Large-scale characterization of sex pheromone communication systems in Drosophila

Abstract

Insects use sex pheromones as a reproductive isolating mechanism to attract conspecifics and repel heterospecifics. Despite the profound knowledge of sex pheromones, little is known about the coevolutionary mechanisms and constraints on their production and detection. Using whole-genome sequences to infer the kinship among 99 drosophilids, we investigate how phylogenetic and chemical traits have interacted at a wide evolutionary timescale. Through a series of chemical syntheses and electrophysiological recordings, we identify 52 sex-specific compounds, many of which are detected via olfaction. Behavioral analyses reveal that many of the 43 male-specific compounds are transferred to the female during copulation and mediate female receptivity and/or male courtship inhibition. Measurement of phylogenetic signals demonstrates that sex pheromones and their cognate olfactory channels evolve rapidly and independently over evolutionary time to guarantee efficient intra- and inter-specific communication systems. Our results show how sexual isolation barriers between species can be reinforced by species-specific olfactory signals.

Fig. 1. Phylogenetic relationships and chemical variations among drosophilids.

a Phylogeny of 99 species within the family Drosophilidae inferred from 13,433,544 amino acids sites that represent 11,479 genes (See Supplementary Data 1 and Methods for details). Using four species in the Colocasiomyini subgenus as outgroups (purple), 95 species are distributed in four subgenera belonging to the Drosophilini tribe (Drosophila, light green branches; Zaprionus, gray branch; Dorsilopha, brown branch; Sophophora, dark green branches). Species names are color coded according to their relationships in nine different species groups, with black species depicting individual representatives of species groups. Scale bar for branch length represents the number of substitutions per site. Maximum likelihood (ML) phylogenetic analyses display strong rapid bootstrap support (100% support indicated by black circle at the nodes) for most relationships among the different species. For divergence times, see Supplementary Fig. 1D. b The first two principal components of male chemical profiles (data in Supplementary Fig. 1A) of the 583 replicates across the 99 male species (>5 replicates per species) based on difference in peak areas of 248 male chemical features present across these replicates (see Methods for details). Data points of each group are enclosed within the line. The lines’ fill is colored according to the group identities in Fig. 1a. b’ The first two principal components of female chemical profiles (data in Supplementary Fig. 1A’) of the 528 replicates across the 99 female species (>5 replicates per species) based on the difference in peak areas of 256 female chemical features present across these replicates. c Heat map showing pairwise correlations between male chemical profiles of the 99 species (ordered on each axis according to their phylogenetic relationships from Fig. 1a). Overall peak areas of 248 male chemical features across the 99 species were compared using Pearson correlation coefficient (R²); Color codes in the heat map illustrate the pairwise correlations, which range from dark blue (Perfect correlation between chemical profiles) through white (no correlation) to dark red (perfect anticorrelation). The diagonal of the correlation matrix is the correlations between each species and itself (values of 1). Note that the male correlation matrix displays frequent dark blue cells mainly around the diagonal, i.e., high correlation coefficients are observed mostly between closely related species. c’ Pairwise correlation analysis between female chemical profiles of the 99 species arranged according to their phylogeny from Fig. 1a. Overall peak areas of 256 female chemical features across the 99 species were compared using the Pearson correlation coefficient (R²). See Supplementary Data 2 for the statistical Pagel’s lambda correlation analysis.

Fig. 2. Newly identified potential sex pheromones.

a Representative gas chromatograms of virgin male (♂), and virgin (v♀), and mated (m♀) female flies obtained by solvent-free thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS). Five replicates or more of each sex were analyzed, yielding more than 580, 520, and 500 replicates of males, and virgin and mated females of all 99 species, respectively. Left panel, example of a monomorphic species, whose males exhibit a chemical profile identical to that of virgin and mated females. Right panel, example of a dimorphic species that displays sexually dimorphic profiles. Colored peaks indicate male-specific compounds (green, compounds transferred to females during mating; red, non-transferred compounds). Drawings made by Mohammed A. Khallaf. b Distribution of 43 male-specific compounds among different drosophilids; 81 species are dimorphic species (in black), while 18 species (in grey) are monomorphic species. Phylogeny on the left side is identical to the tree in Fig. 1a; the branches are colored according to group identities. Numbers on the right side represent the sum of male-specific compounds present per species, while numbers at the bottom of the table represent number of times each male-specific compound appeared in the different species. Cell colors refer to transferred (green) and non-transferred (red) compounds. See Supplementary Fig. 2 for female-specific compounds. c Chemical structures and names of the male-specific compounds according to the International Union of Pure and Applied Chemistry (IUPAC). Out of 43 male-specific compounds, 40 compounds were chemically identified. Compound size ranges between 10 to 32 carbon atoms, with 23 esters, 4 ketones, 8 alkenes, 2 terpenes, 1 ether, and 1 alcohol. See Supplementary Data 3 for Kovat’s Index, chemical formula, exact mass, mass spectrum (M/Z), and boiling temperature of these compounds.

Fig. 3. Drosophilids communicate intra- and inter-specifically through rapidly evolving olfactory channels.

a Left: schematics of single sensillum recordings (SSR) from the antennal trichoid (at1 and at4) sensilla. Right: Names of the different chemicals used to screen the trichoid sensilla. Note that all chemicals are male-specific compounds identified in this study (Fig. 2b), except compounds# 25, 26, 27, 28, 29, 32, and 36, which were described as flies’ pheromones in refs. ^{, ,} . b Color-coded electrophysiological responses towards heterospecific compounds (grey bubbles) and conspecific compounds (colored bubbles) in at1 (top) and at4 (bottom) sensilla of females of 54 species. Compound names are depicted in Fig. 3a. Red and green bubbles represent species-specific male untransferred and transferred compounds, respectively. Bubble size corresponds to the average of response values (n = 3–10) ranging from 25 to 125 spikes per second. Responses less than or equal to 10 spikes per second were excluded from the bubble chart (see Supplementary Fig. 3A, A’, and “Methods” for more details). Species names are arranged on the top according to their phylogenetic relationship; the tree branches are colored according to the group identities. Numbers on the right side represent the sum of species that can detect each of the male-specific compounds, while the number below the table represents the sum of chemicals that can be detected by each species. Note that the compounds’ vapor pressures have no impact on the number of the olfactory responses (Supplementary Fig. 3D). c A summary of female’s abilities to detect their own male-specific compounds through olfaction in 47 dimorphic species (two species, D. robusta and D. neocordata, whose compounds were not included among the 36 compounds, was excluded). Black numbers, undetected male-specific compounds; orange, detected by at1 neuron(s); blue, detected by at4 neurons; green, detected by both. See Supplementary Fig. 3A for more details about the number of neurons in at1 sensillum. Note that, out of 47, females of 36 species detect their conspecific male cues through at1 and/or at4. d Top: A schematic example of how to calculate the olfactory clustering coefficient of a given species (yellow circle) to communicate with heterospecific species (black circles) through at1 (orange lines) and at4 (blue lines). The olfactory clustering coefficient is the number of other species-specific compounds that are detected by a given species through at1 or at4 (colored lines) divided by the total number of detected and undetected species (colored + grey dashed lines). The clustering coefficient of a species is a number between 0 (i.e., no species detected) and 1 (i.e., all species detected). Below: scatter plot indicates olfactory clustering coefficients of the 54 species and their mean through at1 (orange) and at4 (blue); Two-sided Mann–Whitney U test, ***P < 0.001 (n = 54 species). Note that species exhibit more olfactory intra- and inter-specific communication through at1 than at4. See Supplementary Fig. 3B, B’ and “Methods” for more details on communication network analyses. e Frequency histogram of Pagel’s lambda estimates, which explain the correlation between the olfactory responses of at1 (orange) and at4 (blue) among the different species and their phylogenetic relationships. Note that responses of both at1 and at4 display low phylogenetic signals (i.e., do not correlate with the phylogeny). In addition, their phylogenetic signals are comparable to each other; Two-sided Mann–Whitney U test, ns P = 0.27 between at1 and at4 responses.

Fig. 4. Male-specific compounds regulate intraspecific sexual behaviors and interspecific sexual isolation.

a Top left: Names of the compounds that are exclusively produced by males of 54 species (left below) and detected by conspecific females through at1 or/and at4 (right below). These compounds were used for the behavioral experiments in Fig. 4b–d. b Top: Schematic of a mating arena where females of each species had the choice to mate with two conspecific males perfumed with their olfactory-detected male-specific compound (indicated, in Fig. 4a, on the left side of the horizontal dashed line) or solvent (dichloromethane, DCM). For consistency of perfuming and correspondence to biologically relevant amounts see “Methods”. Below: bar plots represent the percentages of copulation success of the rival males. Results from females that were only courted by one male were excluded. In this and other panels, filled bars indicate a significant difference between the tested groups; ns P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001, chi-square test. Number of replicates are stated on the left side on the bar plots. See Supplementary Fig. 4A for details regarding the differences and similarities of sexual behaviors among the 54 species. Note that in 11 instances, females displayed a preference to copulate with the male-specific compound-perfumed males over the control ones, while 6 compounds resulted in avoidance, and 29 turned out to be neutral. See Supplementary Data 7 for raw data and statistical analyses. See Supplementary Fig. 4B for the effect of perfuming on the males’ courtship behavior. Drawings made by Mohammed A. Khallaf. c Top: Competition courtship arenas where a male of each species had the choice to court two decapitated conspecific females perfumed with the male-transferred compound. Note that we tested only transferred compounds (green). Below: bar plots represent the percentage of the first copulation attempts towards perfumed and control females. Results from males that only courted one female were excluded; see Methods. Note that 15 compounds inhibited courtship, 1 compound increased courtship and 16 compounds turned out to be neutral. Drawings made by Mohammed A. Khallaf. d Top: Schematic of a mating arena where a female of each species had the choice to mate with two conspecific males perfumed with olfactory-detected heterospecific cVA or solvent (DCM). Note that we only tested the species that do not produce but still detect cVA. Below: bar plots represent the percentages of copulation success of the rival males. Results from females that were only courted by one male were excluded. Drawings made by Mohammed A. Khallaf.