1. Neuroscience

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

  1. Minsik Yun
  2. Do-Hyoung Kim
  3. Tal Soo Ha
  4. Kang-Min Lee
  5. Eungyu Park
  6. Markus Knaden
  7. Bill S Hansson
  8. Young-Joon Kim  Is a corresponding author
  1. School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Republic of Korea
  2. Department of Biomedical Science, College of Natural Science, Daegu University, Republic of Korea
  3. Department of Evolutionary Neuroethology, Max Planck Institute for Chemical Ecology, Germany
  4. Next Generation Insect Chemical Ecology, Max Planck Centre, Max Planck Institute for Chemical Ecology, Germany
https://doi.org/10.7554/eLife.96013.3
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Cite this article
  1. Minsik Yun
  2. Do-Hyoung Kim
  3. Tal Soo Ha
  4. Kang-Min Lee
  5. Eungyu Park
  6. Markus Knaden
  7. Bill S Hansson
  8. Young-Joon Kim
(2024)
Male cuticular pheromones stimulate removal of the mating plug and promote re-mating through pC1 neurons in Drosophila females
eLife 13:RP96013.
https://doi.org/10.7554/eLife.96013.3
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8 figures, 2 tables and 2 additional files

Figures

Figure 1
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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, w1118 females that are kept alone after mating exhibit an EHP of approximately 90 min, whereas females that are incubated with a naïve CS male exhibit an EHP of approximately 60 min. In this study, we refer to this phenomenon as MIES. (B–F) Normalized 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. Genotype and sample size are shown in Table 1.

Figure 2 with 1 supplement
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The function of Or47b and Or47b-positive olfactory receptor neurons (ORNs) is essential for male-induced EHP shortening (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>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) 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 min). Numbers below the horizontal bar represent the mean of the EHP differences between the indicated treatments. EHP, ejaculate holding period. Genotype and sample size are shown in Table 1.

Figure 2—figure supplement 1
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The identification of trichoid and intermediate sensilla olfactory receptor neurons (ORNs) that are necessary for the production of male-induced EHP shortening (MIES).

ΔEHP of females of the indicated genotypes, incubated with or without naive males immediately after mating. The female genotypes are as follows from left to right:+>TNTactive, Or13a>TNTactive, Or19a>TNTactive, Or23a>TNTactive, Or43a>TNTactive, Or47b>TNTactive, Or65a>TNTactive, Or65b>TNTactive, Or65c>TNTactive, Or67d>TNTactive, Or83c>TNTactive, Or88a>TNTactive. Mann-Whitney test (n.s. p>0.05; *p<0.05; **p<0.01; ***p<0.001). 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 kept alone after mating (‘-’) from the EHP of individual females in comparison. Numbers below the horizontal bar represent the mean of the EHP differences between the indicated treatments. EHP, ejaculate holding period. Genotype and sample size are shown in Table 1.

Figure 3 with 3 supplements
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2-Methyltetracosane (2MC) can induce ejaculate holding period (EHP) shortening through Or47b.

(A–D) ΔEHP 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 olfactory receptor neurons (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 min). Numbers below the horizontal bar represent the mean of the EHP differences between the indicated treatments. Genotype and sample size are shown in Table 1.

Figure 3—figure supplement 1
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Known odorant ligands for Or47b, methyl laurate and trans-palmitoleic acid, were unable to induce ejaculate holding period (EHP) shortening.

ΔEHP of w1118 females incubated with a piece of filter paper perfumed with solvent vehicle or with the indicated amounts of two known Or47b odorant ligands, methyl laurate (A) and trans-palmitoleic acid (B) immediately after mating. Mann-Whitney test (n.s. p>0.05). The ΔEHP is calculated by subtracting the mean of the reference EHP of females incubated with vehicle-perfumed paper (the leftmost column) from the EHP of individual females in comparison. Gray circles indicate the 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 vehicle and odorant treatments. Genotype and sample size are shown in Table 1.

Figure 3—figure supplement 2
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Ejaculate holding period (EHP) shortening is induced by males with feminized oenocytes, females with masculinized oenocytes, and males of other closely related Drosophila species.

(A) ΔEHP of w1118 females incubated with males with feminized oenocytes (Oe Fem male; PromE(800)-Gal4/UAS-Tra) or virgin females with masculinized oenocytes (Oe Mas female; PromE(800)-Gal4/UAS-Tra-RNAi). (B) ΔEHP of w1118 females incubated with naive males of the indicated Drosophila species. D. mel (D. melanogaster), D. sim (D. simulans), D. sec (D. sechellia), D. ere (D. erecta), D. yak (D. yakuba). Mann-Whitney test (n.s. p>0.05; ****p<0.0001). 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. Gray circles indicate the ΔEHP of individual females, and the mean ± SEM of data is presented. Numbers below the horizontal bar represent the mean EHP differences between the indicated treatments. Genotype and sample size are shown in Table 1.

Figure 3—figure supplement 3
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2-Methyltetracosane (2MC) shortens ejaculate holding period (EHP) at a specific concentration.

ΔEHP of w1118 females incubated with a piece of filter paper perfumed with solvent vehicle or the indicated amounts of 2MC. 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 incubated with vehicle-perfumed paper (the leftmost column) from the EHP of individual females in comparison. Gray circles indicate the 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 vehicle and odorant treatments. Genotype and sample size are shown in Table 1.

Figure 4 with 1 supplement
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7-Tricosene (7-T) present in mated females and males reduces ejaculate holding period (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 11-cis-vaccenyl acetate (cVA) (C), or naive males (D) after mating. The female genotypes are as follows: (A–C) w1118, (D) control (ppk23-Gal4/UAS-TNTinactive), ppk23 silencing (ppk23-Gal4/UAS-TNTactive). (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 min). Numbers below the horizontal bar represent the mean of the EHP differences between the indicated treatments. Genotype and sample size are shown in Table 1.

Figure 4—figure supplement 1
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7-Tricosene (7-T) induces ejaculate holding period (EHP) shortening at physiological concentrations, but DEG/ENaC channels expressed in ppk23 neurons are not required for male-induced EHP shortening (MIES).

(A, B) ΔEHP of w1118 females incubated with a piece of filter paper perfumed with solvent vehicle or the indicated amounts of 7-T (A), or 7-pentacosene (B) after mating. Incubation with a specific concentration of 7-T significantly shorten EHP, but 7-pentacosene does not. Unpaired t-test (n.s. p>0.05; *p<0.05). (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: (C) control 1 (w1118), control 2 (ppk23-/+), and ppk23- (ppk23-/ppk23-); (D) control 1 (w1118), control 2 (ppk28-/+), and ppk28- (ppk28-/ppk28-); (E) control 1 (w1118), control 2 (ppk29-/+), and ppk29- (ppk29-/ppk29-). 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 incubated with vehicle-perfumed paper (the leftmost column in A, B) or kept alone after mating (‘-’ in C–E) from the EHP of individual females in comparison. Gray circles indicate the 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. Genotype and sample size are shown in Table 1.

Figure 5 with 3 supplements
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A subset of pC1 neurons, comprising pC1b and pC1c subtypes, regulates ejaculate holding period (EHP) and exhibits CRE-luciferase reporter activity in response to 2-methyltetracosane (2MC) and 7-tricosene (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 min). 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 (Or47b2/2 or Or47b3/3) 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. Genotype and sample size are shown in Table 1.

Figure 5—figure supplement 1
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Characterization of pC1a-split-Gal4.

(A, B) Z-projection confocal images of the brain (A) and VNC (B) of a female carrying pC1a-split-GAL4 and UAS-myrEGFP, stained with anti-EGFP (green) and anti-nc82 (magenta). Scale bars, 50 μm. In the brain, only the pC1a cells are labeled, but in the VNC, several cells are labeled in the abdominal ganglion. (C) An anatomical comparison between pC1a-split-GAL4 neurons (above; pC1a-ss) in the brain and a pC1a neuron (below; NeuPRINT body ID, 5813046951). The panel above shows the maximum intensity projection image (MIP) of an aligned confocal image of the brain from a female carrying pC1a-split-GAL4 and UAS-myrEGFP stained with anti-EGFP and anti-nc82. (D) Mating frequencies of pC1a>GtACR1 (pC1a-split-Gal4/UAS-GtACR1) females during optogenetic silencing, scored as the percentage of females that copulate within 1 hr. Females were cultured on food with or without all trans-retinal (ATR) prior to the mating assay. The optogenetic silencing of pC1a neurons with GtACR1 was observed to suppress mating receptivity almost completely. Chi-square test (****p<0.0001).

Figure 5—figure supplement 1—source data 1

Raw image file for the confocal Z-projection image of pC1a-split-GAL4 neurons in a female brain.

https://cdn.elifesciences.org/articles/96013/elife-96013-fig5-figsupp1-data1-v1.zip
Figure 5—figure supplement 2
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Incubation with 2-methyltetracosane (2MC) or 7-tricosene (7-T) increases cAMP levels in pC1 neurons.

The relative CRE-luciferase reporter activity of pC1 neurons in females incubated with a piece of filter paper perfumed with the indicated amounts of 2MC (A) and 7-T (B). It is noteworthy that the concentration range within which 2MC or 7-T increases cAMP levels in pC1 neurons is narrow. To calculate the relative luciferase activity, the average luminescence unit values of the female incubated with the vehicle are set to 100%. Gray circles indicate the relative luciferase activity (%) of individual females, and the mean ± SEM of data is presented. Mann-Whitney test (n.s. p>0.05; **p<0.01; ***p<0.001; ****p<0.0001). Genotype and sample size are shown in Table 1.

Figure 5—figure supplement 3
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Incubation with 2-methyltetracosane (2MC) or 7-tricosene (7-T) increases cAMP levels in pC1a as well as pC1b, c neurons in virgin females.

The relative CRE-luciferase reporter activity of pC1 neurons in virgin females of the indicated genotypes, incubated with a piece of filter paper perfumed with the indicated odorants. 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. Genotype and sample size are shown in Table 1.

Figure 6 with 1 supplement
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Elevated cAMP levels in pC1 neurons reduce ejaculate holding period (EHP) and increase the responsiveness of pC1 neurons to male courtship cues, thereby promoting subsequent re-mating.

(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 11-cis-vaccenyl acetate (cVA), before and after photoactivation of PhotoAC expressed in pC1 neurons. The calcium response was measured at specific time points after photoactivation: after 1 min (blue dots and box) or 10 min (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 Canton-S (CS) male within 6 hr 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). Genotype and sample size are shown in Table 1.

Figure 6—figure supplement 1
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The knockdown of Dh44R1 and Dh44R2 in pC1 neurons has a limited impact on male-induced EHP shortening (MIES).

ΔEHP of females of the indicated genotypes, incubated with or without naive males immediately after mating. The female genotypes are as follows from left to right: Gal4 control (UAS-Dcr2/+; GMR71G01-Gal4/+), UAS control (UAS-Dh44R1-RNAi/+; UAS-Dh44R2-RNAi/+), Dh44R1-RNAi, Dh44R2-RNAi in pC1 (UAS-Dcr2/+; GMR71G01-Gal4/Dh44R1-RNAi; Dh44R2-RNAi/+). Mann-Whitney test (*p<0.05; ***p<0.001; ****p<0.0001). The ΔEHP is calculated by subtracting the mean of the reference EHP of females kept alone after mating (‘-’) 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 (>120 or <-120 min). Numbers below the horizontal bar represent the mean of the EHP differences between the indicated treatments. EHP, ejaculate holding period. Genotype and sample size are shown in Table 1.

Figure 7
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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 ejaculate holding period (EHP) than those that do not encounter new male partners. This phenomenon, referred to as male-induced EHP shortening (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 2-methyltetracosane (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.

Author response image 1
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The prolonged optogenetic activation of pC1b, c neurons increases EHP, mimicking silencing of pC1b, c neurons.

Females of the indicated genotypes were cultured on food with or without all-trans-retinal (ATR). The ΔEHP is calculated by subtracting the mean of the reference EHP of females cultured in control ATR- food from the EHP of individual females in comparison. The female genotypes are as follows: (A) 71G01-GAL4/UAS-CsChrimson, (B) pC1a-split-Gal4/UAS-CsChrimson, (C) pC1b,c-split-Gal4/UAS-CsChrimson, (D) pC1d-split-Gal4/UAS-CsChrimson, and (E) pC1e-split-Gal4/UAS-CsChrimson. Gray circles indicate the ΔEHP of individual females, and the mean ± SEM of data is presented. 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.

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Genetic reagent (D. melanogaster)Canton SBDSCRRID:BDSC_64349
Genetic reagent (D. melanogaster)w1118VDRCVDRC #60000
Genetic reagent (D. melanogaster)R71G01 (pC1-Gal4)BDSCRRID:BDSC_39599
Genetic reagent (D. melanogaster)Orco1BDSCRRID:BDSC_23129
Genetic reagent (D. melanogaster)Or13a-Gal4BDSCRRID:BDSC_9946
Genetic reagent (D. melanogaster)Or19a-Gal4BDSCRRID:BDSC_9948
Genetic reagent (D. melanogaster)Or23a-Gal4BDSCRRID:BDSC_9955
Genetic reagent (D. melanogaster)Or43a-Gal4BDSCRRID:BDSC_9974
Genetic reagent (D. melanogaster)Or47b-Gal4BDSCRRID:BDSC_9983
Genetic reagent (D. melanogaster)Or47b-Gal4BDSCRRID:BDSC_9984
Genetic reagent (D. melanogaster)Or65a-Gal4BDSCRRID:BDSC_9993
Genetic reagent (D. melanogaster)Or65b-Gal4BDSCRRID:BDSC_23901
Genetic reagent (D. melanogaster)Or65c-Gal4BDSCRRID:BDSC_23903
Genetic reagent (D. melanogaster)Or67d-Gal4BDSCRRID:BDSC_9998
Genetic reagent (D. melanogaster)Or83c-Gal4BDSCRRID:BDSC_23131
Genetic reagent (D. melanogaster)Or88a-Gal4BDSCRRID:BDSC_23137
Genetic reagent (D. melanogaster)UAS-Or47bBDSCRRID:BDSC_76045
Genetic reagent (D. melanogaster)Or47b2/2BDSCRRID:BDSC_51306
Genetic reagent (D. melanogaster)Or47b3/3BDSCRRID:BDSC_51307
Genetic reagent (D. melanogaster)UAS-TNT activeBDSCRRID:BDSC_28837
Genetic reagent (D. melanogaster)UAS-TNT inactiveBDSCRRID:BDSC_28839
Genetic reagent (D. melanogaster)UAS-dTRPA1BDSCRRID:BDSC_26263
Genetic reagent (D. melanogaster)UAS-CsChrimsonBDSCRRID:BDSC_55135
Genetic reagent (D. melanogaster)UAS-GCaMP6mBDSCRRID:BDSC_42748
Genetic reagent (D. melanogaster)R52G04-ADBDSCRRID:BDSC_71085
Genetic reagent (D. melanogaster)SAG-Gal4 (VT50405)VDRCRRID:Flybase_FBst0489354,
VDRC #200652
Genetic reagent (D. melanogaster)UAS-Dh44R1-RNAiVDRCRRID:Flybase_FBst0482273,
VDRC #110708
Genetic reagent (D. melanogaster)UAS-Dh44R2-RNAiVDRCRRID:Flybase_FBst0465025,
VDRC #43314
Genetic reagent (D. melanogaster)UAS-Dicer2VDRCVDRC #60007
Genetic reagent (D. melanogaster)PromE(800)-Gal4Billeter et al., 2009N/A
Genetic reagent (D. melanogaster)UAS-FLP, CRE-F-lucTanenhaus et al., 2012N/A
Genetic reagent (D. melanogaster)LexAop-FLPBussell et al., 2014N/A
Genetic reagent (D. melanogaster)UAS-CsChrimsonKlapoetke et al., 2014N/A
Genetic reagent (D. melanogaster)UAS-GtACR1Mohammad et al., 2017N/A
Genetic reagent (D. melanogaster)UAS-PhotoAC (PACα)Schröder-Lang et al., 2007N/A
Genetic reagent (D. melanogaster)ppk23-Gal4Thistle et al., 2012N/A
Genetic reagent (D. melanogaster)ppk23-Thistle et al., 2012N/A
Genetic reagent (D. melanogaster)ppk28-Thistle et al., 2012N/A
Genetic reagent (D. melanogaster)ppk29-Thistle et al., 2012N/A
Genetic reagent (D. melanogaster)pC1-ADeutsch et al., 2020N/A
Genetic reagent (D. melanogaster)pC1-SDeutsch et al., 2020N/A
Genetic reagent (D. melanogaster)Dh44-pC1-Gal4Kim et al., 2024N/A
Genetic reagent (D. melanogaster)Orco-Gal4Yu et al., 2018N/A
Genetic reagent (D. melanogaster)UAS-EGFP-OrcoYu et al., 2018N/A
Genetic reagent (D. melanogaster)dsx-DBDWang et al., 2020N/A
Genetic reagent (D. melanogaster)pC1a-split-Gal4This studyN/A
Strain, strain background (Drosophila simulans)Drosophila simulansEHIME-Fly, KYORIN-FlyN/A
Strain, strain background (Drosophila sechellia)Drosophila sechelliaEHIME-Fly, KYORIN-FlyN/A
Strain, strain background (Drosophila erecta)Drosophila erectaEHIME-Fly, KYORIN-FlyN/A
Strain, strain background (Drosophila yakuba)Drosophila yakubaEHIME-Fly, KYORIN-FlyN/A
AntibodyMouse monoclonal
anti-Bruchpilot		DSHBCat# Nc82; RRID:AB_23148661:50
AntibodyRabbit Polyclonal
Anti-Green
Fluorescent Protein (GFP)		Thermo Fisher
Scientific
(Invitrogen)		Cat# A-11122, RRID:AB_2215691:1000
AntibodyAlexa 488-conjugated
goat anti-rabbit		Thermo Fisher
Scientific
(Invitrogen)		Cat# A-11008, RRID:AB_1431651:1000
AntibodyAlexa 568-conjugated
goat anti-mouse		Thermo Fisher Scientific (Invitrogen)Cat# A-11004, RRID:AB_25340721:1000
Chemical compoundPhoto-curable
UV glue		ThreeBondA16A01
Chemical compoundAll trans-retinalSigma-AldrichCat# R2500
Chemical compoundVectashieldVector LaboratoriesCat# H-1000
Chemical compoundMethyl laurateSigma-AldrichCat# W271500
Chemical compound7(Z)-TricoseneCayman ChemicalCat# 9000313
Chemical compoundtrans-palmitoleic acidCayman ChemicalCat# 9001798
Chemical compound11-cis-vaccenyl acetate (cVA)Cayman ChemicalCat# 10010101
Chemical compound7(Z)-PentacoseneCayman ChemicalCat# 9000530
Chemical compoundBeetle Luciferin,
Potassium Salt		PromegaCat# E1601
Chemical compoundTriton X-100,
laboratory grade		Sigma-AldrichCat# X100
Chemical compound2-MethyltetracosaneKIPN/A>98%, purity
Software and algorithmsFijihttps://imagej.net/software/fiji/downloadsRRID:SCR_002285
Software and algorithmsGraphPad Prism9https://www.graphpad.com/scientific-software/prism/RRID:SCR_002798
Software and algorithmsMetamorph softwarehttps://www.moleculardevices.com/products/cellular-imaging-systems/acquisition-and-analysis-software/metamorph-microscopyRRID:SCR_002368
Software and algorithmsNeuronbridgehttps://neuronbridge.janelia.org/N/A
Software and algorithmsComputational
Morphometry
Toolkit (CMTK)		https://github.com/jefferis/fiji-cmtk-gui;
Jefferis, 2015		RRID:SCR_002234
Version number: v0.1.1
Software and algorithmsColorMIP_Mask
_Search plugin		https://github.com/JaneliaSciComp/ColorMIP_Mask_Search;
Otsuna et al., 2020		Version number: v1.0.1
OtherDigital camcorderSONYHDR-CX405Behavior recording device
OtherSmart phoneXiaomiRedmi Note 10Behavior recording device
OtherMulti-channel
LED lights		NeoPixelCat# WS2812Light activation device;
red light, 620–625 nm,
390–420 mcd; green light,
522–525 nm, 660–720 mcd;
blue light, 465–467 nm,
180–200 mcd
OtherElectron-multiplying
CCD camera		Andor
Technology		LucaEM R 604MCalcium imaging
assay device
OtherStimulus
Controller		SyntechType CS-55Pheromone
delivery device
OtherMicroplate
luminometer		Berthold TechnologiesCentro XS3 LB 960Luciferase assay
device
Table 1
Table of D. melanogaster genotypes or Drosophila species used to generate the figures and figure supplements in this paper.
FigureD. melanogaster genotypes or Drosophila species ofN numbers
Tested femalesMating partnerIncubation partnerFrom left to right
Figure 1
Figure 1Bw[1118]Canton-SCanton-S59, 69
Figure 1Cw[1118]Canton-SCanton-S18, 15
Figure 1Dw[1118]Canton-SCanton-S12, 12
Figure 1Ew[1118]Canton-SCanton-S20, 18, 23
Figure 1Fw[1118]; TI{w[+mW.hs]=TI}Orco[1]Canton-SCanton-S55, 47
Figure 2
Figure 2Aw[1118];Or47b-Gal4/UAS-TNTinactive(P{UAS-TeTxLC.(-)Q}A2)Canton-SCanton-S14, 14
w[1118];Or47b-Gal4/UAS-TNTactive (P{w[+mC]=UAS-TeTxLC.tnt}E2)Canton-SCanton-S16, 18
Figure 2Bw[1118];Or47b-Gal4/+Canton-SCanton-S28, 31
w[1118];;UAS-dTRPA1/+Canton-SCanton-S21, 16
w[1118];Or47b/+;UAS-dTRPA1/+Canton-SCanton-S11, 15
Figure 2Cw[1118]; TI{w[+mW.hs]=TI}Orco [1]Canton-SCanton-S12, 12
w[1118]; Or47b-Gal4>UAS-EGFP-Orco; Orco[1]/Orco[1]Canton-SCanton-S13, 14
Figure 2Dw[1118]; Or47b[2]/+Canton-SCanton-S12, 15
w[1118]; Or47b[3]/+Canton-SCanton-S13, 14
w[1118]; Or47b[2]/Or47b[3]Canton-SCanton-S13, 12
Figure 2Ew[1118]; Or47b[2]/Or47b[2]Canton-SCanton-S14, 15
w[1118]; Or47b-Gal4>P{w[+mC]=UAS-Or47b.MYC}2; Or47b[2]/Or47b[2]Canton-SCanton-S11, 11
Figure 3
Figure 3Aw[1118]Canton-S13, 16
Figure 3Bw[1118]; TI{w[+mW.hs]=TI}Orco[1]Canton-S11, 12
Figure 3Cw[1118]; TI{w[+mW.hs]=TI}Or47b[2]Canton-S14, 17
Figure 3Dw[1118];Or47b-Gal4/+;Orco[1]/Orco[1]Canton-S22, 22
w[1118];UAS-Orco/+; Orco[1]/Orco[1]Canton-S15, 14
w[1118];Or47b-Gal4/UAS-Orco;Orco[1]/Orco[1]Canton-S18, 19
Figure 4
Figure 4Aw[1118]Canton-SCanton-S18, 22
Figure 4Bw[1118]Canton-S23, 31
Figure 4Cw[1118]Canton-S16, 17
Figure 4Dw[1118];ppk23-Gal4/UAS-TNTinactive(P{UAS-TeTxLC.(-)Q}A2)Canton-SCanton-S18, 13
w[1118];ppk23-Gal4/UAS-TNTactive (P{w[+mC]=UAS-TeTxLC.tnt}E2)Canton-SCanton-S17, 17
Figure 5
Figure 5Aw[1118];pC1(R71G01)-AD/+;Dsx-DBD/UAS-GtACR1Canton-S22, 21
Figure 5Bw[1118];VT25602-AD/+;UAS-GtACR1/VT2064-DBDCanton-S18, 18
Figure 5Cw[1118];R52G04-AD/+;UAS-GtACR1/Dsx-DBDCanton-S15, 14
Figure 5Dw[1118];;Dh44-pC1 (Dsx-DBD, Dh44A-AD)-GAL4/UAS-GtACR1Canton-S17, 20
Figure 5Ew[1118];UAS-FLP/+; GMR71G01-Gal4, CRE-F-Luc/+Canton-S12, 12, 12
w[1118];R52G04-AD/+;UAS-FLP, CRE-F-Luc/Dsx-DBDCanton-S12, 12, 12
w[1118];;UAS-FLP, CRE-F-Luc/Dh44-pC1 (Dsx-DBD, Dh44A-AD)-GAL4Canton-S16, 16, 16
w[1118];VT25602-AD/+;UAS-FLP, CRE-F-Luc/VT2064-DBDCanton-S12, 12, 12
Figure 5Fw[1118]; Or47b[2]/+; pC1-FLP, CRE-F-LucCanton-S8, 8, 8
w[1118]; Or47b[2]/Or47b[2]; pC1-FLP, CRE-F-LucCanton-S8, 8, 8
w[1118]; Or47b[3]/+; pC1-FLP, CRE-F-LucCanton-S8, 8, 8
w[1118]; Or47b[3]/Or47b[3]; pC1-FLP, CRE-F-LucCanton-S8, 8, 8
Figure 6Aw[1118]; R52G04-AD/+;UAS-PhotoAC/Dsx-DBDCanton-S18, 22
w[1118];;UAS-PhotoAC/Dh44-pC1 (Dsx-DBD, Dh44A-AD)-GAL4Canton-S22, 28
w[1118]; VT25602-AD/+;UAS-PhotoAC/VT2064-DBDCanton-S21, 20
Figure 6Bw[1118];UAS-GCaMP6m/+; pC1(GMR71G01)-GAL4/UAS-PhotoACCanton-S9, 9, 9
Figure 6Cw[1118];;+/UAS-PhotoACCanton-S
(1st, 2nd)		60
w[1118];;Dh44-pC1 (Dsx-DBD, Dh44A-AD)-GAL4/UAS-PhotoACCanton-S
(1st, 2nd)		18
Figure 2—figure supplement 1w[1118];+/P{w[+mC]=UAS-TeTxLC.tnt}E2Canton-SCanton-S27, 27
w[1118];Or13a-Gal4/P{w[+mC]=UAS-TeTxLC.tnt}E2Canton-SCanton-S9, 12
w[1118];+/P{w[+mC]=UAS-TeTxLC.tnt}E2;+/Or19a-Gal4Canton-SCanton-S8, 6
w[1118];+/P{w[+mC]=UAS-TeTxLC.tnt}E2;+/Or23a-Gal4Canton-SCanton-S11, 11
w[1118];+/P{w[+mC]=UAS-TeTxLC.tnt}E2;+/Or43a-Gal4Canton-SCanton-S18, 16
w[1118];+/P{w[+mC]=UAS-TeTxLC.tnt}E2;+/Or47b-Gal4Canton-SCanton-S12, 11
w[1118];Or65a-Gal4/P{w[+mC]=UAS-TeTxLC.tnt}E2Canton-SCanton-S13, 16
w[1118];Or65b-Gal4/P{w[+mC]=UAS-TeTxLC.tnt}E2Canton-SCanton-S18, 14
w[1118];+/P{w[+mC]=UAS-TeTxLC.tnt}E2;+/Or65c-Gal4Canton-SCanton-S20, 18
w[1118];Or67d-Gal4/P{w[+mC]=UAS-TeTxLC.tnt}E2Canton-SCanton-S15, 19
w[1118];Or83c-Gal4/P{w[+mC]=UAS-TeTxLC.tnt}E2Canton-SCanton-S20, 17
w[1118];Or88a-Gal4/P{w[+mC]=UAS-TeTxLC.tnt}E2Canton-SCanton-S21, 19
Figure 3—figure supplement 1
Figure 3—figure supplement 1Aw[1118]Canton-S26, 14, 18, 13
Figure 3—figure supplement 1Bw[1118]Canton-S22, 14, 12, 12
Figure 3—figure supplement 2
Figure 3—figure supplement 2Aw[1118]Canton-S33
w[1118]Canton-S+;PromE(800)-Gal4/UAS-Tra36
w[1118]Canton-S+;PromE(800)-Gal4/UAS-Tra-RNAi17
Figure 3—figure supplement 2Bw[1118]Canton-S20
w[1118]Canton-SD. melanogaster23
w[1118]Canton-SD. simulans21
w[1118]Canton-SD. sechellia20
w[1118]Canton-SD. erecta19
w[1118]Canton-SD. yakuba21
Figure 3—figure supplement 3w[1118]Canton-S18, 18, 14, 17, 15, 13
Figure 4—figure supplement 1
Figure 4—figure supplement 1Aw[1118]Canton-S17, 17, 18, 18, 17, 18
Figure 4—figure supplement 1Bw[1118]Canton-S15, 15, 15, 16, 14, 15
Figure 4—figure supplement 1Cw[1118]Canton-SCanton-S21, 18
w[1118]/ppk23-Canton-SCanton-S17, 21
ppk23-Canton-SCanton-S13, 15
Figure 4—figure supplement 1Dw[1118]Canton-SCanton-S18, 14
w[1118]/ppk28-Canton-SCanton-S23, 25
ppk28-Canton-SCanton-S22, 17
Figure 4—figure supplement 1Ew[1118]Canton-SCanton-S17, 14
w[1118];ppk29-/+Canton-SCanton-S19, 20
ppk29-Canton-SCanton-S16, 17
Figure 5—figure supplement 1A–Cw[1118];R52G04-AD/UAS-myrGFP;Dsx-DBD/UAS-myrGFP
Figure 5—figure supplement 1Dw[1118];R52G04-AD/+;Dsx-DBD/UAS-GtACR1Canton-S82, 60
Figure 5—figure supplement 2Aw[1118];UAS-FLP/+; GMR71G01-Gal4, CRE-F-Luc/+8, 8, 8, 12, 8, 4
Figure 5—figure supplement 2Bw[1118];UAS-FLP/+; GMR71G01-Gal4, CRE-F-Luc/+8, 8, 8, 12, 8, 4
Figure 5—figure supplement 3w[1118];UAS-FLP/+; GMR71G01-Gal4, CRE-F-Luc/+12, 12, 12
w[1118]; R52G04-AD/+;UAS-FLP, CRE-F-Luc/Dsx-DBD12, 12, 12
w[1118];;UAS-FLP, CRE-F-Luc/Dh44-pC1 (Dsx-DBD, Dh44A-AD)-GAL412, 12, 12
w[1118]; VT25602-AD/+;UAS-FLP, CRE-F-Luc/VT2064-DBD10, 10, 10
Figure 6—figure supplement 1w[1118]/UAS-Dcr2;;GMR71G01-Gal4/+Canton-SCanton-S27, 26
w[1118];UAS-Dh44R1-RNAi/+; UAS-Dh44R2-RNAi/+Canton-SCanton-S18, 17
w[1118]/UAS-Dicer2;UAS-Dh44R1-RNAi1/+; GMR71G01-GAL4/UAS-Dh44R2-RNAi2Canton-SCanton-S35, 30

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https://cdn.elifesciences.org/articles/96013/elife-96013-mdarchecklist1-v1.docx
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https://cdn.elifesciences.org/articles/96013/elife-96013-data1-v1.xlsx

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  1. Minsik Yun
  2. Do-Hyoung Kim
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  4. Kang-Min Lee
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