The presence of males shortens the ejaculate holding period (EHP) in females through olfactory or gustatory sensation

A,Schematic of the experimental procedure to measure male-induced EHP shortening (MIES). A female mates with a wild-type Canton-S (CS) male. Immediately after mating, the female is incubated with a naïve male. Typically, females kept alone exhibit an EHP of approximately 90 min, whereas females incubated with a naïve male exhibit an EHP of approximately 60 min. In this study, we refer to this phenomenon as male-induced EHP shortening (MIES).

B-F, Ejaculation holding period (EHP) or ΔEHP of the females of the indicated genotypes, incubated under the indicated conditions after mating. Normalized EHP (ΔEHP) is calculated by subtracting the mean of reference EHP of females kept alone after mating (leftmost column) from the EHP of females incubated with other flies.

Mann-Whitney Test (n.s. p > 0.05; ****p < 0.0001). Gray circles indicate the EHP or ΔEHP of individual females, and the mean ± SEM of data is presented. Numbers below the horizontal bar represent the mean of EHP differences between treatments.

Or47b and Or47b-positive ORNs are required for MIES

A, C-E, ΔEHP of females of the indicated genotypes, incubated with or without naive males. Female genotypes are as follows from left to right: (A) control (Or47b>TNTinactive), Or47b ORN silencing (Or47b>TNTactive); (C) Orco mutant (Orco1/Orco1), Orco rescue in Or47b ORNs of Orco mutant (Orco1/Orco1; Or47b>Orco); (D) control 1 (Or47b2/+), control 2 (Or47b3/+), Or47b mutant (Or47b2/Or47b3); (E) Or47b mutant (Or47b2/Or47b2), Or47b rescue (Or47b>Or47b; Or47b2/Or47b2).

B, Thermal activation of Or47b-positive ORNs shortens EHP in the absence of naive males. ΔEHP is calculated by subtracting the mean of reference EHP of females incubated at 21°C control conditions from the EHP of individual females. Female genotypes are as follows from left to right: control 1 (Or47b-Gal4/+), control 2 (UAS-dTRPA1/+), Or47b>dTRPA1 (Or47b-Gal4/UAS-dTRPA1). Mann-Whitney Test (n.s. p > 0.05; *p <0.05; **p <0.01; ****p < 0.0001). Gray circles indicate the ΔEHP of individual females, and the mean ± SEM of data is presented. Gray circles with dashed borders indicate ΔEHP values beyond the axis limits (>90 or <-90 min). Numbers below the horizontal bar represent the mean of EHP differences between treatments.

2-methyltetracosane (2MC) can induce EHP shortening through Or47b A-D,

Δ EHP of mated females of the indicated genotypes, incubated in solvent vehicle or 2MC. Mated females were incubated with a piece of filter paper perfumed with vehicle (-) or 750 ng 2MC (+). Female genotypes are as follow: w1118, (B) Orco mutant (Orco1/Orco1), (C) Or47b mutant (Or47b2/Or47b2), (D) Gal4 control (Or47b-Gal4/+; Orco1/Orco1), UAS control (UAS-Orco/+; Orco1/Orco1), Orco rescue in Or47b ORN (Orco1/Orco1; Or47b-Gal4/UAS-Orco).

A-C, Mann-Whitney Test (n.s. p > 0.05; *p <0.05), D, One-way analysis of variance (ANOVA) test (n.s. p > 0.05; *p < 0.05). Gray circles indicate the ΔEHP of individual females and the mean ± SEM of data is presented. Normalized EHP (ΔEHP) is calculated by subtracting the mean of reference EHP of females incubated with vehicle (A-C) or mean of Gal4 control and UAS control female incubated with vehicle (D) after mating from the EHP of females incubated with chemical perfumed paper. Gray circles with dashed borders indicate ΔEHP values beyond the axis limits (>90 or <-90 min). Numbers below the horizontal bar represent the mean of EHP differences between treatments.

7-Tricosene present in mated females or naive males shorten EHP via ppk23 neurons A-D,

ΔEHP of females of the indicated genotypes, incubated with mated females (A), a piece of filter paper perfumed with 150 ng 7-T (B) or 200 ng cVA (C), or naive males (D) immediately after mating. Female genotypes are: (A-C) w1118, (D) control (ppk23-Gal4/UAS-TNTinactive), ppk23 silencing (ppk23-Gal4/UAS-TNTactive).

Unpaired t-Test (n.s. p > 0.05; * p < 0.05). Gray circles indicate the ΔEHP of individual females, and the mean ± SEM of data is presented. Gray circles with dashed borders indicate ΔEHP values beyond the axis limits (>90 or <-90 min). Numbers below the horizontal bar represent the mean of EHP differences between treatments.

pC1 neuron subset comprising pC1b and c subtypes regulates EHP in response to 2MC and 7-T, both of which upregulate cAMP activity and excitability of pC1 neurons

A-D, Optogenetic silencing of a pC1 neuron subset comprising pC1b and c subtypes increases EHP. Females of the indicated genotypes were cultured on food with or without all-trans-retinal (ATR). ΔEHP is calculated by subtracting the mean of reference EHP of females cultured in control ATR-food from the EHP of individual test females. Female genotypes are: (A) pC1a,b,c>GtACR1 (pC1-S-Gal4/UAS-GtACR1), (B) pC1d,e>GtACR1 (pC1-A-Gal4/UAS-GtACR1), (C) pC1a>GtACR1 (pC1a-split-Gal4 /UAS-GtACR1), and (D) pC1b,c>GtACR1 (Dh44-pC1-Gal4/UAS-GtACR1). Gray circles indicate the ΔEHP of individual females, and the mean ± SEM of data is presented. Gray circles with dashed borders indicate ΔEHP values beyond the axis limits (>120 min). Mann-Whitney Test (n.s. p > 0.05; *p <0.05; ****p < 0.0001). Numbers below the horizontal bar represent the mean of EHP differences between treatments.

E, Relative CRE-Luciferase reporter activity of pC1 neurons in mated females of the indicated genotypes, incubated with a piece of filter paper perfumed with solvent vehicle control or the indicated odorants. To calculate the relative luciferase activity, we set the average luminescence unit values of female incubated with the vehicle to 100%. One-way ANOVA test (n.s. p > 0.05; ***p < 0.001; ****p < 0.0001). Gray circles indicate the relative luciferase activity (%) of individual females, and the mean ± SEM of data is presented.

F, Optogenetic production of cAMP in the pC1 b and c neurons shortens EHP, whereas the same treatment in pC1a or pC1d and e neurons does not. ΔEHP is calculated by subtracting the mean of reference EHP of females incubated in the control illumination (Dim light), which does not activate a photoactivatable adenylate cyclase (PhotoAC), from the EHP of individual test females. Mann-Whitney Test (n.s. p > 0.05, ****p < 0.0001).

G, Optogenetic production of cAMP increases excitability of pC1 neurons transiently. Left, schematic of the experimental procedure. Right, peak ΔF/F in the LPC projections of pC1 neurons from freshly mated females in response to the pheromone cVA, before and after photoactivation of PhotoAC expressed in pC1 neurons. Calcium response was measured at specific time points: after 1 minute (Blue dots and box, 1 TAA = 1 Time After Activation) or 10 minutes (Purple dots and box, 10 TAA) after activation. Repeated measures (RM) one-way ANOVA test with the Geisser-Greenhouse correction followed by Tukey’s multiple comparisons test (*p < 0.05; ***p < 0.001; ****p < 0.0001).

H, Left, schematic of the experimental procedure. Right, re-mating rate of females during optogenetic cAMP production in pC1b and c, scored as the percentage of females that copulate with naive male within 6 h after end of first mating. Female genotypes are control (+/UAS-PhotoAC), pC1b,c>UAS-PhotoAC (Dh44-pC1-Gal4/UAS-PhotoAC). Chi-square test (*p <0.05).