The Drosophila male reproductive system.

A) schematic diagram showing paired testes, SVs (green), and AGs (purple), and a singular sphincter (red), ED (gray), and ejaculatory bulb. B) actual male reproductive system. Te-testes, SV-seminal vesicle, AG-accessory gland, Sph-sphincter, ED-ejaculatory duct, EB-ejaculatory bulb. Scale bar: 200μm.

© 1950, Cold Spring Harbor. Panel A is a coloured version of the male reproductive system from Miller, 1950. The authors would like to thank Cold Spring Harbor press for their kind permission in allowing use of this copyrighted image. This panel is not covered by the CC-BY 4.0 licence and further reproduction would need permission from the copyright holder.

Gal4 and Split-GAL4 expression patterns in the Drosophila male reproductive system.

A) Tdc2-GAL4; B) TRH-GAL4; C) vGlut-GAL4; D) Tdc2-AD ∩ vGlut-GAL4-DBD; E) vGlut-AD ∩ Tdc2-GAL4-DBD; F) TRH-AD ∩ vGlut-GAL4-DBD; G) vGlut-AD ∩ TRH-GAL4-DBD; H) Tdc2-AD ∩ TRH-GAL4-DBD. Scale bar: 200μm.

Expression of TRH-GAL4 and Tdc2-LexA in the Drosophila male reproductive system.

A, D, G, J, M, P) TRH-GAL4, UAS-6XGFP; B, E, H, K, N, Q) Tdc2-LexA, LexAop-6XmCherry. C, F, I, L, O, R) overlay. Scale bars: O-50μm; R-10μm.

Expression of vGlut, TβH-GFP, Tdc2, and 5-HT in the Drosophila male reproductive system.

A) vGlut-40XV5; B) TβH-GFP; C) 5-HT; D) vGlut-40XV5, TβH-GFP overlay; E) vGlut-40XV5, 5-HT overlay; F) TβH-GFP, 5-HT overlay. G) vGlut-40XV5; H) Tdc2; I) 5-HT; J) vGlut-40XV5, Tdc2 overlay; K) vGlut-40XV5, 5-HT overlay; L) Tdc2, 5-HT overlay. Scale bar: 200μm.

Co-conditional expression of vGlut-40XMYC and 6XV5-vMAT in the SV of serotonergic (TRH) or octopaminergic (Tdc2) neurons of the Drosophila male reproductive system.

A-D) 63X. A) vGlut-40XMYC; B) 6XV5-vMAT; C) Tdc2; D) TRH>CD8-mCherry. E-L) 63X zoom 4X. E) vGlut-40XMYC; F) 6XV5-vMAT; G) Tdc2; H) TRH>CD8-mCherry; I) vGlut-40XMYC, 6XV5-vMAT overlay; J) vGlut-40XMYC, 6XV5-vMAT, Tdc2 overlay; K) 6XV5-vMAT, Tdc2 overlay; L) Tdc2, TRH>CD8-mCherry overlay. M-P) 63X. M) vGlut-40XMYC; N) 6XV5-vMAT; O) 5-HT; P) Tdc2>CD8-mCherry. Q-X) 63X zoom 4X. Q) vGlut-40XMYC; R) 6XV5-vMAT; S) 5-HT; T) Tdc2>CD8-mCherry; U) vGlut-40XMYC, 6XV5-vMAT overlay; V) vGlut-40XMYC, 6XV5-vMAT, 5-HT overlay; W) 6XV5-vMAT, 5-HT overlay; X) Tdc2, Tdc2>CD8-mCherry overlay. Scale bars: P-50μm; X-10μm.

Expression of the synaptic vesicle marker Synapsin in combination with vGlut-40XMYC and 6XV5-vMAT in the SV, ED, and AG of the Drosophila male reproductive system.

A-I) SV. A) vGlut-40XMYC; B) 6XV5-vMAT; C) Synapsin; D) vGlut-40XMYC; E) 6XV5-vMAT; F) Synapsin; G) vGlut-40XMYC, 6XV5-vMAT overlay; H) vGlut-40XMYC, Synapsin overlay; I) 6XV5-vMAT, Synapsin overlay. A’-I’) ED. A’) vGlut-40XMYC; B’) 6XV5-vMAT; C’) Synapsin; D’) vGlut-40XMYC; E’) 6XV5-vMAT; F’) Synapsin; G’) vGlut-40XMYC, 6XV5-vMAT overlay; H’) vGlut-40XMYC, Synapsin overlay; I’) 6XV5-vMAT, Synapsin overlay. A’’-I’’) AG. A’’) vGlut-40XMYC; B’’) 6XV5-vMAT; C’’) Synapsin; D’’) vGlut-40XMYC; E’’) 6XV5-vMAT; F’’) Synapsin; G’’) vGlut-40XMYC, 6XV5-vMAT overlay; H’’) vGlut-40XMYC, Synapsin overlay; I’’) 6XV5-vMAT, Synapsin overlay. Scale bars: C’’-50μm; I’’-10μm.

Glutamate receptor GAL4 expression patterns in Drosophila male reproductive system.

A) GluRIIB; B) GluRIIC. Scale bar: 200μm.

GluRIIA-GFP expression in the SV, ED, and AG of the Drosophila male reproductive system.

A-F) SV. A, D) vGlut-40XV5; B,E) GluRIIA-GFP; C, F) vGlut-40XV5, GluRIIA-GFP overlay. G-L) ED. G, J) vGlut-40XV5; H, K) GluRIIA-GFP; I, L) vGlut-40XV5, GluRIIA-GFP overlay. M-R) AG. M, P) vGlut-40XV5; N, Q) GluRIIA-GFP; O, R) vGlut-40XV5, GluRIIA-GFP overlay. Scale bars: O-50μm; R-10μm.

Male and female fertility phenotypes.

Low resolution images of nuclear expression of VT019028-GAL4-DBD in combination with TRH-AD and AbdB-AD in Drosophila male and female adult nervous system relative to 5-HT and Tdc2.

A-D) nuclear expression using HIS2A-GFP marker. A) male VT019028-GAL4-DBD/TRH-AD; B) female VT019028-GAL4-DBD/TRH-AD; C) male VT019028-GAL4-DBD/AbdB-AD; B) female VT019028-GAL4-DBD/AbdB-AD. E-H) 5-HT expression. E) male VT019028-GAL4-DBD/TRH-AD; F) female VT019028-GAL4-DBD/TRH-AD; G) male VT019028-GAL4-DBD/AbdB-AD; H) female VT019028-GAL4-DBD/AbdB-AD. Tdc2 expression. I) male VT019028-GAL4-DBD/TRH-AD; J) female VT019028-GAL4-DBD/TRH-AD; K) male VT019028-GAL4-DBD/AbdB-AD; L) female VT019028-GAL4-DBD/AbdB-AD. Scale bar: 200μm.

Comparison of mating duration between genotypes.

Bar plots show the mean ± SD for each group with individual data points overlaid on each bar. Statistical comparisons were made using unpaired two-tailed t tests where appropriate. Asterisks above indicate significance levels: p < 0.001 (**), p < 0.0001 (***), ns = not significant. (A) Columns from left to right: TRH ∩ AbdB > BONT-C: n= 10, mean= 19.11, SD=4.251. Tdc2 ∩ AbdB > BONT-C: n=14, mean=21.80, SD= 3.155. AbdB ∩ VT019028 > BONT-C: n=12, mean=24.75, SD=4.247. TRH ∩ VT019028 > BONT-C: n=11, mean=28.74, SD=2.155. AbdB; BONT-C: n=8, mean=27.07, SD=4.907. BONT-C: n=7, mean=28.75, SD=4.809. WT Canton-S: n=9, mean=26.91, SD=3.473.

Expression of vGlut-LexA and TRH-GAL4 in the Drosophila male reproductive system.

A, D, G, J, M, P) vGlut-LexA, LexAop-6XmCherry; B, E, H, K, N, Q) TRH-GAL4, UAS-6XGFP. C, F, I, L, O, R) overlay. Scale bars: O-50μm; R-10μm.

Expression of vGlut-LexA and Tdc2-GAL4 in the Drosophila male reproductive system.

A, D, G, J, M, P) vGlut-LexA; B, E, H, K, N, Q) Tdc2-GAL4, LexAop-6XmCherry. C, F, I, L, O, R) overlay. Scale bars: O-50μm; R-10μm.

Fruitless and Doublesex expression in the Drosophila male reproductive system.

A) fru-GAL4, UAS-6XGFP; B) vGlut-LexA, LexAop-6XmCherry; C) overlay; D) Tdc2-AD ∩ dsx-GAL4; E) TRH-AD ∩ dsx-GAL4. Scale bar: 200μm.

Expression of vGlut, TβH-GFP, Tdc2, and 5-HT in the SV of the Drosophila male reproductive system.

A, G) vGlut-40XV5; B, H) TβH-GFP; C, I) 5-HT; D, J) vGlut-40XV5, TβH-GFP overlay; E, K) vGlut-40XV5, 5-HT overlay; F, L) TβH-GFP, 5-HT overlay; M, S) vGlut-40XV5; N, T) Tdc2; O, U) 5-HT; P, V) vGlut-40XV5, Tdc2 overlay; Q, W) vGlut-40XV5, 5-HT overlay; R, X) Tdc2, 5-HT overlay. Scale bars: R-50μm; X-10μm.

Expression of vGlut, TβH-GFP, Tdc2, and 5-HT in the ED of the Drosophila male reproductive system.

A, G) vGlut-40XV5; B, H) TβH-GFP; C, I) 5-HT; D, J) vGlut-40XV5, TβH-GFP overlay; E, K) vGlut-40XV5, 5-HT overlay; F, L) TβH-GFP, 5-HT overlay; M, S) vGlut-40XV5; N, T) Tdc2; O, U) 5-HT; P, V) vGlut-40XV5, Tdc2 overlay; Q, W) vGlut-40XV5, 5-HT overlay; R, X) Tdc2, 5-HT overlay. Scale bars: R-50μm; X-10μm.

Expression of vGlut, TβH-GFP, Tdc2, and 5-HT in the AG of the Drosophila male reproductive system.

A, G) vGlut-40XV5; B, H) TβH-GFP; C, I) 5-HT; D, J) vGlut-40XV5, TβH-GFP overlay; E, K) vGlut-40XV5, 5-HT overlay; F, L) TβH-GFP, 5-HT overlay; M, S) vGlut-40XV5; N, T) Tdc2; O, U) 5-HT; P, V) vGlut-40XV5, Tdc2 overlay; Q, W) vGlut-40XV5, 5-HT overlay; R, X) Tdc2, 5-HT overlay. Scale bars: R-50μm; X-10μm.

Expression of vGlut, TβH-GFP, and 5-HT at the junction of the SV and AGs with the ED of the Drosophila male reproductive system.

A) vGlut-40XV5; B) TβH-GFP; C) 5-HT; D) vGlut-40XV5, TβH-GFP overlay; E) vGlut-40XV5, 5-HT overlay; F) TβH-GFP, 5-HT overlay. A) vGlut-40XV5; B) Tdc2; C) 5-HT; D) vGlut-40XV5, Tdc2 overlay; E) vGlut-40XV5, 5-HT overlay; F) Tdc2, 5-HT overlay. Scale bar: 50μm.

Co-conditional expression of vGlut-40XMYC and 6XV5-vMAT in the ED of serotonergic (TRH) or octopaminergic (Tdc2) neurons of the Drosophila male reproductive system.

A-D) 63X. A) vGlut-40XMYC; B) 6XV5-vMAT; C) Tdc2; D) TRH>CD8-mCherry. E-L) 63X zoom 4X. E) vGlut-40XMYC; F) 6XV5-vMAT; G) Tdc2; H) TRH>CD8-mCherry; I) vGlut-40XMYC, 6XV5-vMAT overlay; J) vGlut-40XMYC, 6XV5-vMAT, Tdc2 overlay; K) 6XV5-vMAT, Tdc2 overlay; L) Tdc2, TRH>CD8-mCherry overlay. M-P) 63X. M) vGlut-40XMYC; N) 6XV5-vMAT; O) 5-HT; P) Tdc2>CD8-mCherry. Q-X) 63X zoom 4X. Q) vGlut-40XMYC; R) 6XV5-vMAT; S) 5-HT; T) Tdc2>CD8-mCherry; U) vGlut-40XMYC, 6XV5-vMAT overlay; V) vGlut-40XMYC, 6XV5-vMAT, 5-HT overlay; W) 6XV5-vMAT, 5-HT overlay; X) Tdc2, Tdc2>CD8-mCherry overlay. Scale bars: P-50μm; X-10μm.

Co-conditional expression of vGlut-40XMYC and 6XV5-vMAT in the AG of serotonergic (TRH) or octopaminergic (Tdc2) neurons of the Drosophila male reproductive system.

A-D) 63X. A) vGlut-40XMYC; B) 6XV5-vMAT; C) Tdc2; D) TRH>CD8-mCherry. E-L) 63X zoom 4X. E) vGlut-40XMYC; F) 6XV5-vMAT; G) Tdc2; H) TRH>CD8-mCherry; I) vGlut-40XMYC, 6XV5-vMAT overlay; J) vGlut-40XMYC, 6XV5-vMAT, Tdc2 overlay; K) 6XV5-vMAT, Tdc2 overlay; L) Tdc2, TRH>CD8-mCherry overlay. M-P) 63X. M) vGlut-40XMYC; N) 6XV5-vMAT; O) 5-HT; P) Tdc2>CD8-mCherry. Q-X) 63X zoom 4X. Q) vGlut-40XMYC; R) 6XV5-vMAT; S) 5-HT; T) Tdc2>CD8-mCherry; U) vGlut-40XMYC, 6XV5-vMAT overlay; V) vGlut-40XMYC, 6XV5-vMAT, 5-HT overlay; W) 6XV5-vMAT, 5-HT overlay; X) Tdc2, Tdc2>CD8-mCherry overlay. Scale bars: P-50μm; X-10μm.

Co-conditional expression of vGlut-40XMYC and 6XV5-vMAT in serotonergic neurons of the Drosophila ED using expansion microscopy.

A, D) 6XV5-vMAT; B, E) vGlut-40XMYC; C, F) overlay. A-C) 6XV5-vMAT and vGlut-40XMYC expression above the threshold intensity values. D-F) Complete 6XV5-vMAT and vGlut-40XMYC expression. G) Mander’s plot of 6XV5-vMAT and vGlut-40XMYC. Vertical line indicates intensity threshold value for 6XV5-vMAT. Horizontal line indicates intensity threshold value for vGlut-40XMYC. H) Mander’s coefficients. Grayscale signal in A-C) denotes the CD8-mCherry plasma membrane marker. Scale bars: 500nm. Scale bars have been corrected by 10X to reflect the empirically determined 10.5X expansion factor.

Co-conditional expression of vGlut-40XMYC and 6XV5-vMAT in octopaminergic neurons of the Drosophila ED using expansion microscopy.

A, D) 6XV5-vMAT; B, E) vGlut-40XMYC; C, F) overlay. A-C) 6XV5-vMAT and vGlut-40XMYC expression above the threshold intensity values. D-F) Complete 6XV5-vMAT and vGlut-40XMYC expression. G) Mander’s plot of 6XV5-vMAT and vGlut-40XMYC. Vertical line indicates intensity threshold value for 6XV5-vMAT. Horizontal line indicates intensity threshold value for vGlut-40XMYC. H) Mander’s coefficients. Grayscale signal in A-C) denotes the CD8-mCherry plasma membrane marker. Scale bars: 500nm. Scale bars have been corrected by 10X to reflect the empirically determined 10.5X expansion factor.

Nuclei of unexpanded and expanded EDs.

A) DAPI-stained nuclei from an unexpanded ED. B) DAPI-stained nuclei of an expanded ED. C) Bar graph plot of nuclei area (mean ± SE-black bars). For the experiment shown the calculated expansion factor (post-ExM nuclear area/pre-ExM nuclear area) was 10.47, p-value < 0.00001. Fij/ImageJi open-source software was used to segment images and calculate nuclear area. Scale bars: 25um.

Expression of the active zone marker Brp in combination with vGlut-40XMYC and 6XV5-vMAT in the SV, ED, and AG of the Drosophila male reproductive system.

A-H) SV. A) vGlut-40XMYC; B) 6XV5-vMAT; C) Brp; D) 6XV5-vMAT, Brp overlay; E) vGlut-40XMYC; F) 6XV5-vMAT; G) Brp; H) 6XV5-vMAT, Brp overlay. I-P) ED. I) vGlut-40XMYC; J) 6XV5-vMAT; K) Brp; L) 6XV5-vMAT, Brp; M) vGlut-40XMYC; N) 6XV5-vMAT; O) Brp; P) 6XV5-vMAT, Brp overlay. I-P) AG. Q) vGlut-40XMYC; R) 6XV5-vMAT; S) Brp; T) 6XV5-vMAT, Brp; U) vGlut-40XMYC; V) 6XV5-vMAT; W) Brp; X) 6XV5-vMAT, Brp overlay. Scale bars: P-50μm; X-10μm.

Expression of the post-synaptic density marker Dlg in combination with vGlut-40XMYC and 6XV5-vMAT in the SV, ED, and AG of the Drosophila male reproductive system.

A-D) Complete reproductive system. A) vGlut-40XMYC; B) 6XV5-vMAT; C) Dlg; D) 6XV5-vMAT, Dlg overlay; E-L) SV. E) vGlut-40XMYC; F) 6XV5-vMAT; G) Dlg; H) 6XV5-vMAT, Dlg overlay; I) vGlut-40XMYC; J) 6XV5-vMAT; G) Dlg; H) 6XV5-vMAT, Dlg overlay. I-P) ED. I) vGlut-40XMYC; K) 6XV5-vMAT; L) Dlg; M-P) ED muscle surface. M) vGlut-40XMYC; N) 6XV5-vMAT; O) Dlg; P) 6XV5-vMAT, Dlg overlay Q) Dlg in the ED epithelial cells. R-U) AG muscle surface. R) vGlut-40XMYC; S) 6XV5-vMAT; T) Dlg; U) 6XV5-vMAT, Dlg overlay; V) Dlg in AG epithelial cells. Scale bars: D-200μm; H-50μm; V-10μm.

Expression of the large core dense vesicle marker IA2-GFP in combination with vGlut-40XMYC and 6XV5-vMAT in the SV of the Drosophila male reproductive system.

A-C) Complete reproductive system. A) vGlut-40XMYC; B) 6XV5-vMAT; C) IA2-GFP; D-O) SV. D, J) vGlut-40XMYC; E, K) 6XV5-vMAT; F, L) IA2-GFP; G, M) vGlut-40XMYC, 6XV5-vMAT overlay; H, N) vGlut-40XMYC, IA2-GFP overlay; I, O) 6XV5-vMAT, IA2 overlay. Scale bars: C-200μm; F-50μm; O-10μm.

Expression of the large core dense vesicle marker IA2-GFP in combination with vGlut-40XMYC and 6XV5-vMAT in the ED and AGs of the Drosophila male reproductive system.

A-L) ED. A, G) vGlut-40XMYC; B, H) 6XV5-vMAT; C, I) IA2-GFP; D, J) vGlut-40XMYC, 6XV5-vMAT overlay; E, K) vGlut-40XMYC, IA2-GFP overlay; F, L) 6XV5-vMAT, IA2 overlay. M-X) AG. M, S) vGlut-40XMYC; N, T) 6XV5-vMAT; O, U) IA2-GFP; P, V) vGlut-40XMYC, 6XV5-vMAT overlay; Q, W) vGlut-40XMYC, IA2-GFP overlay; R, X) 6XV5-vMAT, IA2 overlay. Scale bars: R-50μm; X-10μm.

Expression of vGlut-40XMYC, 6XV5-vMAT, 7XMYC-vAChT, and 9XV5-vGAT in the SV, ED, and AG of the Drosophila male reproductive system.

A-D) SV. A) vGlut-40XV5; B) 6XV5-vMAT; C) 7XMYC-vAChT; D) 9XV5-vGAT. E-H) ED. E) vGlut-40XV5; F) 6XV5-vMAT; G) 7XMYC-vAChT; H) 9XV5-vGAT. I-L) AG. I) vGlut-40XV5; J) 6XV5-vMAT; K) 7XMYC-vAChT; L) 9XV5-vGAT. Scale bar: 50μm.

OA receptor GAL4 expression patterns in Drosophila male reproductive system.

A) OAMB; B) OAα2R; C) Oct-TyrR; D) OAβ1R; E) OAβ2R; F) OAβ3R. Scale bar: 200μm.

5-HT receptor GAL4 expression patterns in Drosophila male reproductive system.

A) 5-HT1A; B) 5-HT2A; C) 5-HT1B; D) 5-HT2B; E) 5-HT7. Scale bar: 200μm.

OAMB expression in the Drosophila male reproductive system.

A-C) Complete male reproductive system. A) OAMB-10XV5; B) Syn; C) OAMB-10XV5, Syn overlay. D-I) SV. D, G) OAMB-10XV5; E, H) Syn; F, I) OAMB-10XV5, Syn overlay. J-R) ED. J-L) muscle surface. J) OAMB-10XV5; K) Syn; L) OAMB-10XV5, Syn overlay. M-O) muscles and epithelial layers. M) OAMB-10XV5; N) Syn; O) OAMB-10XV5, Syn overlay. P-R) epithelial layer. P) OAMB-10XV5; Q) Syn; R) OAMB-10XV5, Syn overlay. Scale bars: C-200μm; F, O-50μm; I, L, R-10μm.

OAMB expression in the Drosophila male reproductive system.

A-C) AG. A) OAMB-10XV5; B) Syn; C) OAMB-10XV5, Syn overlay. D-F) Epithelial layer of AG. D) OAMB-10XV5; E) Syn; F) OAMB-10XV5, Syn overlay. G-I) Muscle layer of AG. G) OAMB-10XV5; H) Syn; I) OAMB-10XV5, Syn overlay. J-L) Ejaculatory bulb. J) OAMB-10XV5; K) Syn; L) OAMB-10XV5, Syn overlay. M-O) muscle layer of ejaculatory bulb. M) OAMB-10XV5; N) Syn; O) OAMB-10XV5, Syn overlay. P-Q) Epithelial layer of ejaculatory bulb. P) OAMB-10XV5; Q) Syn; R) OAMB-10XV5, Syn overlay. Scale bars: C, L-50μm; F, I, R-10μm.

OAα2R-20XV5 expression in the Drosophila male reproductive system.

A-C) Complete male reproductive system. A) OAα2R-20XV5; B) Syn; C) OAα2R-20XV5, Syn overlay. D-I) SV. D, G) OAα2R-20XV5; E, H) Syn; F, I) OAα2R-20XV5, Syn overlay. Cross-section of SV. J) OAα2R-20XV5: K) Syn; L) OAα2R-20XV5, Syn overlay. M-R) AG. M, P) OAα2R-20XV5; N, Q) Syn; O, R) OAα2R-20XV5, Syn overlay. Scale bars: C-200μm; O-50μm; R-10μm.

OAα2R-20XV5 expression in the Drosophila male reproductive system.

A-C) SV/ED junction. A, D) OAα2R-20XV5; B, E) Syn; C, F) OAα2R-20XV5, Syn overlay. G-L) Proximal anterior ED. G, J) OAα2R-20XV5; H, K) Syn; I, L) OAα2R-20XV5, Syn overlay. M-U) Distal anterior ED. M, P, S) OAα2R-20XV5; N, Q, T) Syn; O, R, U) OAα2R-20XV5, Syn overlay. Scale bars: C, I, O-50μm; F-25μm; L, U-10μm.

OAβ2R-40XV5 expression in the Drosophila male reproductive system.

A-C) Complete male reproductive system. A) OAβ2R-40XV5; B) Syn; C) OAβ2R-40XV5, Syn overlay. D-I) SV. D, G,) OAβ2R; E, H) Syn; F, I) OAβ2R-40XV5, Syn overlay. J-L) Cross section of SV. J) OAβ2R-40XV5; K) Syn; L) OAβ2R-40XV5, Syn overlay. M-R) ED. M, P) OAβ2R; N, Q) Syn; O, R) OAβ2R-40XV5, Syn overlay. Scale bars: C-200μm; O-50μm; R-10μm.

OAβ2R-40XV5 expression in the Drosophila male reproductive system.

A-C) Junction of SV and ED. A) OAβ2R-40XV5; B) Syn; C) OAβ2R-40XV5, Syn overlay. D-F) Cross section of ED. D) OAβ2R-40XV5; E) Syn; F) OAβ2R-40XV5, Syn overlay. G-I) Junction of SV and ED. G) OAβ2R-40XV5; H) Syn; I) OAβ2R-40XV5, Syn overlay. J-L) Distal SV. J) OAβ2R-40XV5; E) Syn; F) OAβ2R-40XV5, Syn overlay. M-O) Cross section of distal SV. M) OAβ2R-40XV5; N) Syn; O) OAβ2R-40XV5, Syn overlay. Scale bars: C, I-50μm; O-10μm.

OAβ2R-40XV5 expression in the Drosophila male reproductive system.

A-C) Distal anterior ED. A) OAβ2R-40XV5; B) Syn; C) OAβ2R-40XV5, Syn overlay. D-F) Cross section of distal anterior ED. D) OAβ2R-40XV5; E) Syn; F) OAβ2R-40XV5, Syn overlay. G-L) AG. G, J) OAβ2R-40XV5; H, K) Syn; I, L) OAβ2R-40XV5, Syn overlay. M-O) Ejaculatory bulb. M, O) OAβ2R-40XV5; N, Q) Syn; O, R) OAβ2R-40XV5, Syn overlay. Scale bars: O-50μm; R-10μm.

5-HT7-20XV5 expression in the Drosophila male reproductive system.

A-C) Complete male reproductive system. A) 5-HT7-20XV5.; B) Syn; C) 5-HT7-20XV5, Syn overlay. D-I) SV. D, G) 5-HT7-20XV5.; E, H) Syn; F, I) 5-HT7-20XV5, Syn overlay. J-O) ED. J, M) 5-HT7-20XV5.; K, N) Syn; L, O) 5-HT7-20XV5, Syn overlay. Scale bars: C-200μm; L-50μm; O-10μm.

5-HT7-20XV5 expression in the Drosophila male reproductive system.

A-I) SV. A, D, G,) 5-HT7-20XV5.; B, E, H,) Syn; C, F, I,) 5-HT7-20XV5, Syn overlay. J-L) Cross section of SV. J) 5-HT7-20XV5.; K) Syn; L) 5-HT7-20XV5, Syn overlay. M-O) SV terminus. M) 5-HT7-20XV5.; N) Syn; O) 5-HT7-20XV5, Syn overlay. P-R) SV/ED junction. P) 5-HT7-20XV5.; Q) Syn; R) 5-HT7-20XV5, Syn overlay. Scale bars: C, G-50μm; F, O-10μm; R-25μm.

5-HT7-20XV5 expression in the Drosophila male reproductive system.

A-C) AG. A) 5-HT7-20XV5.; B) Syn; C) 5-HT7-20XV5, Syn overlay. D-F) Epithelial layer of AG. D) 5-HT7-20XV5.; E) Syn; F) 5-HT7-20XV5, Syn overlay. G-I) Cross-section of epithelial layer of AG. G) 5-HT7-20XV5.; H) Syn; I) 5-HT7-20XV5, Syn overlay. J-L) Muscle layer of AG. J) 5-HT7-20XV5.; K) Syn; L) 5-HT7-20XV5, Syn overlay. M-O) Ejaculatory bulb. M) 5-HT7-20XV5.; N) Syn; O) 5-HT7-20XV5, Syn overlay. Scale bars: C, O-50μm; L-10μm.

High resolution images of nuclear expression of VT019028-GAL4-DBD in combination with TRH-AD in posterior ventral nerve cord of Drosophila male adult nervous system relative to 5-HT and Tdc2.

A-E) Dorsal view of complete stack of confocal images.

A) His2A-GFP; B) 5-HT; C) Tdc2; D) His2A-GFP, 5-HT overlay; E) His2A-GFP, Tdc2 overlay. F) Ventral view of His2A-GFP/5-HT overlay of subset of slices containing the dorsal cluster of 5-HT neurons; G-K) Dorsal view of subset of slices containing the dorsal cluster of 5-HT neurons. G) His2A-GFP; H) 5-HT; I) Tdc2; J) His2A-GFP, 5-HT overlay; K) His2A-GFP, Tdc2 overlay. L-P) Dorsal view of subset of slices containing the ventral cluster of 5-HT neurons. L) His2A-GFP; M) 5-HT; N) Tdc2; O) His2A-GFP, 5-HT overlay; P) His2A-GFP, Tdc2 overlay. Scale bar: 50μm.

High resolution images of nuclear expression of VT019028-GAL4-DBD in combination with AbdB-AD in posterior ventral nerve cord of Drosophila male adult nervous system relative to 5-HT and Tdc2.

A-E) Dorsal view of complete stack of confocal images. A) His2A-GFP; B) 5-HT; C) Tdc2; D) His2A-GFP, 5-HT overlay; E) His2A-GFP, Tdc2 overlay. F-J) Dorsal view of subset of slices containing the dorsal cluster of 5-HT neurons. F) His2A-GFP; G) 5-HT; H) Tdc2; I) His2A-GFP, 5-HT overlay; J) His2A-GFP, Tdc2 overlay. K-O) Dorsal view of subset of slices containing the ventral cluster of 5-HT neurons. K) His2A-GFP; L) 5-HT; M) Tdc2; N) His2A-GFP, 5-HT overlay; O) His2A-GFP, Tdc2 overlay. Scale bar: 50μm.

Expression of ProtB-GFP fluorescent marker of sperm nuclei in male reproductive system of control and TRH-AD/VT019028-GAL4-DBD neuron-silenced flies interrupted mid-mating.

A) control. B) TRH-AD/VT019028-GAL4-DBD neurons silenced with BONT-C. Prot-GFP-Protamine GFP. SV-SV, ED-ED. Scale bar: 75μm.

Comparison of male drosophila fecundity between genotypes.

Bar plots show the mean ± SD for each group with individual data points overlaid on each bar. Figure 10S1A males were aged 5 days before copulation. Figure 10S1B males were aged 30 days before copulation. (A) Columns from left to right: TRH ∩ VT019028 → vGlut-: n=34, mean=173.2, SD=24.22. AbdB ∩ TRH, Tdc2 → vGlut-: n=20, mean=175.3, SD=22.92. TRH ∩ VT019028: n=32, mean=149.0, SD=26.10. TRH: n=28, mean=172.8, SD=21.11. VT019028: n=25, mean=148.6, SD=20.81. (B) TRH ∩ VT019028 → vGlut-: n=14, mean=133.9, SD=37.64. TRH ∩ VT019028: n=19, mean=123.7, SD, 25.02. TRH: n=13, mean=106.3, SD=41.93. VT019028: n=9, mean=131.0, SD=38.88.

vGlut expression in AGs of control and vGlut conditional mutant.

A-C) control male. A) vGlut; B) 6XmCherry; C) overlay. D-F) experimental male conditionally silenced for vGlut in TRH-AD/VT019028-GAL4 neurons. Scale bar: 50μm.