Neuroscience

The function of juvenile-adult transition axis in female sexual receptivity of Drosophila melanogaster

Jing Li author has email address
Chao Ning
Yaohua Liu
Bowen Deng
Bingcai Wang
Kai Shi
Rencong Wang
Ruixin Fang
Chuan Zhou

Institute of Molecular Physiology, Shenzhen Bay Laboratory, Shenzhen, China
State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
National Laboratory of Biomacromolecules, New Cornerstone Science Laboratory, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
CAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China
Department of Plant Protection, Shanxi Agricultural University, Jinzhong, China
Chinese Institute for Brain Research, Peking-Tsinghua Center for Life Sciences, Zhongguancun Life Sciences Park, Beijing, China
University of Chinese Academy of Sciences, Beijing, China

https://doi.org/10.7554/eLife.92545.2

Open access
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Figures and data

Ptth null mutants have increased virgin female receptivity.
(A-C) Generation and validation of a 974 bp deletion mutant of the Ptth gene. The 5’ UTR and almost all coding sequence were deleted. The deletion was confirmed through PCR analysis at the PTTH locus in genomic DNA samples (B), and through RT-PCR to identify the loss of PTTH transcripts in cDNA samples of wandering larvae (C). (D-G) Virgin female receptivity of Ptth null mutants on the 1^st (D), 2^nd (E), 3^rd (F) and 6^th day (G) respectively. The comparison referred to Ptth^Delete/Ptth^Delete. (H) Enhanced virgin female receptivity of ΔPtth null mutants was rescued by elav-Gal4 driving UAS-PTTH. The increased copulation rate and decreased latency to copulation on the 1^st day after eclosion were rescued to the comparable level of control. The comparison referred to elav-Gal4/+; Ptth^Delete/Ptth^Delete;UAS-PTTH/+. The copulation latency and copulation rate of elav-Gal4/+; Ptth^Delete/Ptth^Delete is higher and lower than Ptth^Delete/Ptth^Delete;UAS-PTTH/+ respectively. 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.

PTTH expression, weight, attractiveness and locomotion behavior of Ptth null mutant virgin females.
(A) Brain of indicated genotype, immunostained with anti-PTTH antibody (green) and counterstained with nc82 (magenta). Arrows show signals (green) stained with anti-PTTH antibody. Female flies were within 10 hours after eclosion. Scale bars, 50 μm. (B) The weights of 24h-old adult Ptth^Delete/Ptth^Delete null mutant females were significantly higher than that of wild type females (Mann-Whitney U test, n=8 groups, 10 flies in each group). (C) 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). (D-E) Mean velocity had no significant change in Ptth^Delete/Ptth^Delete null mutant females on the 1^st day (D) and the 6^th day (E) compared with control females (Kruskal-Wallis ANOVA and post hoc Mann-Whitney U tests, n = 7-12). The comparison referred to Ptth^Delete/Ptth^Delete. Female flies for behavioral assay were 4-6-days-old adults. Error bars indicate SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant.

Effect of the expression of PTTH on female receptivity.
(A) Overexpressing PTTH in PTTH neurons. The comparison referred to flies Ptth-Gal4/+;UAS-PTTH/+. Female flies for behavioral assay were 4-6 days-old. (B-C) Decreasing PTTH in PTTH neurons. The comparison referred to flies Ptth-Gal4/+;UAS-PTTH-RNAi/+ and Dsx-Gal4/+;UAS-PTTH-RNAi/+. Female flies for behavioral assay were 2-days-old adults. (D) The fly brain of elav-Gal4>UAS-GFP were stained with PTTH antibody. The larvae flies were the wandering ones. The adult flies were within 10 hours after eclosion. Representative of 5 female brains. Scale bars, 50 μm.

PTTH neurons are doublesex-positive neurons.
(A-B) Expression pattern of Ptth-Gal4 revealed by anti-GFP in larvae central nervous system (CNS) (A) and adult brain (B). Representative of five female flies. Scale bars, 50 μm. (C) All PTTH neurons were colabeled by dsx-Gal4 driving UAS-GFP-Stinger (red) and Ptth-LexA driving LexAop-tomato (green). Representative of five female brains. Scale bars, 50 μm and 5 μm (zoom-in). (D) All PTTH neurons were Ptth and Dsx co-expressing, labeled by intersectional strategy. The larvae flies were the wandering ones. The adult flies were within-10 hours-old adults. Representative of 5 female brains. Scale bars, 50 μm.

The function of PTTH neurons in female receptivity.
Expression pattern of Ptth-LexA in the brain revealed by anti-GFP (green) in wandering larvae CNS (A) and within-10-hours adult brain (B). Representative of five female flies. Scale bars, 50 μm. (C) The female receptivity when deceasing the expression of Dsx^F in PTTH neurons. The comparison referred to Ptth-Gal4/+;UAS-Dsx^F-RNAi/+. Female flies were 2-days-old adults. (D) PTTH neurons were inactivated during the whole larval, pupal and adult stages respectively by kir2.1, restricted by shifts from 18°C to 30°C. When the experiment was done at the larval stage is the only situation when the controls were both different from the experimental. The comparison referred to Ptth-Gal4/tub-Gal80ts;kir2.1/+. Female flies were 2-days-old adults. 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.

The anatomical pattern of PTTH neurons expressing PTTH at different developmental stages.
Expression pattern of Ptth-Gal4 in the brain revealed by anti-GFP from the 3^rd larval stage to the 4^th day after eclosion. Arrows show PTTH signals (green) stained with anti-GFP antibody. L3, the 3^rd -instar larvae; wander, the wandering larvae; P4, the 4^th day of the pupal stage; A0h, the 1^st hour of the adult stage; A3h, the 3^rd hour of the adult stage; A6h, the 6^th hour of the adult stage; A9h, the 9^th hour of the adult stage; A12h, the 12^th hour of the adult stage; A4d, the 4^th day of the adult stage. Representative of five female flies. Scale bars, 50 μm.

PTTH neurons expressing PTTH do not regulate virgin female copulation rate during adult stage.
PTTH 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. Female flies were 4-days-old adults. 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.

Activation of PTTH neurons expressing PTTH during the 3^rd-instar larvae inhibits virgin female receptivity.
(A) Four developmental stages of Drosophila before eclosion when PTTH 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 1^st and 2^nd day of pupal stage. (B-E) Ptth-Gal4 driving UAS-dTrpA1 activated PTTH neurons at 29°C. Activation of PTTH 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 PTTH neurons at the stage 2 significantly increased the latency to copulation. (G) Mean velocity had no significant change when PTTH 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 comparison referred to Ptth-Gal4/UAS-dTrpA1. Female flies were 4-days-old adults. 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 3^rd-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 comparison referred to Ptth^Delete/Ptth^Delete + 20E. Female flies were 1-day-old. 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 3^rd - 4^th day after eclosion when EcR-A was knocked down in pC1 neurons (n = 17-36). The UAS-EcR-A-RNAi control causes a massive decrease in female fertility. (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 the whole larval (I), pupal (J) and adult (K) stages respectively. When the experiment was done at the pupal stage is the only situation when the controls were both different from the experimental (J). The comparison referred to flies with decreased EcR isoform in pC1 neurons. Female flies for behavioral assay were 4-6-days-old adults. 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.

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). Female flies were during prepupae stage. 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 comparison referred to pC1-ss2-Gal4/UAS-EcR-RNAi (A) and pC1-ss2-Gal4/UAS-EcR-A-RNAi (B). Female flies were 4-6-days-old adults. 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 comparison referred to flies with decreased EcR isoform in vpoDN neurons. Female flies were 4-6-days-old adults. 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 comparison referred to pC1d-Gal4/UAS-EcR-A-RNAi. Female flies were 4-6-days-old adults. 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 pC1 neurons induces the morphological changes.
(A1-A2) pC1-ss2-Gal4 expressing 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 2^nd day of the pupal stage (B1-B2) and retained to the adult stage including the 1^st day (C1-C2) and the 4^th day (D1-D2) of the adult stage. p0, the 1^st day of the pupal stage; p2, the 2^nd day of the pupal stage; A1, the 1^st day of the adult stage; A4, the 4^th day of the adult stage. (E-F) Fluorescence intensity of EVP in pC1d neurons on the 2^nd day of the pupal stage (E) and the 4^th 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 4^th 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). The comparison referred to flies with decreased EcR isoform in pC1 neurons. Female flies in (G-H) were 4-days-old adults. 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.

The function of PTTH on EcR-A and pC1 neurons.
(A) qRT-PCR for EcR-A when PTTH was deleted. Bars represent mean ± SEM. p values are from Mann-Whitney U test (n = 8 for Ptth^Delete/+ and n = 6 for Ptth^Delete/Ptth^Delete, each sample contains about 10 bodies of newly formed prepupae). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns indicates no significant difference. (B) Deletion of PTTH induced less elaborated morphologies of pC1d axons, especially the extra vertical projection (EVP). The EVP regions of pC1d neurons was indicated by arrows. Female flies were 4-days-old adults. (C) Fluorescence intensity of EVP in pC1d neurons on the 4^th day was quantified when PTTH was deleted (n=7). The quantified EVP regions were marked in (B1) and (B2) with red ellipses. (D) Torso-Gal4 and Dsx-LexA were labeled by stinger GFP and stingerRFP respectively. Arrows indicated the overlap of GFP and RFP signals. Representative of five female brains. Scale bars, 50 μm. (E-F) Knock-down of Torso in pC1 neurons inhibited virgin female copulation rate and enhanced latency to copulation. Females had similar locomotion speeds between groups (F). The pC1-ss2-Gal4 control causes a massive decrease in female receptivity. The comparison referred to pC1-ss2-Gal4/UAS-torso-RNAi. Female flies were 4-6-days-old adults. 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.

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