Fig. 4. R-HDEA provokes divergent sexual behaviors through activation of homologous sensory neurons.

(A) Left: Schematic of mating arena where a perfumed male courts a virgin con-subspecific female. Black droplet, hexane (control); gray droplet, one of the other three male-transferred compounds. Right: Copulation success of D. moj. wrigleyi and D. moj. sonorensis males perfumed with hexane or male-specific acetates. Fisher’s exact test. ns, P > 0.05 (n = 40 assays per treatment). See fig. S4A for OCDA impact on copulation success and fig. S3E for courtship indices of hexane- and acetates-perfumed males toward females of the same subspecies. (B) Copulation latencies of the same males as in (A). Filled circles indicate significant differences from the solvent. Kruskal-Wallis test with Dunn’s post hoc correction. ns, P > 0.05; *P < 0.05 [n from left to right represent successful copulations in (A) = 29, 28, 29, 24, 32, 28, 30, and 26 assays]. See fig. S4B for OCDA impact on females’ copulation latencies. (C) Competition between two con-subspecific males, perfumed with one of the three compounds (colored droplet) or with the solvent hexane (black droplet), to copulate with a virgin female of the same subspecies. Pie charts represent copulation success of the rival males. Filled droplets indicate significant difference between the tested groups. χ² test; ns, P > 0.05; **P < 0.01; from top to bottom = 124, 120, 124, 104, 112, and 116 assays. See fig. S4C for the competition results of OCDA-perfumed males and fig. S4D for D. moj. mojavensis and D. moj. Baja competition experiments. (D) Scanning electron micrograph of antennal surface, showing different sensillum types (intermediate, trichoid, coeloconic, and basiconic). Scale bar, 2 μm (Photo credit: V.G., Max Planck Institute for Chemical Ecology). (E) Electrophysiological responses toward R-HDEA, S-HDEA, and HDA in all types of olfactory sensilla on antenna and maxillary palp of D. moj. wrigleyi (pink) and D. moj. sonorensis (turquoise). Mann Whitney U test; ns, P > 0.05; ***P < 0.001 (n = 3 to 6 neurons). ab, antennal basiconic; ac, antennal coeloconic; at, antennal trichoid; ai, antennal intermediate; pb, palp basiconic. See fig. S4 (E to H) for similarity between male and female responses to R-HDEA, OCDA responses, representative SSR traces, and responses of R-HDEA among D. mojavensis subspecies, respectively. (F) Dose-dependent responses of at4 neurons in D. moj. wrigleyi and D. moj. sonorensis toward R-HDEA (in red) and HDA (in blue) (mean ± SEM). Two-way analysis of variance (ANOVA) followed by Sidak’s multiple comparison test between the two subspecies’ responses to the same stimulus; ns, P > 0.05 (n = 4 to 6 neurons). See fig. S4I for D. melanogaster at1 and at4 responses to D. moj. wrigleyi–specific acetates and fig. S4J for alignments of D. moj. wrigleyi–OR65a and D. melanogaster–OR65a/b/c protein sequences.