Fly stocks were raised in vials containing standard fly medium made of yeast, corn and agar. The stocks were maintained in fly incubators at 25 °C and 60% humidity on a 12h:12h light-dark circle. In most cases, flies for behavioral assays were collected within 8 h after eclosion and were raised individually (“tester” males), or at 30 males/vial (“target” males) for 5–7 d before behavioral assays.
Canton-S flies were obtained from M. Heisenberg. Gr32a−/−
, the genomic rescue strains, and the Gr32a-GAL4
were from T. M. and H. A.. The fly strains for the ablation of oenocytes were from J-C. B. and J. D. L.. The Or47b
mutant alleles were generated and characterized by J. M. at L. Vosshall’s laboratory, and were backcrossed into the Canton-S background by J-C. B. and J. D. L..
Genetic elimination of male CHs
Ablation of male oenocytes6
was achieved by crossing male “+; PromE(800)-GAL4, tub-GAL80TS; +
” flies to female “+; UAS-StingerII, UAS-hid/CyO; +
” or female “+; UAS-StingerII; +
” at 18 °C to generate oe−
male progeny, respectively. Adult male progeny were collected within 8 h after eclosion and kept at 25 °C for 1 d. Subsequently, both oe+
males were maintained at 30 °C during the daytime and at 25 °C during the nighttime for 3 more d. The flies were then maintained at 25 °C for 1–2 d before use. The expression of UAS-hid44
in oenocytes specifically in the adult stage (under the control of tub-GAL80TS
eliminates most if not all male CHs.
Unless otherwise indicated, the behavioral assays were performed using mixed fly pairs consisting of one single-housed “tester” male and one group-housed “target” male. As previously shown, group housing suppresses both aggression and courtship by male flies2, 46
. Using a single-housed tester and a group-housed target male forces aggression and courtship to be conducted predominantly by the tester males. This uni-directional bias in the initiation of aggressive or courtship behaviors by the tester towards the target male facilitated analysis of the effects of experimental manipulations on the tester vs. the target.
To measure social interactions, one tester male and one target male were introduced into the behavior chamber (see below) by gentle aspiration. Their behavioral interactions were videotaped for 20 min and analyzed manually or by using custom CADABRA software2
. To distinguish testers and targets, a blue dot was painted on the thorax of target males under CO2
anesthesia, 1–2 d before behavioral assays were performed.
The design of the behavior arena was adapted from previous reports2, 47
. Briefly, a rectangular chamber (4 cm × 5 cm × 12 cm) was placed on top of an acrylic base. In the center of the base, a 1 cm × 1 cm × 0.5 cm hole was filled with apple juice-agar-sucrose medium, surrounded by a 0.5 cm wide border containing 1% agar medium. The behavior arena was illuminated by a ring-shaped fluorescent lamp. Videotaping was performed using a commercial camcorder (Sony DCR-HC38) placed on top of the arena.
For experiments involving synthetic cVA, the experimental design was as previously reported13
. Briefly, before and during behavioral assays, a small piece of filter paper containing 5 μl solvent (acetone) or 5 μl solvent containing 500 μg cVA was placed at one corner of the behavior arena. Male flies of the indicated genotypes were housed at 10 flies/vial for 5–7 d before behavioral assays. A pair of male flies of identical genotype and age was introduced into the behavior arena and their behavioral interactions were recorded for 20 min and analyzed by using custom CADABRA software.
For courtship assays between males and females, virgin Canton-S females were group-housed (10 flies/vial) for 5–7 d before behavioral assays. One tester male fly and one virgin Canton-S female were introduced into the behavior arena and their behavioral interactions were recorded for 10 min. The latency to copulate and the occurrence of one wing extensions were scored if applicable.
Chemical synthesis of CH molecules
(Z)-7-pentacosene (7-P) was synthesized as follows. n-BuLi (2.5 M in hexanes, 8.86 ml, 22.2 mmol) was added drop-wise to a solution of 1-octyne (2.22 g, 20.2 mmol) in THF (100 ml) at 78 °C and the mixture was stirred at 78 °C for 30 min and at 0 °C for 30 min. The resulting solution was treated with a solution of 1-bromoheptadecane (5.10 g, 16.0 mmol) in THF (10 ml), tetrabutylammonium iodide (0.74 g, 2.02 mmol), and refluxed for 15 h. The reaction mixture was quenched with saturated aqueous NH4Cl and extracted with ether. The combined ether extracts were washed with brine, dried (MgSO4), and concentrated under vacuum. The residue was chromoatographed (hexanes) to give 7-pentacosyne as a colorless viscous oil (2.93 g, 42 %).
To a solution of 7-pentacosyne (0.920 g, 2.64 mmol) in hexanes (35 ml) was added quinoline (0.853 g, 6.60 mmol) and Lindlar catalyst (0.562 g). The resulting suspension was vigorously stirred under a hydrogen balloon for 2 h. The catalyst was filtered off through a pad of Celite. The solvent was evaporated and the residue was purified by flash chromatography (hexanes) to afford (Z)-7-pentacosene as a colorless oil (0.823 g, 89%) with greater than 98% purity by gas chromatography.
(Z)-7-tricosene (7-T, greater than 98% purity by gas chromatography) was synthesized from 1-octyne and 1-bromopentadecane employing a procedure analogous to that used to synthesize (Z)-7-pentacosene.
was prepared employing a modification of published procedures48, 49
. A solution of methylmagnesium bromide (3.0 M in ether, 2.11 ml, 6.34 mmol) was added drop-wise to a stirring suspension of (dppp)NiCl2
(1.56 g, 2.88 mmol) in benzene (40 ml) and the mixture was refluxed for 15 min. A solution of hexylmagnesium bromide (2.0 M in ether, 36.0 ml, 72.0 mmol) was added and most of the ether was removed by distillation under N2
. Benzene (100 mL) and dihydropyran (9.09 g, 108 mmoL) were added and mixture was refluxed for 16 h. The reaction mixture was quenched with saturated aqueous NH4
Cl and extracted with ether. The combined ether extracts were washed with brine, dried (MgSO4
), and concentrated under vacuum. The residue was twice chromatographed (hexanes/EtOAc = 20/1 to 10/1), the second time on silica gel embedded with AgNO3
(10% w/w) to give (Z
)-4-undecan-1-ol as a colorless viscous oil (0.675 g, 6 %).
A mixture of (Z)-4-undecan-1-ol (0.635 g, 3.73 mmol) and PCC (1.21 g, 5.60 mmol) was stirred in CH2Cl2 (10 ml) for 1 h. The suspension was diluted with ether, filtered through a pad of Celite, and concentrated under vacuum. The residue was chromoatographed (hexanes/EtOAc=10/1) to give (Z)-4-undecanal as a colorless oil (0.338 g, 54 %).
n-BuLi (2.5 M in hexanes, 0.63 ml, 1.57 mmol) was added drop-wise to a solution of hexadecyltriphenylphosphonium bromide (0.975 g, 1.72 mmol) in THF (30 ml) at −30 °C. The reaction mixture was allowed to warm to room temperature for 20 min, then cooled to −30 °C, HMPA (5 ml) was added, and cooled to −60 °C. A solution of (Z)-4-undecanal (0.240 g, 1.43 mmol) in THF (10 ml) was added drop-wise and the mixture was allowed to warm to room temperature. The reaction mixture was quenched with water and extracted with ether. The combined ether extracts were washed with brine, dried (MgSO4), and concentrated under vacuum. The residue was chromoatographed (hexanes) to give (Z, Z)-7,11-heptacosadiene as a colorless oil (0.361 g, 67%) with greater than 98% purity by gas chromatography.
Quantification of male CHs
The quantification method was adapted from earlier reports6, 14
. Briefly, individual male flies were CO2
anesthetized and washed for 5 min in 25 μl of iso-octane containing 20 ng/μl of octadecane as an internal standard. The iso-octane extracts were analyzed by gas chromatography. 1 μl of each male CH extract was injected into a Hewlett-Packard 5890 II gas chromatograph coupled with a HP 5972 mass selective detector system. The injector was held at 300 °C and operated in splitless mode for 0.75 min after injection. A 30 m × 0.25 mm ID × 0.25 μm film thickness RTX-5MS column from Restek Corporation (Bellafonte, Pennsylvania, US) was operated with a flow of 0.9 ml/min helium corresponding to a linear velocity of 34.4 cm/sec. The oven temperature began at 55 °C for 1.5 min and was ramped at 40 °C/min to 135 °C and then at 25 °C/min to 235 °C and then at 3 °C/min to 275 °C where it was held for 1 min. Electron impact spectra (70 eV electron energy) were recorded from 50 to 550 m/z at a rate of 1.5 scans per sec. HP Chemstation G1701 BA version B.01.00 software was used to calculate the retention time, the total peak area, and the identity of each compound.
Perfuming of male flies with CH molecules
The procedure for perfuming live oe−
males with male CHs was based on a passive transfer protocol adapted from a previous report7
. Briefly, ten 5–6 d old oe−
male flies were mixed with 100 oe+
males in small vials (~10 cm3
). The vials were placed upside-down in a 25 °C incubator for 1 d before behavioral assays or gas chromatography. Such a protocol ensured that a wild type-equivalent amount of male CH molecules was transferred to individual oe−
males. For behavioral assays, these oe−
males were marked by a blue dot on the thorax, which could be used to sort them out from oe+
males without anesthesia. For gas chromatography, these males were instead marked by cutting off one wing before mixing.
The procedure for perfuming male flies with synthetic CH molecules was also adapted from a previous report6
. Briefly, the compound of interest (2.5 μl for 7-T, 1 μl for 7-P and 1.5 μl for 7,11-HD, or no compound for control) was applied directly onto a small piece of filter paper in a 5 ml glass vial. Groups of 5–8 males were introduced into the vial by gentle aspiration, and vortexed twice at medium speed, each for 20 sec. The male flies were then transferred to fresh vials containing fly food. The vials were placed upside-down in the 25 °C incubator for 24 h before behavioral assays or gas chromatography. Such a protocol ensured that a wild type-equivalent amount of 7-T, 7-P or 7,11-HD was transferred to individual males.
To perfume different amounts of synthetic 7-T, an identical procedure was followed, except that the oe− males carrying synthetic 7-T were allowed to recover in vials for 6 h, 24 h, 72 h and 96 h before behavioral assays or gas chromatography, resulting in the oe− males carrying progressively smaller amounts of 7-T as a function of recovery time. Target males flies were used at a comparable age for behavioral assays or gas chromatography, independent of their post-transfer recovery times.
For experiments involving TRPV1, capsaicin (Sigma M2028) was dissolved in ethanol at 400mM, and subsequently diluted at 1:25 in acetone (adapted from ref. 24
). 0.5 μl of this solution was carefully pipetted onto the abdomen of individual male flies under CO2
anesthesia. For control males, the same procedure was applied, except that no capsaicin was added in the ethanol:acetone solution. Flies were transferred back to vials and were allowed to recover for ~12 h before behavioral experiments.
Generation of Or47b mutant alleles
Mutants for Or47b
were generated by homologous recombination, using the “ends-out” technique50
, which replaces the exons of interest with a selectable marker, in this case the eye color pigmentation gene, white
. Regions 5′ and 3′ of the gene were amplified by PCR from genomic DNA as follows:
- 5′ ARM 5.218 kb: Or47b.up-for and Or47b.up-rev
- 3′ ARM 3.369 kb: Or47b.dn-for and Or47b.dn-rev
Fragments were subcloned into the CM105 (S. Chen and G. Struhl) vector which contains two polylinkers flanking the mini-white gene with a unique I-SceI site 5′ of the white gene, flanked by FRT sites and containing conventional P element repeats. The 5′ arm was cloned into the AvrII site and the 3′ arm was cloned into the NotI site. The construct was designed to delete sequences containing the first two exons and 1kb of DNA upstream of the translation start site. Virgin female flies carrying one targeting construct were crossed to w118, 70FLP, 70I-SceI, Sco/CyO and 3-day-old progeny were heat shocked at 38oC for 60 min. Homozygous transgenic lines were created by standard techniques. To check that the targeted homologous recombination took place, PCR primers Or47b.2-for and Or47b.2-rev were used to amplify sequence containing the first 2 exons of the Or47b gene, which were deleted in the null mutant. Primers Or47b.3-for and Or47b.3-rev were used to amplify sequence containing the last 4 exons of the Or47b gene, which is intact in the Or47b null mutants.
Most of the behavioral data were non-parametrically distributed. Mann–Whitney U tests (for pair-wise comparisons) and Kruskal-Wallis analysis of variance (ANOVA) (for comparisons among >2 groups) were applied. Significant difference among groups detected by Kruskal-Wallis ANOVA was analyzed using Dunn’s post hoc test (with corrections for multiple comparisons) to identify groups with statistically significant differences. Two-way ANOVA was applied for comparisons among cumulative copulation latency curves.