Abdominal-B neurons control Drosophila virgin female receptivity

Abstract

Background: Female sexual receptivity offers an excellent model for complex behavioral decisions. The female must parse her own reproductive state, the external environment, and male sensory cues to decide whether to copulate. In the fly Drosophila melanogaster, virgin female receptivity has received relatively little attention, and its neural circuitry and individual behavioral components remain unmapped. Using a genome-wide neuronal RNAi screen, we identify a subpopulation of neurons responsible for pausing, a novel behavioral aspect of virgin female receptivity characterized in this study.

Results: We show that Abdominal-B (Abd-B), a homeobox transcription factor, is required in developing neurons for high levels of virgin female receptivity. Silencing adult Abd-B neurons significantly decreased receptivity. We characterize two components of receptivity that are elicited in sexually mature females by male courtship: pausing and vaginal plate opening. Silencing Abd-B neurons decreased pausing but did not affect vaginal plate opening, demonstrating that these two components of female sexual behavior are functionally separable. Synthetic activation of Abd-B neurons increased pausing, but male courtship song alone was not sufficient to elicit this behavior.

Conclusions: Our results provide an entry point to the neural circuit controlling virgin female receptivity. The female integrates multiple sensory cues from the male to execute discrete motor programs prior to copulation. Abd-B neurons control pausing, a key aspect of female sexual receptivity, in response to male courtship.

Figure 1. Abd-B is required in neurons for virgin female receptivity

(A,C,H) Schematic of food vial mating assay with 2 males. (B, D) Receptivity of virgin females with elav- (B, D) or nsyb- (D) Gal4-driven RNAi against the indicated gene [***p <0.0001 or *p <0.005 compared to control (B) or ***p <0.0001 compared to parental control (D): pairwise Fischer’s exact test with Bonferroni correction; mean and 95% confidence interval are shown, n = 30-300 (B), n = 30-273 (D)]. Abd-B RNAi 1 is the original hairpin from the Vienna screen and was used in all other experiments. For unknown reasons, RNAi 2 when driven by nsyb was lethal, precluding further analysis. (E) Schematic of the fly nervous system (gray) indicating the abdominal ganglion (red). (F,G) Immunofluorescence of Abd-B (green) and nuclei (DAPI, magenta) in abdominal ganglia from females of the indicated genotype and mating status. Scale bar: 50 μm. (I) Receptivity of virgin females with Abd-B RNAi temporally restricted by shifts from 18°C to 30°C (***p <0.0001, pairwise Fischer’s exact test with Bonferroni correction; mean and 95% confidence interval are shown, n = 23-32). See also Figures S1 and S2.

Figure 2. Neuronal Abd-B RNAi does not induce the post-mating response

(A, C, E) Schematics of assays in B, D, F. (B) Courtship index of wild-type males during the first 5 min of courtship of a female of the indicated genotype and mating status (n.s. = not significant, one-way ANOVA with Bonferroni correction, mean ± SEM, n = 8). (D) Female ovipositor extrusion during assays in A (n.s. = not significant, one-way ANOVA with Bonferroni correction, mean ± SEM, n = 8). (F) Egg-laying during the first 48 h after mating (n.s. = not significant; bars labeled with different letters are significantly different: p <0.01, one-way ANOVA with Bonferroni correction, mean ± SEM, n = 24-32).

Figure 3. Adb-B^LDN-Gal4 labels Abd-B receptivity neurons in the abdominal ganglion and reproductive tract

(A) Co-localization of Abd-B (magenta) and nuclear β-gal (green) driven by Abd-B^LDN-Gal4 in the virgin female abdominal ganglion. Scale bar: 50 μm. (B) Receptivity of virgin females with RNAi against Abd-B driven by Abd-B^LDN-Gal4 and UAS-Dcr2, limited to neurons by nsyb-lexA, lexAop-FLP, and tub-FRT-Gal80-FRT-STOP (***p <0.001, Fischer’s exact test; mean and 95% confidence interval are shown, n = 16-40). See Supplemental Experimental Methods for complete genotypes. (C-Z) Immunofluorescence of GFP (green) and nc82 or rhodamine-phalloidin (magenta) in the indicated tissue in virgin females of the indicated genotype. Insets are separate z-stacks at higher magnification of approximate areas indicated. Arrowheads in G and H indicate neuronal cell bodies. Scale bars: 50 μm. See also Figure S3.

Figure 4. Abd-B^LDN neurons are functionally required for virgin female receptivity

(A-B) Temperature-shift receptivity of virgin females (A) or females 48 h after mating (B). (C-D) Receptivity of virgin females. In A-D, *p <0.05, **p <0.01, ***p <0.001 compared to parental controls (at the same temperature in A-B), Fischer’s exact test; mean and 95% confidence interval are shown. Sample sizes: n = 33-40 (A), n = 23-35 (B), n = 20-59 (C), n = 29-56 (D). (E-P) Immunofluorescence of GFP (green) and nc82 or rhodamine-phalloidin (magenta) in the indicated tissue in virgin females of the indicated genotype. Insets are separate z-stacks at higher magnification of approximate areas indicated. Scale bars: 50 μm. See also Figures S4 and S5.

Figure 5. Silencing Abd-B^LDN neurons decreases pausing during courtship

(A) Tracking arena with fly positions during the last 60 s before copulation between Abd-B^LDN-Gal4 mature virgin female and wild-type male. (B) Receptivity of virgin females in the tracking arena (**p <0.01 compared to parental controls, Fischer’s exact test; mean and 95% confidence interval are shown, n = 8-10). (C) Per-frame parameters calculated from tracks in (A). (D) Female pausing during courtship (Student’s t-test, mean ± SEM, n = 8-10, ***p <0.001). (E) Female pausing in the presence of a male (Student’s t-test, mean ± SEM, n = 10, **p <0.01). (F) Female pausing (Student’s t-test, mean ± SEM, n = 8-10, ***p <0.05). See also Figure S6 and Movies S1 and S2.

Figure 6. Pausing is a response to multiple male courtship cues

(A) Cross-correlation between female pausing and male wing extension or male touch during courtship tracking assays with Abd-B^LDN-Gal4 mature virgin females and wild-type males (n = 5). (B) Schematic of sound playback assay. (C) Receptivity of Abd-B^LDN Gal4 mature virgin females during sound playback with wild-type males with and without wings as indicated (***p <0.001, Fischer’s exact test; mean and 95% confidence interval are shown, n = 23-24 (D-E) Pausing of mature virgin females with wild-type males with and without wings as indicated (D) or in the absence of a male (E) during sound playback [n.s. = not significant, *p <0.05, one-way ANOVA with Bonferroni correction, mean ± SEM; n = 6-7 (D), n = 22-23 (E)].

Figure 7. Activating Abd-B neurons induces pausing

(A) Schematic of assay in B. (B) Temperature-shifted pausing of mature virgin females in the absence of a male (n.s. = not significant, **p <0.01, Student’s t-test, mean ± SEM, n = 7-10). (C) Schematic of assay in D. (D) Temperature-shifted pausing of mature virgin females during courtship from wild-type males (n.s. = not significant, **p <0.01, Student’s t-test, mean ± SEM, n = 7-11). (E) Video frame of Abd-B^LDN-Gal4 mature virgin female and wild-type male without wings in tracking arena. (F-G) Tracked fly positions during the fourth min of courtship between Abd-B^LDN-Gal4>TrpA1 mature virgin female and wild-type male without wings at 22°C (F) or 30°C (G). (H) Schematic of assay in I. (I) Temperature-shifted female speed excluding frames classified as pausing during courtship from a male without wings (n.s. = not significant, **p <0.01, Student’s t-test, mean ± SEM, n = 12-16). (J) Schematic of assay in (K). (K) Temperature-shifted pausing of virgin females during courtship from males without wings (n.s. = not significant, *p <0.05, Student’s t-test, n = 12-16). See also Figure S7 and Movie S2.