DOI: 10.5061/dryad.rbnzs7hhd
title: Data from: A mosaic of endogenous and plant-derived courtship signals in moths

description: Insects rely on olfaction to guide a wide range of adaptive behaviors,
 including mate- and food-localization, mate choice, oviposition site
 selection, kin recognition and predator avoidance. In nocturnal
 insects, such as moths and cockroaches, mate finding is stimulated
 predominantly by long-range species-specific sex pheromones, typically
 emitted by females. During courtship, at close-range, males in most moth
 species emit a blend of pheromone compounds from an everted, often large,
 pheromone gland. While long-distance communication with sex pheromones has
 been remarkably well characterized in thousands of moth
 species, close-range chemosensory sexual communication remains
 poorly understood. We reveal that in the moth Chloridea virescens, the
 male pheromone consists of three distinct classes of compounds: de novo
 biosynthesized alcohols, aldehydes, acetates and carboxylic acids that
 resemble the female’s emissions, and newly identified compounds that are
 unique to the male pheromone: aliphatic polyunsaturated hydrocarbons and
 sequestered plant secondary compounds and hormone derivatives, including
 methyl salicylate (MeSA). Thus, males employ a mosaic pheromone blend of
 disparate origins that may serve multiple functions during courtship. We
 show that two olfactory receptors in female antennae are tuned to MeSA,
 which facilitates female acceptance of the male. Because MeSA is emitted
 by plants attacked by pathogens and herbivores, the chemosensory
 system of female moths was likely already tuned to this plant volatile,
 and males appear to exploit the female’s preadapted sensory bias.
 Interestingly, while female moths (largely nocturnal) and butterflies
 (diurnal) diverged in their use of sensory modalities in sexual
 communication, MeSA is used by males of both lineages.
description: Chloridea rearing We used the standard
 laboratory YDK strain of <em>C. virescens</em> for our
 experiments, which was originally collected in 1988 from seven tobacco
 fields in Yadkin County, and one field each in neighboring Stokes and
 Forsythe Counties, North Carolina, USA.<sup>44</sup> Eggs were
 briefly rinsed in a 1.3% bleach solution, and after drying eggs were
 placed individually in 30 ml diet cups (DART, Mason, MI, USA) each
 containing 7.5 ml of artificial diet45 and maintained at 26 ± 1°C with a
 photoperiod of 14:10 (L: D). Pupae were separated by sex and placed in
 separate plastic cups. Emerged adults were checked daily and provided with
 a 10% sucrose solution. Male and female pupae and adults were kept in
 separate incubators at 26 ± 1°C and on a reversed photocycle (14 L:10 D)
 with L-off at 09:00. GC-EAD analysis of male HP
 components Gas chromatography (GC) coupled to
 electroantennogram detection (EAD) was performed as described
 previously.46 A Hewlett-Packard 5890 Series II GC (Agilent, Folsom, CA,
 USA), equipped with either a non-polar capillary column (EC-5, 30 m × 0.25
 mm ID, 0.25 μm film thickness; Alltech, Deerfield, IL, USA) or a polar
 capillary column (EC-Wax, 30 m × 0.25 mm ID, 0.25 μm film thickness;
 Alltech) was used for GC-EAD analyses. The oven temperature was held at
 50°C for 2 min, then increased by 15°C/min to 250°C. Injector and detector
 temperatures were both set to 250°C. Splitless injection was used with
 hydrogen as carrier gas at a flow of 1.2 ml/min. The column effluent was
 split 1:1 to a flame ionization detector (FID) and through a heated
 transfer line (250°C) to the EAD. Moth antennae were
 excised with micro-scissors from 1-3-day-old virgin females and positioned
 between two gold wire electrodes within saline-filled glass capillaries.
 The physiological saline solution was adjusted specifically for
 <em>Chloridea</em> based on Hayashi and Hildebrand47: 8.77
 mg/ml NaCl, 0.3 mg/ml KCl, 0.67 mg/ml CaCl<sub>2</sub>, 2.4
 mg/ml HEPES, and 0.9 mg/ml D-(+)-glucose were dissolved in
 diH<sub>2</sub>O, and the pH adjusted to ~7 with 1 N NaOH. Aliquots of the saline
 solution were stored at –30°C. The electrodes were held in a
 custom-made acrylic holder that was placed inside a humidified cooling
 condenser maintained at ~15°C. Pure humidified air at
 ambient temperature was continuously blown over the antenna preparation in
 the cooled condenser. The output signal from the antennae was amplified
 10× by a custom high-input impedance DC amplifier and filtered by a
 high-pass filter with a cutoff frequency of 0.5
 Hz.<sup>46</sup> The amplifier output was routed through a
 signal acquisition board within the GC and displayed along with the FID
 signal in Agilent OpenLab CDS (version A.01.07). GC-MS
 analysis of male HP components For compound
 identification we used a 6890N GC coupled with a 5975 mass-selective
 detector (MS, Agilent, Folsom, CA, USA) operating in electron impact
 ionization mode (MS ion source set to 230°C, MS quadruple set to 150°C).
 Samples were injected in splitless mode with an initial temperature of
 280°C. Separation of compounds was performed on a non-polar column
 (HP-5ms, 30 m x 0.25 mm x 0.25 μm (5%-phenyl)-methyl-siloxane stationary
 phase, Agilent). The temperature program started at 50°C for 1 min and
 then increased by 10°C per min to a final temperature of 320°C. Compounds
 were identified according to their Kovats indices, diagnostic ions, and
 their respective mass spectra. The Wiley 7/NIST 05 database was used for
 reference, and the identity of novel compounds was confirmed with
 synthetic standards (Sigma-Aldrich, St. Louis, MO, USA) processed in the
 GC-MS as well as on the GC-EAD-FID; both non-polar and polar columns were
 used. Behavioral assays Observations
 of moth mating behavior were conducted in a dark room at 26 ± 1°C.,
 illuminated with dim red lights. Two-day old adult moths, which are best
 suited for behavioral assays,48 were used in all mating behavior
 experiments. The moths were taken out of the incubators and allowed to
 acclimate in the dark room 1 hr before L-off. Thirty min before L-off, one
 male and one female (2-days old) were placed in an observation arena
 (cylinder, 9 cm diameter x 7.5 cm high) (Fabri-Kal Corp, Kalamazoo, MI,
 USA) and moths were observed for mating every 30 min for 5 hrs. Moths mate
 for several hours, so pairs that remained in copula for at least 60 min
 were considered mated.<sup>21</sup> Effects
 of HP ablation on mating Application of gentle pressure
 to the abdomen caused the hairpencil (HP) complex to extend. HP structures
 were ablated by removing only the hairs associated with structure-2
 (Figure S1A–D) with fine forceps. Sham-operated males were squeezed to
 expose the HPs, but no hairs were removed. All sham-operations and
 ablations were conducted before the onset of the photophase, about 14 hrs
 before the behavioral experiments, to give adequate time for the males to
 recover after the surgery. Two groups of male moths
 were set up: (1) HP<sup>+</sup>: Sham-operated <em>C.
 virescens</em> males; (2) HP<sup>–</sup>:
 HP-ablated<em> C. virescens</em> males. The mating rate of
 these two groups of males with 2-day-old virgin female moths was monitored
 in single pair assays. Extraction of HP chemicals after
 HP-ablation The hairs removed during HP ablation are
 largely storage and emission sites, whereas their underlying cells
 presumably biosynthesize the male sex pheromone. To determine whether HP
 ablation in fact reduced the amount of male sex pheromone, the HPs were
 extracted, and the quantity of male sex pheromone was determined by GC-FID
 analysis. In separate groups of males, the structure-2 hairs of the HPs
 (Figure S1A–D) were excised, and either 1 hr or 14 hrs later the whole HP
 complex was removed and extracted. Two extractions were performed. Short
 (10 sec) extractions were used to represent the chemicals on the surface
 of the HP gland. Long (20 min) extractions were designed to extract the
 chemicals from both the surface and interior of the HP glands. Each HP was
 extracted in 100 μl hexane containing 40 ng pentadecyl acetate (15:OAc) as
 internal standard. All extracts were kept at −30°C prior to chemical
 analysis and analyzed individually on an Agilent 7890A GC equipped with a
 30 m × 0.25 mm ID DB-5 capillary column (Agilent). The amount of 16:OAc,
 the main component of the HPs of <em>C. virescens</em>, was
 quantified in each extract. Effects of HP extract and
 MeSA on mating A large pool of HP extracts was produced
 by excising the structure-2 hairs of 2–4-day-old <em>C.
 virescens</em> males in 100 μl hexane per male. For behavioral
 assays, one male-equivalent (1 ME; 100 μl of extract) was applied to a
 42.5 mm diameter filter paper disc (Whatman #1, Sigma, St. Louis, MO,
 USA). The hexane was allowed to evaporate, and the filter paper was added
 to the behavioral assay arena. The effect of HP extract
 and MeSA on mating of <em>C. virescens</em> was tested using
 HP-ablated males and 2-day old virgin females. Hair pencil extract and a
 range of MeSA doses were loaded onto filter papers and the mating rates in
 different groups were assessed. Four concentrations of MeSA were tested
 (100 ng, 10 ng, 1 ng and 0.1 ng) along with a hexane control for a total
 of six treatment groups: (1) Hexane: 100 μl hexane; (2) HP extract: 100 μl
 (1 ME) of C. virescens HP extract; (3) MeSA 100 (100 ng MeSA: 100 μl of 1
 ng/μl); (4) MeSA 10 (10 ng MeSA: 100 μl of 0.1 ng/μl); (5) MeSA 1 (1 ng
 MeSA: 100 μl of 0.01 ng/μl); (6) MeSA 0.1 (0.1 ng MeSA: 100 μl of 0.001
 ng/μl). Cloning and sequence analysis of MeSA
 ORs The full ORF sequences of CvirOR27 and CvirOR43
 were obtained in the <em>C. virescens</em> antennal
 transcriptomes based on the basic local alignment search tool (BLAST)
 using the HarmOR27 and HarmOR43 sequences as queries. Full-length coding
 sequences of CvirOR27 and CvirOR43 were obtained from the antennal cDNA of
 C. virescens adults by PCR with specific primer pairs for each gene.
 Phylogenetic analysis was performed using 20 ORs from <em>C.
 virescens</em> (including 18 previously reported ORs and the two ORs
 we identified in this study) and 65 ORs from <em>H.
 armigera</em>. Amino acid sequences were aligned using ClustalW and
 neighbor-joining tree was constructed using Poisson model as implemented
 in MEGA X software. Functional characterization of
 ORs The Functional characterization of individual ORs
 was performed by heterologous expression in <em>Xenopus</em>
 oocytes combined with a two-electrode voltage-clamp
 system.<sup>49</sup> Briefly, the full-length gene of each OR
 and Orco were subcloned into the eukaryotic expression vector pT7TS. cRNAs
 were generated from linearized expression vectors using the mMESSAGE
 mMACHINE T7 kit (Ambion, Austin, TX, USA). Then, the cRNA mixture of ORx
 and Orco (27.6 ng each) was injected (Nanoliter 2010, WPI Inc., Sarasota,
 FL) into Xenopus oocytes. After incubation in nutrient solution at 18°C
 for 3–5 days, the response profile of each oocyte to multiple plant
 odorants was recorded via a two-electrode voltage clamp (OC-725C oocyte
 clamp, Warner Instruments, Hamden, CT, USA) at a holding potential of -80
 mV. Data were acquired by using a Digidata 1440 A and were analyzed by
 pCLAMP 10.2 software (Molecular Devices, San Jose, CA, USA).
 Stock solutions of each odorant were prepared at 1 M using DMSO
 as a solvent, and each odorant was diluted in 1× Ringer’s solution (96 mM
 NaCl, 2 mM KCl, 5 mM MgCl<sub>2</sub>, 0.8 mM
 CaCl<sub>2</sub>, and 5 mM HEPES, pH 7.6) to the indicated
 concentrations for electrophysiological recording. Information on the 66
 odorants used in this study is listed in Table S5.
 QUANTIFICATION AND STATISTICAL ANALYSIS All
 quantification and statistical analysis methods are described in the
 Figure legends and Table legends. Quantification of compounds in HP
 extracts was done using Agilent OpenLab CDS software (version
 A.01.07). The distribution of 16:OAc and MeSA in various structures of the
 HPs and the effects of extraction duration were assessed using ANOVA
 followed by Tukey’s Honestly Significant Difference test (JMP). The
 time-course of cumulative mating success was analyzed using survival
 analysis. A Cox Proportional Hazards model was fitted and the Wald test
 was used to compare treatments. Dunnett’s multiple comparison test was
 used to compare supplementation with various doses of MeSA to the
 no-supplementation control (0-ng treatment). For all statistical analyses
 α = 0.05.