Fig. 5. Conserved peripheral sensory pathways for R-HDEA detection among D. mojavensis subspecies.

(A) Expression of olfactory receptor genes (OrX: Or47b, Or88a, Or65a, and Or67d) in D. moj. wrigleyi and D. moj. sonorensis female antennae. See Materials and Methods and fig. S5A for the receptor terminology. Because of the high degree of sequence identity (99.1%) of the Or47b-related loci (data file S1), cross-hybridization between probes and mRNAs is likely to occur. (B) Number of the Or-expressing cells (OrX: Or47b, Or88a, Or65a, and Or67d) in D. moj. wrigleyi and D. moj. sonorensis females. Filled circles indicate significant differences between species. Mann Whitney U test; ns, P > 0.05; *P < 0.05 (n = 10 antennae). (C) Schematic of voltage-clamp recordings from X. laevis oocytes ectopically expressing the different ORs (ORX) together with ORCO. (D) Electrophysiological responses of oocytes expressing different OR genes to R-HDEA (1 mM). In this panel and in (F), filled circles indicate significant differences from the solvent response. Mann Whitney U test; ns, P > 0.05; *P < 0.05; ***P < 0.001 (n = 4 to 8 recordings). See fig. S5B for responses to other compounds. Right: Traces of electrophysiological responses of D. moj. wrigleyi–OR65a to DMSO and R-HDEA. (E) Schematic of ORs (ORX) expressed in D. melanogaster at1 neurons. (F) Electrophysiological responses of five ORs expressed in D. melanogaster at1 neurons to R-HDEA (10 μg). Mann Whitney U test; ns, P > 0.05; ***P < 0.001 (n = 7 recordings). See fig. S5C for responses to other compounds. (G) Electrophysiological responses of D. moj. wrigleyi or D. moj. sonorensis OR65a toward increasing concentrations of R-HDEA (n = 8 recordings). Two-way ANOVA followed by Sidak’s multiple comparison test between the two subspecies’ responses to the same stimulus; ns, P > 0.05. (H) Schematics of the D. moj. Or65a locus illustrating the single guide RNA (sgRNA) binding sites. Scissors denote the presumed cutting site. The Or65a loss-of-function allele carries a 2–base pair (bp) deletion in exon 1, resulting in a premature stop codon (highlighted in red). See fig. S5 (E and F) for details on generating white gene knockouts using CRISPR-Cas9. (I) Electrophysiological responses of Or65a heterozygous (left) and homozygous (right) animals to hexane, R-HDEA, and methyl laurate [ML; diagnostic odor for OR47b and OR88a (13)]. The responses to ML show an intact neuronal excitation of the Or65a-neighboring neuron(s). See fig. S5D regarding quantification of SSR responses. (J) Competition between two males of D. moj. wrigleyi, perfumed with R-HDEA (red droplet) or hexane (black droplet), to copulate with a transgenic virgin female of the same subspecies. Left: Heterozygous animal at the Or65a locus (n = 148); right: homozygous mutant at the Or65a locus (n = 128). Filled droplets indicate a significant difference between the rival males. χ² test; ns, P > 0.05; **P < 0.01. All males and females used in this and other panels were 10-day-old virgin flies. (J′) Copulation latencies in seconds for Or65a heterozygous (left) and homozygous (right) females courted by wild-type males (single-pair assays). Filled circles indicate significant differences between both transgenic females. Mann Whitney U test; ns, P > 0.05; *P < 0.05 (n = 25). (K) Three-dimensional reconstruction of antennal lobes from representative female brains of D. moj. wrigleyi and D. moj. sonorensis. Neurobiotin-marked neurons in (L) are highlighted: VA1d (green), VA1v (yellow), and VA8 (blue, only present in D. moj. wrigleyi and D. moj. sonorensis). Landmark glomeruli (dark gray, DA1, DL3, VL2p, VL2a, VL1, and V glomeruli) are used to support the homology of these three glomeruli in both D. mojavensis subspecies. VA8 is located ventrally to VA1v and anterior to VL2a in an area far off the DL3 glomerulus, which is targeted by Or65a neurons in D. melanogaster. See fig. S6A for details on the glomeruli’ volumes in both subspecies. Scale bars, 20 μm. (L) Left top: Fluorescence staining for neurobiotin (magenta) and nc82 (gray) of D. moj. wrigleyi antennal lobe, backfilled from the at4 sensillum in D. moj. wrigleyi (identified by electrophysiological recordings; Fig. 4E). Right top: Reconstruction of the neurobiotin-marked neurons and their corresponding glomeruli reveals at4-housed neurons project to three glomeruli (D. mojavensis VA8, VA1v, and VA1d). Left bottom: Neurobiotin-backfilled neurons from at4 sensillum in D. moj. sonorensis that innervate similar regions in antennal lobe. Images in the four panels correspond to a projection of 40 Z-stacks (watch movies S7 and S8). Right bottom: Landmark glomeruli were labeled according to their positions based on the map of the D. melanogaster AL (16) (see Materials and Methods). See fig. S6B for more details. See fig. S6 (C to F) and movie S9 for backfilling of at1 sensilla. (M) Volumes of VA1d, VA1v, and VA8 glomeruli normalized to the total antennal lobe volume in D. moj. wrigleyi and D. moj. sonorensis (males, black; females, gray). Filled circles indicate significant difference between sexes of the same subspecies. Mann Whitney U test; ns, P > 0.05; *P < 0.05 (n = 4 to 6 brains).