bromide gave 13-methyl-13E/Z-hentriacontene in 39% yield, as a ~ 55/45 mixture of geometric isomers. MS (m/z, abundance): 448 (M+, 6), 294 (7), 279 (5), 266 (4), 210 (7), 195 (11), 182 (5), 167 (4), 153 (5), 139 (10), 125 (18), 111 (36), 97 (58), 83 (52), 69 (63), 57 (100), 55 (78), 43 (82), 41 (47). Hydrogenation then gave (±)-13-methyluntriacontane in 97% yield as a white solid. 1H NMR: δ 0.83 (d, 3H, J = 6.4 Hz), 0.88 (t, 6H, J = 6.8 Hz), 1.01–1.14 (m, 2H), 1.18–1.38 (m, 55H). 13C NMR δ 14.35, 19.96, 22.93, 27.32, 29.60, 29.89, 29.93, 29.96, 30.26, 32.16, 32.97, 37.32. MS (m/z, abundance): 435 (M-15) (1), 281 (7), 280 (8), 252 (2), 197 (6), 196 (11), 183 (2), 169 (3), 168 (3), 155 (4), 141 (8), 127 (9), 113 (12), 99 (18), 85 (44), 71(69), 57 (100), 43 (72).

15-Methyl-15E/Z-nonacosene was obtained in 46% yield from 2-hexadecanone and tetradecyltriphenylphosphonium bromide. MS (m/z, abundance): 420 (M+, 8), 238 (13), 223 (17), 182 (9), 167 (3), 153 (8), 139 (11), 125 (21), 111 (43), 97 (63), 83 (58), 69 (69), 57 (100), 55 (89), 43 (83), 41 (58). Hydrogenation gave 15-methylnonacosane in 94% yield as a white solid. 1H NMR: δ 0.83 (d, 3H, J = 6.4 Hz), 0.88 (t, 6H, J = 6.6 Hz), 1.00–1.15 (m, 2H), 1.16–1.40 (m, 51H).13C NMR: δ 14.35, 19.96, 22.93, 27.32, 29.60, 29.90, 29.94, 29.97, 30.27, 32.16, 32.98, 37.33. MS (m/z, abundance): 407 (M-15) (2), 225 (21), 224 (35), 207 (5), 196 (8), 169 (3), 155 (5), 141 (8), 127 (9), 113 (13), 99 (21), 85 (59), 71(76), 57 (100), 43 (62).

15-methyl-15E/Z-hentriacontene was obtained in 52% yield from 2-hexadecanone and hexadecyltriphenylphosphonium bromide. MS (m/z, abundance): 448 (M+, 6), 266 (7), 251 (8), 238 (10), 223 (8), 210 (7), 182 (5), 167 (5), 153 (7), 139 (12), 125 (19), 111 (39), 97 (58), 83 (53), 69 (63), 57 (100), 55 (80), 43 (87), 41 (50). Hydrogenation yielded (±)-15-methylhentriacontane in 95% yield as a white solid. 1H NMR: δ 0.83 (d, 3H, J = 6.6 Hz), 0.88 (t, 6H, J = 7.0 Hz), 1.01–1.14 (m, 2H), 1.18–1.38 (m, 55H). 13C NMR: δ 14.35, 19.96, 22.92, 27.31, 29.60, 28.89, 29.93, 29.96, 30.26, 32.15, 32.97, 37.32. MS (m/z, abundance): 435 (M-15) (2), 253 (6), 252 (9), 225 (6), 224 (12), 183 (3), 169 (4), 155 (5), 141 (7), 127 (8), 113 (12), 99 (19), 85 (46), 71(67), 57 (100), 43 (76).

Electrophysiology

For all single-sensillum recordings, flies were assayed 4–6 days post eclosion. In experiments using the Orco-GAL4 driver, experimental fly genotypes were w + , UAS-HsOr; w +, Orco-GAL4, and control flies were +; w +, Orco-GAL4. For empty neuron experiments, flies were Δhalo; Or22a-GAL4/UAS-HsOr263. Flies were mounted and extracellular recordings from a given sensillum were performed as previously described25. Trials were randomized within the constraint of fly line availability.

Sensillum type was confirmed using a six-odorant panel consisting of paraffin oil, or 2-heptanone, ethyl acetate, geranyl acetate, (E)-2-hexenal, and racemic 1-octen-3-ol dissolved in paraffin oil. Each odorant was diluted 100-fold in paraffin oil and 20 µl of each solution was loaded into a Pasteur pipette delivery cartridge. For hydrocarbons, each compound was diluted to 10 micromolar in neat pentane and 20 nmol (2 µl) were applied to each delivery cartridge (~3 cm from open end of Pasteur pipette). Each hydrocarbon cartridge was then heated for ~1 s (avg temp. 112.5 ± 2.8 °C, n = 10) with a handheld butane torch before being puffed over the fly antenna for 1 s in 6 ml of humidified air. Cartridges containing ant cuticular extracts were loaded with a normalized dose of 1.2 µg total hydrocarbon and heated in a similar fashion before application to the fly antennal preparation. Control cartridges were loaded with pentane only.

Spike frequencies were blindly and manually analysed. Spikes were counted in a 200 ms window between 0.4 and 0.6 s of the 1 s stimulus, and the Δspikes/sec was calculated by subtracting both the pre-stimulus spike frequency and then the response of the solvent (pentane) spike frequencies.

Chemical space analysis

PCA analysis of Or responses to stimuli was performed using JMP software (SAS).

Data availability

All datasets are available from the authors upon request.