Hierarchical chemosensory regulation of male-male social interactions in Drosophila

Abstract

Pheromones regulate male social behaviors in Drosophila, but the identities and behavioral role(s) of these chemosensory signals, and how they interact, are incompletely understood. We found that (z)-7-tricosene, a male-enriched cuticular hydrocarbon that was previously shown to inhibit male-male courtship, was essential for normal levels of aggression. The mechanisms by which (z)-7-tricosene induced aggression and suppressed courtship were independent, but both required the gustatory receptor Gr32a. Sensitivity to (z)-7-tricosene was required for the aggression-promoting effect of 11-cis-vaccenyl acetate (cVA), an olfactory pheromone, but (z)-7-tricosene sensitivity was independent of cVA. (z)-7-tricosene and cVA therefore regulate aggression in a hierarchical manner. Furthermore, the increased courtship caused by depletion of male cuticular hydrocarbons was suppressed by a mutation in the olfactory receptor Or47b. Thus, male social behaviors are controlled by gustatory pheromones that promote aggression and suppress courtship, and whose influences are dominant to olfactory pheromones that enhance these behaviors.

Figure 1. Male CHs are important for the normal balance of male-male social behaviors

(a) Quantification of cVA and major CH molecules from oe⁺ (blue), oe⁻ (orange) or oe⁻ males perfumed with male CHs (green) (n=10). (b, c) Quantification of aggression (b) and courtship (c) performed by Canton-S tester males towards target males of the indicated genotypes and CH perfuming treatments (n=20). (d) Quantification of cVA and major CH molecules from oe⁺ (blue) or oe⁺ males perfumed with synthetic 7,11-HD (red) (n=9). (e, f) Quantification of aggression (e) and courtship (f) performed by Canton-S tester males towards target males of the indicated genotypes and CH perfuming treatments (n=16). Error bars are s.e.m. in this and all subsequent figures. NS: p>0.05, *p<0.05, **p<0.01 and ***p<0.001.

Figure 2. 7-T reciprocally regulates both male-male aggression and courtship

(a) Quantification of cVA and major CH molecules from oe⁺ (blue), oe⁻ (orange) or oe⁻ males perfumed with synthetic 7-T (green) or 7-P (purple) (n=11–12). (b, c) Quantification of aggression (b) and courtship (c) performed by Canton-S tester males towards target males of the indicated genotypes and CH perfuming treatments (n=20). (d) Relative levels of 7-T carried by oe⁻ males incubated after 7-T transfer for various periods of time (green), shown as a percentage of wild-type levels of 7-T (blue) (n=9–11). Absolute quantification is shown in Supplementary Fig. 6. Green shading represents relative amount of synthetic 7-T carried by oe⁻ males (darker = higher). (e, f) Quantification of aggression (e) and courtship (f) performed by Canton-S tester males towards oe⁻ targets (orange), oe⁺ targets (blue), or oe⁻ target males carrying different amounts of synthetic 7-T (green) (n=18). NS: p>0.05, *p<0.05, **p<0.01 and ***p<0.001.

Figure 3. Gr32a mediates the behavioral effects of 7-T and permits the aggression-promoting effect of cVA

(a, b) Quantification of aggression (a) and courtship (b) performed by tester males of the indicated genotypes towards oe⁺ target males (n=26–28). (c, d) Quantification of aggression (c) and courtship (d) performed by tester males of the indicated genotypes towards oe⁻ target males (orange) or oe⁻ males perfumed with synthetic 7-T (green) (n=26–30). Dashed lines represent control levels of social behaviors (performed by Gr32a^+/− testers towards oe⁺ targets (from Fig. 3a, b)). (e) Quantification of aggression performed by pairs of males of the indicated genotypes, in the presence of acetone alone (blue), or in the presence of 500 μg synthetic cVA (green) (n=18–20). Note the flies were group-housed prior to the behavioral assays to better reveal the effect of cVA (see Methods). (f) Quantification of aggression performed by tester males of the indicated genotypes towards oe⁻ target males (orange) or oe⁻ males perfumed with 7-T (green) (n=20). NS: p>0.05, *p<0.05, **p<0.01 and ***p<0.001.

Figure 4. Or47b is required for elevated male-male courtship caused by depletion of male CHs

(a) Schematic illustration of the targeting construct (top) and site of homologous recombination (bottom) in Or47b locus. The flanking gene (nompA) included in the targeting construct is not disrupted. (b) PCR validation of two independently recovered mutant alleles lacking the first two exons of Or47b. (c) RNA in situ hybridization for Or47b (green) and Or88a (magenta). (d) Projections of Or47b⁺ ORNs to the VA1lm glomeruli visualized using Or47b-GAL4; UAS-mCD8GFP in flies of the indicated genotypes; nc82, neuropil counter-stain. Scale bar = 50 mm in (c, d). (e, f) Quantification of aggression (e) and courtship (f) performed by tester males of the indicated genotypes towards oe⁺ (blue) or oe⁻ (orange) target males (n=20). (g, h) Cumulative latency to copulation (g; n=20) and unilateral wing extension frequency (h; n=17–18) by males of the indicated genotypes towards virgin females. NS: p>0.05, *p<0.05, **p<0.01 and ***p<0.001.