(A,B) Neither expressing yellow-RNAi nor yellow-cDNA in fru-expressing cells using fruGAL4 (Stockinger et al., 2005) affected male copulation. (C) Expressing yellow-RNAi in dsx-expressing cells using dsxGAL4 (Robinett et al., 2010) significantly inhibited male mating success. (D) Expressing yellow in dsx-expressing cells using dsxGAL4 in a y1 mutant background was sufficient to restore male mating success. (E,F) Expressing yellow-RNAi using pan-neuronal (elav-GAL4 and nsyb-GAL4) and pan-glia (repo-GAL4) drivers did not affect male mating success. (G) Restricting yellow-RNAi expression to dsx-expressing neurons using the split-GAL4 technique, combining dsxGAL4-DBD (Pavlou et al., 2016) with elavVP16-AD (Luan et al., 2006), did not affect male mating success. (H) Restricting yellow-RNAi expression to dsx-expressing glutamatergic neurons using the split-GAL4 technique, combining dsxGAL4-DBD (Pavlou et al., 2016) with vGlutdVP16-AD (Gao et al., 2008) did not affect male mating success. (I) Expressing yellow in dsx-expressing cells restricted outside the CNS using dsxGAL4 and nsyb-GAL80 (courtesy of Julie Simpson) in a y1 mutant background significantly increased male mating success. (J) Brain and ventral nerve cord of adult male ymCherry flies stained with anti-N-Cadherin (N-cad) antibody labeling neuropil (white) and anti-DsRed antibody labeling Yellow::mCherry (red). We observed sparse, inconsistent signal outside the CNS at the top of the brain in males (white arrow), but we were unable to confirm a previous report that ymCherry is expressed in the adult brain (Hinaux et al., 2018). (K) Diagram of the male exon structure of the dsx locus highlighting 10 genomic fragments between 1.7 and 4 kb used to clone Janelia enhancer trap GAL4 drivers (Pfeiffer et al., 2008). Black boxes indicate coding exons. White boxes indicate 5’ and 3’ UTRs, and the arrow in exon two denotes the transcription start site. (L) Expressing yellow-RNAi using each Janelia dsx-GAL4 driver identified 42D04-GAL4 and 40 F03-GAL4 as affecting male mating success when compared with the yellow-RNAi control. (M) A replicate experiment comparing 42D04-GAL4 and 40F03-GAL4 effects on male mating success with both GAL4 and UAS parental controls confirmed the significant effect of 42D04-GAL4 but not 40F03-GAL4. We attribute differences in the 40F03-GAL4 effect between (L) and (M) to between experiment variability in the levels of male mating success; each common genotype tested in (L), for example, mated at higher levels in (M), but 42D04-GAL4 consistently showed a significant effect relative to controls. Sample sizes are shown at the top of each barplot. Significance was measured using Fisher’s exact tests with Bonferroni corrections for multiple comparisons. Comparisons that were statistically (p<0.05) are indicated (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).