A new male sex pheromone and novel cuticular cues for chemical communication in Drosophila

Abstract

Background: In many insect species, cuticular hydrocarbons serve as pheromones that can mediate complex social behaviors. In Drosophila melanogaster, several hydrocarbons including the male sex pheromone 11-cis-vaccenyl acetate (cVA) and female-specific 7,11-dienes influence courtship behavior and can function as cues for short-term memory associated with the mating experience. Behavioral and physiological studies suggest that other unidentified chemical communication cues are likely to exist. To more fully characterize the hydrocarbon profile of the D. melanogaster cuticle, we applied direct ultraviolet laser desorption/ionization orthogonal time-of-flight mass spectrometry (UV-LDI-o-TOF MS) and analyzed the surface of intact fruit flies at a spatial resolution of approximately 200 mum.

Results: We report the chemical and spatial characterization of 28 species of cuticular hydrocarbons, including a new major class of oxygen-containing compounds. Via UV-LDI MS, pheromones previously shown to be expressed exclusively by one sex, e.g., cVA, 7,11-heptacosadiene, and 7,11-nonacosadiene, appear to be found on both male and female flies. In males, cVA colocalizes at the tip of the ejaculatory bulb with a second acetylated hydrocarbon named CH503. We describe the chemical structure of CH503 as 3-O-acetyl-1,3-dihydroxy-octacosa-11,19-diene and demonstrate a behavioral role for this compound as a long-lived inhibitor of male courtship. Like cVA, CH503 is transferred from males to females during mating. Unlike cVA, CH503 remains on the surface of females for at least 10 days.

Conclusions: Oxygenated hydrocarbons comprise a major previously undescribed class of compounds on the Drosophila cuticular surface. A newly discovered long-chain acetate, CH503, serves as a mediator of courtship-related chemical communication.

Figure 1. Cuticular hydrocarbon profiles of adult virgin male and female Drosophila melanogaster using direct UV-LDI MS analysis

(A) Flies are mounted on a custom-made sample plate using adhesive tape. A UV laser is used to scan two primary areas: the anogenital region (AG) and legs (both circled in white). The picture was taken after analysis. Some changes in the structural integrity of the abdomen and eyes occur after the flies are removed from the high vacuum conditions used during analysis. Damage to the cuticle by the UV-laser is not visible. Scale bar: 2 mm. (B) Each CH is detected bearing a potassium adduct and with a series of resolved isotopes. The signal for cVA is shown as an example. For each species, only the monoisotopic [M+K]⁺ peak is used to calculate intensity (indicated by arrow). (C, E) Representative CH profiles of the AG (C) and legs (E) of male flies. Males show large differences in CH expression when comparing the two regions. Novel oxygen-containing CH species are highlighted in blue. Major ion signals are labeled with the proposed elemental composition. All labeled signals correspond to potassiated molecules [M+K]⁺. Peaks attributed to sodiated species are not labeled. (D, F) Representative CH profiles of the AG (D) and legs (F) of female flies. Both regions appear similar in terms of content and relative abundances of individual CHs. Profiles of socially naïve females show low levels of signals corresponding to the male pheromone cVA (inset). The accompanying peaks in the inset represent non-specific chemical noise. (G, H) In-situ MS profiling followed by carbonization with a second laser was used to mark a region of the male terminalia expressing the highest levels of cVA and CH503. Prior to laser ablation, the penis mantle is darkly pigmented (G). The site of interest was located first using the standard UV-laser with a 200 μm flat top beam profile (no visible damage to the cuticle is induced by this laser). A second, more tightly-focused high-intensity UV-laser beam with ca. 100 μm diameter was aimed at the site illuminated by the first UV-laser, thereby causing carbonization of the cuticle. After laser-ablation, the penis mantle pigmentation is no longer present and carbonization of the penis is evident (H). The marked site is indicated by a bracket and the inset shows a mass spectrum taken from this region. Scale bar: 100 μm.

Figure 2. Female Drosophila exhibit changes in CH profile for at least 10 d after successful copulation

(A) Male flies exhibit several stereotyped behaviors during courtship such as wing extension (indicative of singing), following the female, tapping the female with the forelegs, and copulation. (B–D) Representative UV-LDI mass spectra from the anogenital region (AG) of females before and after mating. 24 h after mating, levels of cVA and CH503 are significantly higher. Increases in hydroxylated tricosene (C₂₃H₄₆O) and hydroxylated tricosadiene (C₂₃H₄₄O) abundance also are observed. By 8 d, cVA on the cuticle is found near baseline quantities while the signal for CH503 is still evident. All assigned peaks correspond to potassiated molecules [M+K]⁺. (E, F) Intensity values for cVA, hydroxylated tricosene and tricosadiene, and CH503 in the AG and legs before and after mating are shown normalized to the sum of the values for the major female hydrocarbons (see Experimental Procedures; see Table S4 for values). Changes observed in the AG are greater in both magnitude and duration than those found in the legs. Some variation can be attributed to the method (which samples over a small surface) and pair-to-pair differences in copulation time and intensity. In the AG, cVA exhibits a monotonic decrease in abundance over time. Hydroxylated tricosene (C₂₃H₄₆O) and tricosadiene (C₂₃H₄₄O) increase in relative intensity between 24 h – 2 d. CH503 expression persists for at least 10 d. In the legs, the most striking increases are seen in the levels of hydroxylated tricosene and tricosadiene between 24 h – 2 d after mating. *p < 0.05; **p < 0.01; ***p < 0.001 compared to virgin levels (Dunn’s multiple comparison test). Relative intensity values are not representative of absolute abundances since ionization efficiency can depend partly on chemical structure.

Figure 3. Male Drosophila exhibit time-dependent changes in the CH profile for up to 24 h after successful copulation. All assigned peaks correspond to potassiated molecules [M+K]⁺

(A, B) Profiles of the anogenital region (AG) and legs of virgin males show low levels of the characteristic female hydrocarbons heptacosadiene (C₂₇H₅₂) and nonacosadiene (C₂₉H₅₆) (insets). (C, D) 60 min after mating, the major female CHs heptacosadiene (C₂₇H₅₂) and nonacosadiene (C₂₉H₅₆) are detected above normal intensity levels in both the AG and legs of males. (E, F) Intensity values for heptacosadiene (C₂₇H₅₂) and nonacosadiene (C₂₉H₅₆) in the AG and legs before and after mating are shown normalized to the sum of the values for the major male hydrocarbons (see Experimental Procedures; see Supplemental table 5 for values). Increases in the abundance of these compounds appear greater in the legs than in the AG. In both regions, the highest intensities for these compounds are found 1–2 h after mating and drop precipitously by 24 h. Levels of the major female CHs are no longer significant at 24 h post-mating in the AG and legs. **p < 0.01; ***p < 0.001 compared to virgin levels (Dunn’s multiple comparison test).

Figure 4. Structural characterization of CH503

(A) HPTLC of crude cuticular extract from 10 g of flies separated CH503 from triacylglycerides, phospholipids, and other hydrocarbons. GC/MS analysis did not detect any other hydrocarbons in this fraction (data not shown). (B) NanoESI-QTOF mass analysis of extracts from the HPTLC band containing CH503 shows that CH503 is the major component in the fraction. Due to the high concentration of the compound in the solution, CH503 is detected primarily as a mixture of sodiated monomers and oligomers. Other unassigned signals correspond to the protonated and potassiated forms of CH503 and residual amounts of triacylglycerides. (C) The protonated molecule [M+H]⁺ of CH503 at m/z 465.5 was analyzed from crude cuticular extract using nanoESI-QTOF MS and low-energy CID tandem mass spectrometry. The product mass spectrum reveals the presence of: (i) an acetate group, indicated by the loss of acetic acid (CH₃COOH, MW 60.0 Da); (ii) a hydroxyl group, indicated by the loss of a water molecule (MW 18.0 Da); and (iii) a linear hydrocarbon chain indicated by a series of fragment ions separated by mass increments of 14.0 Da. (D) The proposed chemical structure of CH503 as 3-O-acetyl-1,3-dihydroxy-octacosa-11,19-diene and is shown in a (Z, Z) 11,19 configuration. The actual (Z/E) configuration of the double bonds remains to be determined.

Figure 5. Male courtship behavior is inhibited in the presence of CH503

(A) Courtship initiation is suppressed in a dose-dependent manner with increasing concentration of CH503. The number of trials for each condition is indicated in parentheses; ^†equivalent to the amount extracted from 7 male flies; ***: p<0.001 (Chi-Squared test). (B) The courtship index is not significantly different over time and across dosage concentrations in the trials during which courtship was seen (p>0.05, ANOVA); ^†equivalent to the amount extracted from 7 male flies. (C) The male courtship index decreases in a dose-dependent manner with increasing concentration of CH503. Male courtship indices (CI) were calculated in 10 min time bins for each 30 min trial. CI is defined as the amount of time the male spends following, extending and vibrating one wing, tapping, licking, and copulating divided by the total length of the trial. The number of trials for each condition is indicated in parentheses; ^†equivalent to the amount extracted from 7 male flies; *: p<0.01 (Tukey-Kramer post-hoc analysis); **: p<0.001 (Tukey-Kramer post-hoc analysis).