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. 2016 Nov 15;5:e20713. doi: 10.7554/eLife.20713

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Figure 2. — (a-d) Sexually dimorphic expression of dsx/vGlut neurons in the brain (top) and VNC (bottom) of adult males (a,b) and females (c,d). (a,c) dsx/vGlut cell bodies visualized by vGlut^dVP16-AD/dsx^GAL4-DBD driving UAS-nGFP in (a) male and (c) female CNSs. nGFP stained with anti-GFP (green); neuropil counterstained with anti-nC82 (magenta). (b,d) dsx/vGlut projection patterns visualized by vGlut^dVP16-AD/dsx^GAL4-DBD driving UAS-2XEGFP in (b) male and (d) female CNSs. EGFP stained with anti-GFP (black); sexually dimorphic midline crossing (red arrowhead) and neurons of the Abg and their descending projections (red arrow) are shown. (e) dsx/vGlut driven EGFP expression in the T1 tarsi of the male foreleg. Projections from these neurons form the male-specific contralateral commissural bridge in the mesothoracic gangion of the male VNC (red arrowheads in bottom panels of b,d). (f,g) dsx/vGlut driven EGFP expression in the (f) internal reproductive system and (g) abdomen reveals motor neuron arborizations onto (f) muscles of the aedeagus and (g) dorsal and ventral muscles of the sixth abdominal segment. (g1-2) Higher magnification of ventral (g1) and dorsal (g2) longitudinal muscles of sixth abdominal segment showing dsx/vGlut motor neuron innervations and synaptic termini. EGFP stained with anti-GFP (green). Internal reproductive system and abdominal muscles counterstained with the F-actin specific antibody Phalloidin (phall; magenta). Detail of internal genitalia: testes, ejaculatory bulb (EjB), and aedeagus (Aed) indicated. Scale bar = 50 μm. (h-j) Effects of silencing dsx/vGlut neurons on male copulatory and courtship behaviors. (h) Percentage of successful matings in 1 hr (n = 24–30). (i) Male fertility (n = 30). (j) Courtship index (mean ± S.E.M.; n = 24–30). Genotypes indicate males. See also Video 1. (k,l) Effects of thermoactivating dsx/vGlut neurons on male courtship and copulatory behaviors. (k) Courtship index (mean ± S.E.M.; n = 20–30). (l) Percentage of successful matings in 1 hr (n = 20–30). Statistical comparisons of the experimental genotype at 31°C in (k-l) were made against the same genotype at 23°C and all control genotypes at 31°C. Genotypes indicate males. (m) Effects of thermoactivating male dsx/vGlut neurons 5 min into copulation. Percentage of copulations terminated over a 2 hr period is graphed (n = 22–24). (n) Video still showing ‘stuck’ dsx/vGlut>TrpA1 male at the end of the 2 hr observation period. See also Video 2. (h-l) **p<0.001, ***p<0.0001 by Fisher exact test (h,i,k) or Kruskal-Wallis and Dunn’s test (j,l).

DOI: http://dx.doi.org/10.7554/eLife.20713.005

Figure 2—figure supplement 1. — (a-d) Sexually dimorphic expression of dsx/vGlut neurons in the brain (top) and VNC (bottom) of adult males (a,b) and females (c,d). (a,c) dsx/vGlut cell bodies visualized by vGlut^dVP16-AD/dsx^GAL4-DBD driving UAS-nGFP in (a) male and (c) female CNSs. nGFP stained with anti-GFP (green); neuropil counterstained with anti-nC82 (magenta). (b,d) dsx/vGlut projection patterns visualized by vGlut^dVP16-AD/dsx^GAL4-DBD driving UAS-2XEGFP in (b) male and (d) female CNSs. EGFP stained with anti-GFP (black); sexually dimorphic midline crossing (red arrowhead) and neurons of the Abg and their descending projections (red arrow) are shown. (e) dsx/vGlut driven EGFP expression in the T1 tarsi of the male foreleg. Projections from these neurons form the male-specific contralateral commissural bridge in the mesothoracic gangion of the male VNC (red arrowheads in bottom panels of b,d). (f,g) dsx/vGlut driven EGFP expression in the (f) internal reproductive system and (g) abdomen reveals motor neuron arborizations onto (f) muscles of the aedeagus and (g) dorsal and ventral muscles of the sixth abdominal segment. (g1-2) Higher magnification of ventral (g1) and dorsal (g2) longitudinal muscles of sixth abdominal segment showing dsx/vGlut motor neuron innervations and synaptic termini. EGFP stained with anti-GFP (green). Internal reproductive system and abdominal muscles counterstained with the F-actin specific antibody Phalloidin (phall; magenta). Detail of internal genitalia: testes, ejaculatory bulb (EjB), and aedeagus (Aed) indicated. Scale bar = 50 μm. (h-j) Effects of silencing dsx/vGlut neurons on male copulatory and courtship behaviors. (h) Percentage of successful matings in 1 hr (n = 24–30). (i) Male fertility (n = 30). (j) Courtship index (mean ± S.E.M.; n = 24–30). Genotypes indicate males. See also Video 1. (k,l) Effects of thermoactivating dsx/vGlut neurons on male courtship and copulatory behaviors. (k) Courtship index (mean ± S.E.M.; n = 20–30). (l) Percentage of successful matings in 1 hr (n = 20–30). Statistical comparisons of the experimental genotype at 31°C in (k-l) were made against the same genotype at 23°C and all control genotypes at 31°C. Genotypes indicate males. (m) Effects of thermoactivating male dsx/vGlut neurons 5 min into copulation. Percentage of copulations terminated over a 2 hr period is graphed (n = 22–24). (n) Video still showing ‘stuck’ dsx/vGlut>TrpA1 male at the end of the 2 hr observation period. See also Video 2. (h-l) **p<0.001, ***p<0.0001 by Fisher exact test (h,i,k) or Kruskal-Wallis and Dunn’s test (j,l).

DOI: http://dx.doi.org/10.7554/eLife.20713.005