Neuroscience

Male cuticular pheromones stimulate removal of the mating plug and promote re-mating through pC1 neurons in Drosophila females

Minsik Yun
Do-Hyoung Kim
Tal Soo Ha
Kang-Min Lee
Eungyu Park
Markus Knaden
Bill S Hansson
Young-Joon Kim author has email address

School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Cheomdangwagi-ro 123, Buk-gu, Gwangju, 61005, Republic of Korea
Department of Biomedical Science, College of Natural Science, Daegu University, Gyeongsan 38453, Gyeongsangbuk-do, Korea
Department of Evolutionary Neuroethology, Max Planck Institute for Chemical Ecology, Hans-Knöll-Str. 8, 07745 Jena, Germany
Next Generation Insect Chemical Ecology, Max Planck Centre, Max Planck Institute for Chemical Ecology, Hans-Knöll-Straße 8, D-07745, Jena, Germany

https://doi.org/10.7554/eLife.96013.2

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Figures and data

The presence of males reduces the ejaculate holding period (EHP) in females through olfactory or gustatory sensation
A, Schematic of the experimental procedure employed to measure male-induced EHP shortening (MIES). Immediately after the end of copulation, the female is incubated with a wild-type Canton-S (CS) male that has not been previously exposed to the female. Typically, w¹¹¹⁸females that are kept alone after mating exhibit an EHP of approximately 90 minutes, whereas females that are incubated with a naïve CS male exhibit an EHP of approximately 60 minutes. In this study, we refer to this phenomenon as MIES. B-F, Normalized ejaculate holding period (EHP) or ΔEHP of the females of the indicated genotypes, incubated under the indicated conditions after mating. The ΔEHP is calculated by subtracting the mean of the reference EHP of females kept alone after mating (the leftmost column) from the EHP of individual females in comparison. 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 the EHP differences between the indicated treatments.

The function of Or47b and Or47b-positive ORNs is essential for MIES
A, C-E, ΔEHP of females of the indicated genotypes, incubated with or without naive males after mating. The female genotypes are as follows from left to right: (A) control (Or47b>TNT^inactive), Or47b ORN silencing (Or47b>TNT^active); (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, Thermogenetic activation of Or47b-positive ORNs shortens EHP in females kept alone after mating. The 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). The ΔEHP is calculated by subtracting the mean of the reference EHP of females kept alone after mating (‘-’ in A, C-E) or incubated at 21°C control conditions (B) from the EHP of individual females in comparison. Gray circles indicate the ΔEHP of individual females, and the mean ± SEM of data is presented. The gray circles with dashed borders indicate ΔEHP values that exceed the axis limits (>90 or <-90 minutes). Numbers below the horizontal bar represent the mean of the EHP differences between the indicated treatments.

2-Methyltetracosane (2MC) can induce EHP shortening through Or47b
A-D, Δ EHP of females of the indicated genotypes, incubated in solvent vehicle or 2MC. Mated females were incubated with a piece of filter paper perfumed with either vehicle (-) or 750 ng 2MC (+). The female genotypes are as follows: (A) 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 with Fisher’s LSD multiple comparison (n.s. p > 0.05; *p < 0.05). Gray circles indicate the ΔEHP of individual females and the mean ± SEM of data is presented. The ΔEHP is calculated by subtracting the mean of the reference EHP of females incubated with vehicle-perfumed paper (the leftmost column in A-C) or the mean of the Gal4 control and UAS control female incubated with vehicle-perfumed paper (the two leftmost columns in D) from the EHP of individual females in comparison. Gray circles with dashed borders indicate ΔEHP values that exceed the axis limits (>90 or <-90 minutes). Numbers below the horizontal bar represent the mean of the EHP differences between the indicated treatments.

7-Tricosene present in mated females and males reduces 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), 200 ng cVA (C), or naive males (D) after mating. The female genotypes are as follows: (A-C) w¹¹¹⁸, (D) control (ppk23-Gal4/UAS-TNT^inactive), ppk23 silencing (ppk23-Gal4/UAS-TNT^active). A, Unpaired t-Test, B-D, Mann-Whitney Test (n.s. p > 0.05; *p < 0.05). The ΔEHP is calculated by subtracting the mean of the reference EHP of females kept alone (‘-’ in A, D) or incubated with vehicle-perfumed paper (the leftmost column in B, C) from the EHP of individual females in comparison. Gray circles indicate the ΔEHP of individual females, and the mean ± SEM of data is presented. The gray circles with dashed borders indicate ΔEHP values that exceed the axis limits (>90 or <-90 minutes). Numbers below the horizontal bar represent the mean of the EHP differences between the indicated treatments.

A subset of pC1 neurons, comprising pC1b and pC1c subtypes, regulates EHP and exhibits CRE-luciferase reporter activity in response to 2MC and 7-T
A-D, The optogenetic silencing of a pC1 neuron subset comprising pC1b and pC1c neurons (pC1b, c) increases EHP. Females of the indicated genotypes were cultured on food with or without all trans-retinal (ATR) after eclosion. The ΔEHP is calculated by subtracting the mean of the reference EHP of females cultured in control ATR-food (the leftmost column) from the EHP of individual females in comparison. The female genotypes are as follows: (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. The gray circles with dashed borders indicate ΔEHP values that exceed the axis limits (>120 minutes). Mann-Whitney Test (n.s. p > 0.05; *p <0.05; ****p < 0.0001).
Numbers below the horizontal bar represent the mean of the EHP differences between the indicated treatments.
E, F, Relative CRE-luciferase reporter activity of pC1 neurons in females of the indicated genotypes, incubated with a piece of filter paper perfumed with solvent vehicle control or the indicated pheromones immediately after mating. The CRE-luciferase reporter activity of pC1 neurons of Or47b-deficient females (Or47b^2/2 or Or47b^3/3) was observed to increase in response to 7-T but not to 2MC. To calculate the relative luciferase activity, the average luminescence unit values of the female incubated with the vehicle are set to 100%. Mann-Whitney Test (n.s. p > 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). Gray circles indicate the relative luciferase activity (%) of individual females, and the mean ± SEM of data is presented.

Elevated cAMP levels in pC1 neurons reduce EHP and increase the responsiveness of pC1 neurons to male courtship cues, thereby promoting subsequent remating
A, The optogenetic production of cAMP in the pC1b, c neurons shortens EHP, whereas the same treatment in pC1a or pC1d, e neurons does not. ΔEHP is calculated by subtracting the mean of the 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 females. Mann-Whitney Test (n.s. p > 0.05, ****p < 0.0001).
B, The optogenetic production of cAMP transiently increases the excitability of pC1 neurons. 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. The calcium response was measured at specific time points after photoactivation: after 1 minute (blue dots and box) or 10 minutes (purple dots and box) after activation. Repeated measures 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).
C, Left, schematic of the experimental procedure. Right, re-mating rate of females during optogenetic cAMP production in pC1b, c neurons, scored as the percentage of females that copulate with a naive CS male within 6 hours after the first mating. The female genotypes are as follows: Control (+/UAS-PhotoAC), pC1b,c>UAS-PhotoAC (Dh44-pC1-Gal4/UAS-PhotoAC). Chi-square test (*p <0.05).

The presence of male odorants, which reflect changes in the social-sexual context, stimulates newly mated females to remove the male ejaculate and engage in subsequent re-mating
Following the initial mating, a female that encounters a new courting male removes the male ejaculate after a shorter EHP than those that do not encounter new male partners. This phenomenon, referred to as MIES in this study, is followed by a second mating with the new partner. The production of MIES depends on the functions of the Or47b+ olfactory and ppk23+ gustatory neurons, which are activated by 2MC and 7-T, respectively. These odorants increase cAMP levels in pC1b, c neurons, enhancing their responsiveness to male courtship cues and increasing mating receptivity. Consequently, 2MC and 7-T promote a second mating with a faster removal of the male ejaculate or mating plug.

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