Intraspecific male-male aggression, which is important for sexual selection, is regulated by environment, experience and internal states through largely undefined molecular and cellular mechanisms. To understand the basic neural pathway underlying the modulation of this innate behavior, we established a behavioral assay in Drosophila melanogaster and investigated the relationship between sexual experience and aggression. In the presence of mating partners, adult male flies exhibited elevated levels of aggression, which was largely suppressed by prior exposure to females via a sexually dimorphic neural mechanism. The suppression involved the ability of male flies to detect females by contact chemosensation through the pheromone-sensing ion channel ppk29 and was mediated by male-specific GABAergic neurons acting on the GABAA receptor RDL in target cells. Silencing or activating this circuit led to dis-inhibition or elimination of sex-related aggression, respectively. We propose that the GABAergic inhibition represents a critical cellular mechanism that enables prior experience to modulate aggression.
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- Supplementary Figure 1: Experience with females inhibits sex-related male-male aggression. (180 KB)
(a) Average aggression duration of cs males was quantified in 5 min intervals for 2 hours for naïve male only (blue), experienced males only (purple), naïve males + virgin females (red), experienced males + virgin females (yellow), naïve males + mated females (green), experienced males + mated females (brown). (n = 4 pairs for each condition). (b) Courtship index measured at 70–75 min for naïve male + virgin female and naive male + mated female. The latter showed significantly higher courtship index. (n = 4 pairs for each condition). (c) Average aggression frequency of cs and w1118 males was quantified for the 60-90 min. No obvious difference in the inhibition of aggression is apparent between the two genotypes. (n = 11 and 6 pairs for cs and w1118). **: P < 0.01, ***: P < 0.001 by Student's t test. Error bars denote s.e.m.
- Supplementary Figure 2: Experimental schemes for behavioral analysis. (221 KB)
(a) Detailed experimental scheme for analyzing sex-related male-male aggression and its regulation by prior female experience. (b) The dis-inhibition phenotype was examined after temperature shift to induce silencing of specific group of neurons with enhancer-Gal4 driven UAS-Gal80ts and UAS-Kir2.1.
- Supplementary Figure 3: Conditioning parameters for the prior female experience dependent inhibition of aggression. (89 KB)
Inhibition of aggression were displayed by males conditioned with virgin females for 24 hours, with mated females, with virgin females that express membrane bound sex peptide using fru-Gal4, which would therefore reject males, and with Drosophila pseudoobscura. However, conditioning with virgin females for 10 hours, removing females right after mating during conditioning, conditioning males in vials previously housed virgin females, conditioning males with virgin females that were separated by a nylon mesh, conditioning with Drosophila virilis, or with extra males all failed to elicit inhibition of aggression. On the other hand, removing females after mating during testing abolished aggression, revealing the presence of females during the aggression assay is crucial. (n = 11, 11, 5, 6, 6, 6, 6, 5, 5, 5, 6 and 8 pairs of flies for each condition). ***: P < 0.001 by One-way ANOVA followed by Bonferroni's multiple comparison test. Error bars denote s.e.m.
- Supplementary Figure 4: Previous female contact does not significantly affect the courtship index and locomotion of experienced males. Social hierarchy established between males housed in pairs has little to do with the female-contacts dependent inhibition of sex-related male aggression. (114 KB)
(a) The courtship index and climbing index of experienced vs. naive male flies did not exhibit significant differences. (n = 5 flies for courtship index and 3 groups for climbing index). (b) Two male flies that were housed in pairs but not with each other (mixed group), so that they had social experiences but no established hierarchy with each other, exhibited as much contact-dependent inhibition of the sex-related aggression as did males that were raised together (pair). (n = 5 pairs of flies for each condition). **: P = 0.0012, by Student's t-test. . Error bars denote s.e.m.
- Supplementary Figure 5: Aggression duration measurements corresponding to Fig. 2a, 2b, 2d (n = 11, 6, 7 and 5 pairs of flies for each condition). (340 KB)
***: P < 0.001, by One-way ANOVA followed by Bonferroni's multiple comparison test.. Error bars denote s.e.m.
- Supplementary Figure 6: Sexual dimorphic distribution of fru+, d5-HT1B+ neurons in the brain and VNC of adult male and female flies. (1,172 KB)
(a) fru+, d5-HT1B+ neurons in the adult male brain labeled by mCD8::GFP including γ-neurons of the mushroom bodies and a cluster with soma located below the antennal lobes and above the SOG region. Scale bar = 40 μm. The magnified view is shown in the lower left panel. Scale bar = 10 μm. nc82 co-staining in red shows the neuropil. A 3D rendering of the neuropil is shown in the lower right panel. (b) A subpopulation of fru+, d5-HT1B+ neurons independent of mushroom bodies are required for the prior female-contacts dependent inhibition of aggression. MB-Gal80 co-expression excluded the mushroom body neurons from the fru+, d5-HT1B+ population, indicated by the loss of mCD8::GFP labeling. nc82 staining is in red and marks the neuropil. Scale bar = 40 μm. (c) The fru+, d5-HT1B+ neuron in the VNC is sexually dimorphic, but does not exhibit anti-GAD staining. One fru+, d5-HT1B+ neuron is evident in the male VNC and it does not overlap with anti-GAD immunostaining. No soma was labeled in female VNC. Scale bar = 40 μm.
- Supplementary Figure 8: fru+, GABA+ neurons display sexual dimorphism in the brain and VNC. (1,189 KB)
(a) fru+, GABA+ neurons showed distinct projection patterns, as labeled by Dscam17.1::GFP, and distinct cell distribution in the VNC, as labeled by CD8::GFP between males and females. (b) The relationship between the dendrite branching pattern of fru+, Rdl+ neurons and the GABA distribution in the male fly brain and VNC. The dendritic patterning of fru+, Rdl+ neurons is shown in green using Dscam17.1::GFP and the GABA distribution is shown in red by anti-GABA staining. Scale bar = 40 μm.
- Supplementary Figure 9: GABAergic inhibition underlies the female contact dependent suppression of sex-related male-male aggression. (198 KB)
a) Aggression duration measurements corresponding to Fig. 6a. (b) Rdl transcripts are reduced in the hypomorphic Rdl allele RdlMB08800.Semi-quantitative RT-PCR analyses showed a reduction of Rdl mRNA level in the RdlMB08800 allele as compared to the wild type control, using primers targeting two regions of the transcript. The numbers indicate the base pair location. The levels of α;-tubulin, used as the loading control, are comparable between the two genotypes. (c, d) Aggression duration measurements corresponding to Fig. 6b. Error bars denote s.e.m. (e) A model for GABA inhibition involved in the aggression suppression. Male flies, when encountering females, activated their female pheromone sensing ppk29 neurons through direct contacts, followed by the GABAergic inhibition of central neurons in the aggression circuit, which possibly express the Rdl GABAA receptor and lead to reduced aggressive behavioral output.
- Video 1: Supplementary Movie 1 (2.11 MB, Download)
- Representative one minute long video clips extracted from the 60-90 min period. For all the videos, the configuration is as the following: top left-naive males only, top right-experienced males only, middle left-naive males + virgin females, middle right-experienced males + virgin females, bottom left-naive males + mated females, bottom right-experienced males + mated females. The genotypes are as follows: cs
- Supplementary Text and Figures (5,853 KB)
Supplementary Figures 1–9, Supplementary Table 1 and Supplementary Movies 1–5
- Supplementary Table 1 (337 KB)
Summary of the enhancer-Gal4 lines tested with tub-Gal80ts; UAS-Kir2.1.