The Drosophila male reproductive system.

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

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) 5-HT, 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, LexAop-6XmCherry; B, E, H, K, N, Q) Tdc2-GAL4, UAS-6XGFP. 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-DBD; E) TRH-AD ∩ dsx-GAL4-DBD. 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. 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. distal anterior ejaculatory duct proximal anterior ejaculatory duct seminal vesicle/ ejaculatory duct junction

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.

Male reproductive system expression of VT019028-GAL4-DBD in combination with TRH-AD and AbdB-AD.

A) VT019028-GAL4-DBD ∩ TRH-AD; B) VT019028-GAL4-DBD ∩ AbdB-AD. For both genotypes, there is broad innervation across the SV, AG, and ED. Scale bar: 200μm.

Prot-GFP fluorescent marker in male and female reproductive systems in control and TRH-AD/VT019028-GAL4-DBD neuron-silenced flies.

A) control. B) TRH-AD/VT019028-GAL4-DBD neurons silenced with BONT-C. Matings were interrupted 10 minutes after initiation. No Prot-GFP sperm is observed in the ejaculatory duct of the experimental male. C and D) Wildtype Canton-S female reproductive systems from completed matings to a C) control male; and D) TRH-AD/VT019028-GAL4-DBD male whose neurons were silenced with BONT-C. Abundant Prot-GFP sperm are observed in the mating to the control male (arrow), while no Prot-GFP sperm is observed in the female reproductive system after mating to the experimental male (arrow). Prot-GFP-Protamine GFP. SV-SV, ED-ED. Scale bars: A and B-75μm; C and D-200μm.

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.