Drosophila melanogaster stocks

Drosophila melanogaster stocks Or67d^+[1] Or67d^{GAL4 [1]}, and Or67d^GAL4[1];UAS-HvOR13 (Kurtovic et al. 2007) were provided by A. Widmer, Research Institute of Molecular Pathology (Vienna, Austria). UAS-HvOR13* and UAS-HvOR15 were prepared by subcloning an H. virescens OR13* cDNA and an H. virescens OR15 cDNA, respectively, into pUAS (donated by J. Mahaffey, North Carolina State University, Department of Biological Sciences, Raleigh, NC) using Novagen pSTBlue-1 Acceptor Vector Kit (EMD Millipore,  www.emdmillipore.com). The HvOR13* cDNA contained two amino acid polymorphisms and an early termination codon (Figure 1). Transgenic fly lines containing UAS-HvOR13* and UAS-HvOR15 were generated by Rainbow Transgenic Flies, Inc. ( www.rainbowgene.com). To express the HvOR13* and HvOR15 in the trichoid Tl sensilla, six UAS-HvOR13* lines were crossed to Or67d^{GAL4 [1]} and four UAS-HvOR15 lines were crossed to Or67d^{GAL4 [1]}.

Single sensillum recordings

Recordings were performed as described by Syed et al. (2006). In brief, a D. melanogaster adult was restrained, a glass reference electrode was placed in the eye, and the recording electrode was brought into contact with the base of the trichoid sensillum. Recorded extracellular action potentials (spontaneous and upon stimulation) were amplified, fed into an IDAC4-USB box (Syntech,  www.syntech.nl), recorded, and analyzed with Auto Spike version 3.7 (Syntech). AC signals (action potentials or spikes) were band-pass filtered between 100 and 10,000 Hz. The preparation was held in a humidified air stream delivered at 20 mL/sec, to which a stimulus pulse of 2 mL/sec was added for 500 msec. Unless specified otherwise, signals were recorded for 10 sec starting 2 sec before stimulation, and spikes were counted off-line in a 500 msec period before and during the stimulation. Responses from individual neurons were calculated as the increase in spike frequency (spikes/sec) relative to the pre-stimulus frequency. At least three flies of each of the four genotypes (Or67d^+[1], Or67d^{GAL4 [1]}; UAS-HvOR13, Or67d^{GAL4 [1]};UAS-HvORl3*, and Or67d^{GAL4 [1]}\UAS-HvORl5) were examined, and recordings were made from up to five sensilla from each fly tested. Data were pooled after observing no significant differences between sensilla, sexes, or age groups (1- to 5-day-old flies) were observed.

The following compounds were used as stimuli: (Z)-ll-hexadecenal (Z11-16:Ald), (Z)-9-tetradecenal (Z9-14:Ald), (Z)-9-hexadecenal (Z9-16:Ald), hexadecanal (16:Ald), (Z)-11-hexadecenyl acetate (Z11-16:OAc), (Z)-11-hexadecen-1-ol (Z11-16:OH), and (Z)-9-hexadecen-l-ol (Z9-16:OH) (all 93–95% minimum purity) and cVA ( ⩾98% purity) . All compounds were purchased from Bedoukian ( www.bedoukian.com) except for cVA, which was purchased from Cayman Chemical ( www.caymanchem.com). A 10 μL aliquot of a stimulus dissolved in double distilled hexane at the desired dose was loaded on a filter paper strip, the solvent evaporated for 30 sec, and the strip was placed in a disposable Pasteur pipette. Hexane loaded on a filter paper strip alone and an empty Pasteur pipette served as controls. All pheromone components were tested at 10 μg source dose for initial screening and subsequent recordings were performed with varying doses ranging from 10 ng to 10 μg. Source doses indicated throughout the manuscript for electrophysiology data represent the dilutions of the solution deposited onto the filter paper of the stimulus cartridge. Thus, a source dose of 10 μg means a 10 μL solution of 10 μg/μL was deposited.

Courtship assays

Single pair courtship assays were performed following Kurtovic et al. (2007). In brief, Or67d^+[1] virgin female flies (5–8 days old) were treated by applying 0.2 μL of Z11-16:Ald (4 μg), Z9-14:Ald (2 μg), or acetone (solvent control) onto their dorsal abdomens while lightly anaesthetized with CO₂. Treated females were placed individually in a round chamber (10 mm diameter and 4 mm height) with moistened nitrocellulose paper and paired with single Or67d^+[1], Or67d^{GAL4 [1]};UAS-HvORl3, or Or67d^{GAL4 [1]};UAS-HvOR13* male flies (5*8 days old). A clear acetate sheet prevented contact between female and male flies. Flies were allowed to recover for 1 hr before behavioral assays were performed. Courtship index, the percentage of time for which the male courts the female during a 10-min assay, was used to quantify male courtship behavior. In these assays, Or67d^+[1] male flies were expected to be avid courters in greater than 70% of all treated female flies. In the courtship ritual, the male orients toward and follows the female, taps her with his forelegs, sings a courtship song by extending and vibrating one wing, licks her genitalia, and finally curls his abdomen for copulation (Demir and Dickson 2005). In contrast, Or67d^{GAL4 [1]}; UAS-HvOR13 and Or67d^{GAL4 [1]};UAS-HvORl3* male flies were expected to display a reduced courtship index when paired with females treated with Z11-16:Ald, as this compound would inhibit male courtship behavior in these transgenic flies, which have HvOR13 replacing Or67d, the receptor for cVA, a male sex pheromone that inhibits male courtship.

HvOR13 expression levels by qRT-PCR

HvOR13 mRNA expression levels were measured in heads of Or67d^{GAL4 [1]};UAS-HvOR13 and Or67d^{GAL4 [1]};UAS-HvORl3* male flies by qRT-PCR. Total RNA from each sample (pool of five adult heads) was isolated and purified using the RNeasy Plus Mini Kit (Qiagen,  www.qiagen.com). RNA quality and quantity were determined using a NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies,  www.nanodrop.com). Total RNA (150 ng) was converted to cDNA using Ambion ArrayScript reverse transcriptase (Ambion, Invitrogen,  www.invitrogen.com). Random hexamers (Applied Biosystems, Invitrogen) were used as cDNA synthesis primers in a reaction mix that included 10X Array Script buffer (Ambion), RNAseOUT (Invitrogen), and 10 mM dNTP (Invitrogen). Primers targeting exons of HvOR13 and the housekeeping gene RP49 were designed with PRIMER EXPRESS 2.0 software (Applied Biosystems) set to select for an optimal primer annealing temperature of 59° C (58–60° C), amplicon sizes of 40–150 bp, a -3′GC clamp = 0, and a minimum and maximum GC content of 30% and 80%, respectively. Primers were designed based on H. virescens OR13 and B. mori RP49 mRNA sequences obtained from GenBank database. Quantitative RT-PCR was performed with an ABI Prism 7900 sequence detector and 96-well optical reaction plates (Applied Biosystems). All reactions were performed in triplicate in a total volume of 10 μL containing 5 μL of SYBR Green PCR Master Mix (Applied Biosystems) and 10 μM of each primer under the following conditions: 50° C for 2 min, 95° C for 10 min followed by 40 cycles of denaturation at 95° C for 15 sec, annealing and extension at 60° C for 1 min, followed by 95° C for 15 sec and 60° C for 15 sec. A dissociation curve and negative control (cDNA reaction without reverse transcriptase enzyme) were used to ensure primer specificity and lack of contamination. Six samples per genotype were examined, and the same samples were run on separate plates twice (two runs). A standard curve was generated for each primer set using dilutions of genomic DNA to calculate the relative quantities of mRNA levels. For each sample, the ratio of the expression level of the target gene to that of the control gene (RP49) was calculated (ABI User Bulletin 2, Applied Biosystems) and used for data analysis.

HvOR13 cDNA sequence comparison between Or67d^GAL4[1];UAS-HvOR13 lines

HvOR13 was amplified by RT-PCR from H. virescens male antennae and from heads of Or67d^{GAL4 [1]}; UAS-HvOR13 and Or67d^{GAL4 [1]};UAS-HvORl3* flies using gene specific primers designed based on published H. virescens cDNA sequences (Krieger et al. 2004). H. virescens male antennal RNA, and RNA from heads of Or67d^{GAL4 [1]};UAS-HvOR13 and Or67d^{GAL4 [1]};UAS-HvOR13* lines, were isolated using Qiagen RNeasy Plus. cDNA was synthesized using Qiagen QuantiTect Reverse Transcription kit with a total RNA concentration of 0.5 μg. PCR was performed using the FastStart High-fidelity PCR system (Roche,  www.roche.com) under the following conditions: 94° C for 3 min followed by 19 cycles of denaturation at 94° C for 1 min, annealing at 57° C for 1 min with 0.5° C decreasing per cycle, extension at 72° C for 2 min followed by 19 cycles of denaturation at 94° C for 1 min, annealing at 47° C for 1 min and extension at 72° C for 2 min, followed by 72° C for 7 min. PCR products were purified and sequenced by Genewiz ( www.genewiz.com). Nucleotide and translated sequences were aligned using ClustalW2 (EMBL-EBI,  www.ebi.ac.uk).

Electrophysiological assays

These experiments were designed to examine whether HvOR13 expressed in D. melanogaster Tl sensilla have high specificity and sensitivity for Z11-16:Ald, as compared to Wang et al. (2011). Or67d^{GAL4 [1]};UAS-HvOR13 T1 sensilla responded to varying doses (0.01–10 μg) of Z11-16:Ald in a dosedependent manner (Figure 2A, B) while T1 sensilla of Or67d^{GAL4 [1]};UAS-HvOR13* responded only to the highest dose of Z11-16:Ald tested (10 μg) (Figure 2B). The control Or67d^+[1] did not respond to any of the H. virescens pheromone components, except for Z11-16:OAc at the highest dose (10 μg) tested (87 ± 15.87 spikes/sec, n = 7), and responded to doses of cVA between 0.1–10 μg (Figure 3). In addition, Or67d^{GAL4 [1]};UAS-HvORl3 Tl sensilla responded only to high doses (10 μg) of Z9-14:Ald and Z9-16:Ald, and no response to 16:Ald, Z11-16:OAc, Z11-16:OH, or Z9-16:OH was observed at the same high dose (Figure 4). Or67d^GAL4[1];UAS-HvOR13* Tl sensilla did not respond to any of the other pheromone compounds tested. Or67d^{GAL4 [1]};UAS-HvOR15 T1 sensilla did not respond to any of the pheromone compounds tested.

Table 1.

Courtship indices (mean ± SE) for males of the indicated genotypes paired with Or67d^+[1]; virgin female Drosophila melanogaster treated with acetone, Z11-16:Ald, or Z9-14:Ald.

Behavioral assays

In single pair courtship assays, the genotypes Or67d^+[1]; Or67d^{GAL4 [1]}; UAS-HvOR13, and Or67d^{GAL4 [1]};UAS-HvORl3* had comparable responses when paired with Or67d^+[1] virgin female flies treated with acetone (F_2,40 = 1.60, p = 0.2152) (Table 1). Comparable responses were also recorded when females were treated with Z9-14:Ald (F_2,32 = 0.58, p = 0.5634), a H. virescens pheromone component for which no electrophysiological responses were observed in the transgenic fly strains. In contrast, Or67d^{GAL4 [1]}; UAS-HvOR13 and Or67d^{GAL4 [1]};UAS-HvORl3* males exhibited a lower courtship index than that of the Or67d^+[1] males when paired with female flies treated with Z11-16:Ald (F_2,44.7 = 63.67, p < 0.0001) (Table 1). The lower courtship index indicates that male flies expressing HvOR13 detected Z11-16:Ald and responded accordingly.

HvOR13 expression and cDNA sequence comparison

HvOR13 mRNA expression levels in Or67d^{GAL4 [1]}; UAS-HvOR13 (HvOR13/RP49 = 0.038 ± 0.005) and Or67d^{GAL4 [1]};UAS-HvOR13* (HvOR13/RP49 = 0.043 ± 0.005) flies were examined, but no differences in transcript abundance were found between these lines (t = 0.41, p = 0.91), indicating that differences in electrophysiological and behavioral responses are not related to differences in gene expression. HvOR13 coding sequence comparison between H. virescens and the Or67d^{GAL4 [1]};UAS-HvOR13, and Or67d^GAL4[1];UAS-HvOR13* flies indicated some sequence variation that may represent nucleotide polymorphisms (122A>C; 287C>T; 387C>T; 504A>G; 714T>C; 717C>T;732G>A;861T>C) or single point mutations (160G>C; 261T>C;606C>T;801G>A;877A>G; 947T>A;1150C>T). Point mutations led to two conserved amino acid changes at position 54 (V → L) and 293 (M → V), a major amino acid change at position 316 (L → Q), and an early termination codon at position 384 (Q → stop codon) in Or67d^{GAL4 [1]}; UAS-HR13* flies (Figure 1).