A neuropeptide circuit that coordinates sperm transfer and copulation duration in Drosophila

Abstract

Innate behaviors are often executed in concert with accompanying physiological programs. How this coordination is achieved is poorly understood. Mating behavior and the transfer of sperm and seminal fluid (SSFT) provide a model for understanding how concerted behavioral and physiological programs are coordinated. Here we identify a male-specific neural pathway that coordinates the timing of SSFT with the duration of copulation behavior in Drosophila. Silencing four abdominal ganglion (AG) interneurons (INs) that contain the neuropeptide corazonin (Crz) both blocked SSFT and substantially lengthened copulation duration. Activating these Crz INs caused rapid ejaculation in isolated males, a phenotype mimicked by injection of Crz peptide. Crz promotes SSFT by activating serotonergic (5-HT) projection neurons (PNs) that innervate the accessory glands. Activation of these PNs in copulo caused premature SSFT and also shortened copulation duration. However, mating terminated normally when these PNs were silenced, indicating that SSFT is not required for appropriate copulation duration. Thus, the lengthened copulation duration phenotype caused by silencing Crz INs is independent of the block to SSFT. We conclude that four Crz INs independently control SSFT and copulation duration, thereby coupling the timing of these two processes.

Fig. 1.

Silencing corazonin-GAL4 neurons results in male infertility and extended copulation duration. (A) Schematic illustrating neuropeptide promoter behavioral screen. (B) Fertility of crz-GAL4 UAS-Kir2.1 (0%, n = 42), crz-GAL4 (96%, n = 28), and Kir2.1 (93%, n = 30) males after mating with a wild-type virgin. (C) Copulation durations of crz-GAL4 UAS-Kir2.1 (103 ± 22 min, n = 33), Crz-GAL4 (19 ± 2 min, n = 22), and Kir2.1 (19 ± 2 min, n = 21) males paired with wild-type virgins. (D) Measure of courtship ability by males of the indicated genotype (Materials and Methods), n ≥ 10. ***P < 0.001. Error bars denote SEM. Illustration of Drosophila male and female (A) courtesy NASA.

Fig. 2.

Male-specific corazonin-GAL4 neurons in the abdominal ganglia control sperm transfer. (A) Confocal image of crz-GAL4/UAS-mCD8-GFP; UAS-DsRed (nuclear) whole-mount CNS, triple labeled with antibodies to nc82 (red), GFP (green), and DsRed (blue); higher-magnification view of the male (B and C) and female abdominal ganglia (D). (Scale bar, 50 μm.) Arrows indicate the cell bodies of two pairs of sexually dimorphic crz-GAL4 neurons. (A) A composite of separate images of the brain and VNC to form the full figure. (E–H) Confocal image of a crz-GAL4 UAS-nLacZ male abdominal ganglia triple-labeled with antibodies to Fru^M (green), Crz (red), and LacZ (blue). (I) Percentage of males that ejaculate during TRPA1-induced neuronal activation, n ≥ 25 flies per condition. (J) Copulation duration of infertile males paired with wild-type virgins (Materials and Methods). n ≥ 12 per genotype. (K) Transfer of sperm (dj:GFP) and seminal fluid (SP:GFP) to females during mating. Males of the indicated genotype were mated to wild-type virgins. Following mating cessation, females were dissected and were examined for the presence of GFP in the reproductive organs. (L) Crz peptide promotes ejaculation. Individual male flies were injected with synthetic corazonin (Crz), scrambled corazonin (sCrz), crustacean cardioactive peptide (CCAP), or buffer and observed for ejaculation (Materials and Methods). Error bars denote SEM.

Fig. 3.

Corazonin receptor-GAL4 neurons innervate reproductive organs and control sperm transfer. (A–C) Confocal image of crzR-GAL4; UAS-mCD8-GFP male AG stained with anti-corazonin (red). (D–F) Higher-magnification view (boxed region in C) of the AG showing the intermingling of corazonin and crzR-GAL4 terminals. (G–L) crzR-GAL4; UAS-mCD8-GFP male labeled with antibodies to GFP (green) and 5-HT (red). (G–I) High-magnification view of sexually dimorphic abdominal ganglia neurons expressing both 5-HT and crzR-GAL4. (J and K) Overlapping projections innervating the accessory glands. (M) Fertility of the indicated genotypes after mating with wild-type virgins. n ≥ 30 per genotype. (N) Copulation duration of crzR-GAL4; UAS-Kir2.1 (20 ± 2 min, n = 15), Tph2-GAL4/Kir2.1 (18 ± 2 min, n = 16), and Kir2.1/+ (19 ± 2 min, n = 14) males. (O) Assay for transfer of sperm (dj:GFP) and seminal fluid (SP:GFP) to females during mating. (P) Individual male flies were injected with 5-HT, dopamine, or buffer and observed for ejaculation (Materials and Methods). (Q and R) Z-projection image of a crzR-Gal4; UAS-GCaMP5G abdominal ganglia before (Q) and after (R) Crz bath application. (S) Peak ΔF/F values (n = 3–5 cells from at least three crzR-Gal4; UAS-GCaMP5G flies for each condition) following bath application of buffer (control) or the Crz peptide dissolved in buffer. Crz was applied either to an isolated nerve cord (ex vivo prep) or an intact fly with the AG exposed (in vivo prep). (T) Example ΔF/F responses from ex vivo (red) and in vivo (blue) preparations. Traces have been aligned to the rise of the response. Crz peptide (or buffer alone) was added before time = 0 and washed out 300 seconds later.

Fig. 4.

Role of SSFT in determining copulation duration. (A) Percentage of isolated males of the indicated genotypes that ejaculated during TRPA1-induced neuronal activation; n ≥ 30 flies per condition. (B and C) In copulo activation of INs and PNs causes precocious SSFT. Mating pairs containing males of the indicated genotypes were shifted to the activating temperature (30 °C) 1 min after copulation was initiated. (B) Female fertility was assessed after mechanically separating mating pairs 4 min following the temperature shift; n ≥ 15 per genotype. (C) Copulation duration of undisturbed mating pairs; n ≥ 15 per genotype. Gray bars are controls mock-shifted 1 min after initiation of copulation. ***P < 0.001. Error bars denote SEM. (D) Summary of main experimental results. Red arrows indicate inhibition of SSFT or shortened copulation duration; green arrows indicate promotion of SSFT or increased copulation duration. ∅, no change; *, activation of Crz INs is not neutral but can override shortened copulation duration caused by premature SSFT (see C). (E) Schematic illustrating how the activity of Crz INs (perhaps triggered by sensory signals associated with the initiation of mating) may coordinate both SSFT and the duration of copulation. In the absence of Crz IN function, premature SSFT caused by hyperactivation of PNs shortens copulation duration (D), suggesting that SSFT determines the minimum duration (lower limit) of copulation. When Crz INs are active, copulation duration is extended for ∼10–20 min beyond the normal time required for successful fertilization (∼8 min; Fig. S8; upper limit). This influence of Crz INs may be exerted through another unknown circuit element (?). AG, abdominal ganglion; EOs, ejaculatory organs. ACh? indicates that Crz INs express Cha-GAL4 (Fig. S11), and there is evidence of cholinergic regulation of SSFT (11).