Ptth null mutants have increased virgin female receptivity.

(A-C) Generation and validation of a 974 bp deletion mutant of the Ptth gene. (D-G) Virgin female receptivity of Ptth null mutants on the 1st (D), 2nd (E), 3rd (F) and 6th day (G) respectively. (H) Brain of indicated genotype, immunostained with anti-PTTH antibody (green) and counterstained with nc82 (magenta). Arrows show signals (green) stained with anti-PTTH antibody. Scale bars, 50 μm. (I) Enhanced virgin female receptivity of ΔPtth null mutants was rescued by elavGAL4 driving UAS-PTTH. The increased copulation rate and decreased latency to copulation on the 1st day after eclosion were rescued to the comparable level of control. The number of female flies paired with wild type males is displayed in parentheses. For the copulation rate, chi-square test is applied. For the latency to copulation, Kruskal-Wallis ANOVA and post hoc Mann-Whitney U tests are applied. Error bars indicate SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns indicates no significant difference.

Inactivation of doublesex-positive PG neurons expressing PTTH enhances virgin female receptivity during the larval stage.

(A-B) Expression pattern of PtthGAL4 revealed by anti-GFP in larvae central nervous system (CNS) (A) and adult CNS (B). Representative of five female flies. Scale bars, 50 μm. (C) All PG neurons were colabeled by dsxGAL4 driving UAS-GFPStinger (red) and PtthLexA driving LexAop-tomato (green). Representative of five female brains. Scale bars, 50 μm and 5 μm (zoom-in). (D) All PG neurons were Ptth and Dsx co-expressing, labeled by intersectional strategy. Representative of 5 female brains. Scale bars, 50 μm. (E-G) PG neurons were inactivated during larval (E), pupal (F) and adult (G) stages respectively by kir2.1, restricted by shifts from 18°C to 30°C. The inactivation during the larval stage significantly increased the copulation rate (E). The number of female flies paired with wild-type males is displayed in parentheses. For the copulation rate, chi-square test is applied. Error bars indicate SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p< 0.0001, ns indicates no significant difference.

Activation of PG neurons expressing PTTH during the 3rd-instar larvae inhibits virgin female receptivity.

(A) Four developmental stages of Drosophila before eclosion when PG neurons were thermogenetic activated by dTrpA1. L1, L2, and L3: start of three larval stages, W: start of wandering stage, Pp: puparium formation, P1 and P2: start of the 1st and 2nd day of pupal stage. (B-E) PtthGAL4 driving UAS-dTrpA1 activated PG neurons at 29°C. Activation of PG neurons at the stage 2 significantly decreased copulation rate (C), but not at the stage 1 (B), stage 3 (D) and stage 4 (E). (F) Activation of PG neurons at the stage 2 significantly increased the latency to copulation. (G) Mean velocity had no significant change when PG neurons were activated during the stage 2 compared with control females (ns = not significant, Kruskal-Wallis ANOVA and post hoc Mann-Whitney U tests, mean ± SEM, n = 8-12). The number of female flies paired with wild-type males is displayed in parentheses. For the copulation rate, chi-square test is applied. For the latency to copulation, Mann-Whitney U test is applied. Error bars indicate SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns indicates no significant difference.

Feeding 20E restores virgin female receptivity of Ptth null mutant flies.

(A-B) The increased copulation rate and decreased latency to copulation of the 24h-old ΔPtth flies were rescued to the comparable level of wild type females by feeding 20E to the 3rd-instar larval ΔPtth flies. The wild type larval females fed by 20E had no significantly different copulation rate and latency to copulation compared with the wild type females fed by the same volume of 95% ethanol which is the solvent of 20E. The number of female flies paired with wild-type males is displayed in parentheses. For the copulation rate, chi-square test is applied. For the latency to copulation, Kruskal-Wallis ANOVA and post hoc Mann-Whitney U tests are applied. Error bars indicate SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns indicates no significant difference.

Virgin females with reduced EcR-A in pC1 neurons have reduced sexual receptivity.

(A) Knock-down of EcR-A in pC1 neurons driven by pC1-ss2-GAL4 significantly decreased the copulation rate and increased the latency to copulation. (B) Knock-down of EcR-B1 in pC1 neurons driven by pC1-ss2-GAL4 significantly prolonged the latency to copulation. (C-D) Knock-down of EcR-A (C) or EcR-B1 (D) in pC1 neurons driven by pC1-ss1-GAL4 did not affect the copulation rate or the latency to copulation. (E) Courtship index of wild-type males towards a female with the indicated genotype (n = 8). (F) The number of eggs laid by virgin females during the 3rd - 4th day after eclosion when EcR-A was knocked down in pC1 neurons (n = 17-36). (G) Knock-down of EcR-A in pC1 neurons decreased the opening of vaginal plate of virgin females compared with controls (n = 8). (H) Knock-down of EcR-A in pC1 neurons increased the ovipositor extrusion of virgin females compared with controls (n = 8). (I-K) Virgin female copulation rate when EcR-A was knocked down in pC1 neurons temporally restricted by shifts from 18°C to 30°C. EcR-A was knocked down during larval (I), pupal (J) and adult (K) stages respectively. Knock-down of EcR-A during pupal stage significantly decreased the copulation rate (J). The number of female flies paired with wild-type males is displayed in parentheses. For the copulation rate, chi-square test is applied. For other comparisons, Kruskal-Wallis ANOVA and post hoc Mann-Whitney U tests are applied. Error bars indicate SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns indicates no significant difference.

Reduced EcR-A in pC1 neurons induces the morphological changes.

(A1-A2) pC1 neurons appeared at the start of the pupal stage. (B-D) Reduced EcR-A in pC1 neurons induced more elaborated morphologies of pC1d axons, especially the extra vertical projection (EVP). The EVP regions of pC1d neurons was indicated by arrows. The morphological changes appeared on the 2nd day of the pupal stage (B1-B2) and retained to the adult stage including the 1st day (C1-C2) and the 4th day (D1-D2) of the adult stage. p0, the 1st day of the pupal stage; p2, the 2nd day of the pupal stage; A1, the 1st day of the adult stage; A4, the 4th day of the adult stage. (E-F) Fluorescence intensity of EVP in pC1d neurons on the 2nd day of the pupal stage (E) and the 4th day of the adult stage (F) was quantified when EcR-A was reduced in pC1 neurons (n=7). The quantified EVP regions were marked in (B) and (D) with orange ellipses. (G) pC1 neurons of the 4th day adults had comparable cell body number when EcR-A was reduced in pC1 neurons or not (n = 7). (H) Basal GCaMP6s signals in the LPC region of pC1 neurons when EcR-A was reduced in pC1 neurons (n = 22). LPC regions, the neurites extending from pC1 cell bodies, were marked with orange square in (D1) and (D2). Scale bars are 50 μm. For all comparisons, Mann-Whitney U test is applied. Error bars indicate SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns indicates no significant difference.

Reduced EcR-A in pC1 neurons induces the down-regulated dopamine beta-monooxygenase (DBM) level.

(A) Volcano plot of RNA-seq from virgin female brains in which EcR-A was knocked down in pC1 neurons or not. Each circle represents a protein-coding gene. Differential genes with a p-value < 0.01 are highlighted in blue. Differential genes with a fold change > 4 are highlighted in green. Differential genes with a p-value < 0.01 and fold change > 4 are highlighted in red. DBM, the most down-regulated genes with annotation, are indicated by black arrow. Data are from three replicates, each contains about 100 brains. (B) qRT-PCR for DBM when EcR-A was knocked down in pC1 neurons. Bars represent mean ± SEM. p values are from Mann-Whitney U test (n = 12 based on four replicates for each, each contains about 100 brains). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns indicates no significant difference.

Weight, attractiveness and locomotion behavior of Ptth null mutant virgin females.

(A) The weight of 24h-old adult ΔPtth null mutant females was significantly higher than that of wild type females (Mann-Whitney U test, n=8 groups, 10 flies in each group). (B) Courtship index of wild-type males during the first 5 min of courtship towards a female with the indicated genotype (Kruskal-Wallis ANOVA and post hoc Mann-Whitney U tests, n = 7-9). (C-D) Mean velocity had no significant change in ΔPtth null mutant females on the 1st day (C) and the 6th day (D) compared with control females (Kruskal-Wallis ANOVA and post hoc Mann-Whitney U tests, n = 7-12). Error bars indicate SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant.

PG neurons expressing PTTH labeled by PtthLexA.

Expression pattern of PtthLexA in the brain revealed by anti-GFP (green) in larvae brain (A) and adult brain (B). Representative of five female flies. Scale bars, 50 μm.

The anatomical pattern of PG neurons expressing PTTH at different developmental stages.

Expression pattern of PtthGAL4 in the brain revealed by anti-GFP from the 3rd larval stage to the 4th day after eclosion. Arrows show PTTH signals (green) stained with anti-GFP antibody. L3, the 3rd-instar larvae; wander, the wandering larvae; P4, the 4th day of the pupal stage; A0h, the 1st hour of the adult stage; A3h, the 3rd hour of the adult stage; A6h, the 6th hour of the adult stage; A9h, the 9th hour of the adult stage; A12h, the 12th hour of the adult stage; A4d, the 4th day of the adult stage. Representative of five female flies. Scale bars, 50 μm.

PG neurons expressing PTTH do not regulate virgin female copulation rate during adult stage.

PG neurons were activated during adult stage by dTrpA1 at 29°C. The female copulation rate and the latency to copulation did not change significantly. The number of female flies paired with wild-type males is displayed in parentheses. For the copulation rate, chi-square test is applied. For the latency to copulation, Mann-Whitney U test is applied. Error bars indicate SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns indicates no significant difference.

Expression of EcR-A and EcR-B1 in pC1 and vpoDN neurons.

(A-C) pC1 neurons were colabeled by pC1-ss2 driving UAS-mCD8-GFP (green, A) and EcR-A antibodies (red, B). Magnification of green boxed region in (C) is shown in (D1– D3). (E-G) pC1 neurons were colabeled by pC1-ss2 driving UAS-mCD8-GFP (green, E) and EcR-B1 antibodies (red, F). Magnification of green boxed region in (G) is shown in (H1–H3). (I-K) vpoDN neurons were colabeled by vpo-ss1 driving UAS-mCD8-GFP (green, I) and EcR-A antibodies (red, J). Magnification of green boxed region in (K) is shown in (L1–L3). (M-O) vpoDN neurons were colabeled by vpo-ss1 driving UAS- mCD8-GFP (green, M) and EcR-B1 antibodies (red, N). Magnification of green boxed region in (O) is shown in (P1–P3). Scale bars for magnified regions are 5 μm, for others are 50 μm.

Reduced EcR in pC1 neurons reduces virgin female receptivity.

(A) Knock-down of EcR in pC1 neurons driven by pC1-ss2-GAL4 significantly decreased the copulation rate and increased the latency to copulation. (B) Mean velocity had no significant change when EcR-A was knocked down in pC1 neurons compared with controls (Kruskal-Wallis ANOVA and post hoc Mann-Whitney U tests, n = 8-11). The number of female flies paired with wild-type males is displayed in parentheses. For the copulation rate, chi-square test is applied. For the latency to copulation, Kruskal-Wallis ANOVA and post hoc Mann-Whitney U tests are applied. Error bars indicate SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns indicates no significant difference.

Reduced EcR in vpoDN neurons has no effect on virgin female receptivity.

(A-C) Knock-down of EcR-A in vpoDN neurons driven by vpoDN-ss1-GAL4, vpoDN-ss2-GAL4 and vpoDN-ss3-GAL4 had no effect on virgin female receptivity. (D-F) Knock-down of EcR-B1 in vpoDN neurons driven by vpoDN-ss1-GAL4, vpoDN-ss2-GAL4 and vpoDN-ss3-GAL4 had no effect on virgin female receptivity. The number of female flies paired with wild-type males is displayed in parentheses. For the copulation rate, chi-square test is applied. For the latency to copulation, Kruskal-Wallis ANOVA and post hoc Mann-Whitney U tests are applied. Error bars indicate SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns indicates no significant difference.

Reduced EcR-A in pC1d neurons has no effect on virgin female receptivity.

(A) Knock-down of EcR-A in pC1d neurons had no effect on virgin female copulation rate and latency to copulation. The number of female flies paired with wild-type males is displayed in parentheses. For the copulation rate, chi-square test is applied. For the latency to copulation, Kruskal-Wallis ANOVA and post hoc Mann-Whitney U tests are applied. Error bars indicate SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns indicates no significant difference.