Sexual plasticity, the ability to modify sexual state or reproductive behavior in response to changing social conditions, is observed in both vertebrates and invertebrates (Bruce, 1959; Koene and Ter Maat, 2007; Roberts et al., 2012; Steiger et al., 2008; Yagound et al., 2012). In rodents, exposure to unfamiliar males often leads to the sudden termination of pregnancy, known as the Bruce effect. It is induced by male urinary peptides, such as MHC I peptides, activating the vomeronasal organ (Becker and Hurst, 2008; Leinders-Zufall et al., 2004; Zipple et al., 2019). This effect enhances reproductive fitness of both sexes, by eliminating the offspring of competing males and enabling females to select better mates even after conception. Many species also adapt their reproductive behavior in response to the social-sexual context change (SSCC), involving encounters with new sexual partners or competitors. Understanding the neural circuit mechanisms behind female responses to SSCC emerges as a central focus of neuroscience (Gaspar et al., 2022; Kim et al., 2019; Liu et al., 2022; Wei et al., 2021).

Drosophila melanogaster, the fruit fly, displays various social behaviors like aggregation, aggression, and sexual behavior (Bartelt et al., 1985; Billeter et al., 2006; Billeter and Levine, 2015). Similar to rodents, they primarily use the olfactory system to communicate socially through pheromones (Kohl et al., 2015; Sengupta and Smith, 2014). Some of these pheromones act as aphrodisiacs, while others regulate aggression or foster aggregation. For instance, 11-cis-vaccenyl acetate (cVA) attracts females but repels males and promotes aggregation (Billeter and Levine, 2015; Kurtovic et al., 2007; Mane et al., 1983). 7-Tricosene (7-T), a cuticular hydrocarbon (CHC) present in males, is an aphrodisiac to females and affects social interactions between males (Grillet et al., 2006; Wang et al., 2011). On the other hand, 7,11-heptacosadiene (7,11-HD), a related female-specific pheromone, functions as an aphrodisiac to males, triggering courtship behavior and involving species recognition (Antony et al., 1985; Toda et al., 2012).

The fruit fly’s chemosensory organs, located in various parts of the body, detect these pheromones (Ali et al., 2022; Joseph and Carlson, 2015). Olfactory receptor neurons (ORNs) in the sensilla of the antennae and maxillary palp are responsible for the detection of long-range volatile pheromones like cVA, while short-range pheromones like 7-T are sensed by neurons on the forelegs and labellum (Joseph and Carlson, 2015; Kohl et al., 2015; Sengupta and Smith, 2014).

The olfactory receptor Or47b, expressed in ORNs located in the at4 trichoid sensilla on the third antennal segment, is involved in several sociosexual interactions, including male mating success, mate preference, and female aggression toward mating pairs (Gaspar et al., 2022; Kohlmeier et al., 2021; Lone et al., 2015; Lone and Sharma, 2012; Zhuang et al., 2016). In males, Or47b senses fatty acid methyl esters and fatty acids that affect mating competition and copulation (Dweck et al., 2015; Lin et al., 2016). While the role of Or47b in female aggression is well established (Gaspar et al., 2022), its involvement in female sexual behavior is uncertain. In both sexes, Or47b ORNs project to VA1v glomeruli, where VA1v projection neurons receive their signal and project to the mushroom body calyx and lateral horn. Male Or47b neurons connect to neurons such as aSP5, aSP8, and aSP9, which express a male-specific transcription factor Fru^M (Yu et al., 2010).

CHC pheromones, which function as short-range pheromones, are detected primarily by neurons on the forelegs and the labellum that express gustatory receptors, ionotropic receptors, or the ppk/DEG-ENaC family of sodium channels (Joseph and Carlson, 2015; Kohl et al., 2015; Sengupta and Smith, 2014). CHCs like 7-T and 7,11-HD are sensed by ppk23-expressing M and F cells in the tarsi (Liu et al., 2020). 7-T and cVA are sensed by M cells expressing ppk23, whereas 7,11-HD and 7,11-nonacosadiene are sensed by F cells expressing ppk23, ppk25, and ppk29. In males, 7-T or 7,11-HD affects the neuronal activity of the Fru^M-expressing P1 neurons (Inagaki et al., 2014; Kohatsu et al., 2011; Sato and Yamamoto, 2020). However, how these CHCs signal in the female brain remains unknown.

Sperm ejection is a process by which females can remove the male ejaculate or the mating plug after copulation. This phenomenon has been observed in several animal species including feral fowl (Pizzari and Birkhead, 2000), black-legged kittiwake (Wagner et al., 2004), and dunnock (Davies, 1983). In the fruit fly, it typically occurs approximately 90 min after mating (Lee et al., 2015). This specific interval, referred to as ejaculate holding period (EHP), is thought to affect sperm usage and fecundity (Lee et al., 2015; Manier et al., 2010). The neurosecretory neurons in the brain pars intercerebralis (PI) that produce diuretic hormone 44 (Dh44), an insect orthologue of the corticotropin-releasing factor, regulate EHP (Lee et al., 2015). There is evidence that Drosophila females sense the social-sexual context through sperm ejected by other females. For instance, females were likely to lay more eggs when placed on a food patch containing male ejaculate deposited by other females (Duménil et al., 2016). However, it remains unknown whether the SSCC influences sperm ejection and EHP.

Female pC1 neurons, which express a specific transcription factor Dsx^F, integrate olfactory and auditory cues associated with male courtship (Lee et al., 2002; Zhou et al., 2014). The pC1 neurons, their male counterparts (i.e. P1 neurons), and the ventrolateral subdivision of ventromedial hypothalamus (VMHvl) neurons in mice share conserved circuit configurations and demonstrate functional similarity in coordinating social and sexual behaviors (Anderson, 2016; Jiang and Pan, 2022). There are 14 Dsx-positive pC1 neurons in each hemisphere of the brain, responsive to the male sex-pheromone cVA and courtship songs (Kim et al., 2024; Zhou et al., 2014). Connectome analyses identified 10 pC1 neurons that fall into five subtypes, with pC1a, b, and c subtypes associated with mating behavior and pC1d and e subtypes associated with aggression (Chiu et al., 2023; Deutsch et al., 2020; Han et al., 2022; Kim et al., 2024; Schretter et al., 2020; Wang et al., 2020). Although direct evidence connecting pC1 neurons to sperm ejection is limited, they are promising candidates for regulating sperm ejection or EHP, because sperm ejection allows females to eliminate the mating plug and male ejaculate, thereby restoring sexual attractiveness (Laturney and Billeter, 2016).

In this study, we demonstrated that two male pheromones, 2-methyltetracosane (2MC) and 7-T, significantly reduced the EHP through Or47b neurons and ppk23 neurons, respectively. These pheromone pathways converge on pC1 neurons, where they increase cAMP levels. The elevated cAMP in pC1 neurons resulted in a reduction of the EHP to a degree that was comparable to the effects of the male pheromones. It also enhanced the excitability of pC1 neurons, making them more responsive to both olfactory and auditory male courtship cues and promoting further mating following the earlier removal of the mating plug. These findings establish a novel behavioral paradigm that sheds light on the intricate molecular and neuronal pathways underlying female sexual plasticity.

Males employ a diverse range of strategies to enhance their reproductive fitness. One such strategy involves the formation of a ‘mating plug’, a mechanism that prevents females from engaging in further mating, thereby increasing fertilization success rates (Dixson, 1998; Parker, 1970; Schneider et al., 2016). As a means of intra-sexual competition, rival males often promote the removal or precocious expulsion of the mating plug. The evolution of this strategy is driven by intersexual interactions with polyandrous females, who often remove the mating plug to engage in additional mating with males of superior traits or higher social status than their previous partners (Dean et al., 2011; Pizzari and Birkhead, 2000). In the dunnock Prunella modularis, a small European passerine bird, the male often engages in cloacal pecking of mated females, inducing the expulsion of the previous mate’s sperm and mating plug, thereby increasing their chances of successful mating (Davies, 1983). In this study, we discovered that in D. melanogaster, freshly mated females exhibit an earlier removal of the mating plugs or a shorter EHP when kept with actively courting males. This behavior is primarily induced by the stimulation of females via male sex pheromones. In addition, our study has revealed that the neural circuit that processes male courtship cues and controls mating decisions plays an important role in regulating this behavior. This fly circuit has recently been proposed to be homologous to VMHvl in the mouse brain (Anderson, 2016; Jiang and Pan, 2022). By delving into the molecular and neuronal mechanisms underlying MIES, our study provides valuable insights into the broader aspect of behaviors induced by changes in the social-sexual context.

Our findings highlight the involvement of the Or47b receptor and Or47b ORNs in MIES. These OR and ORNs have been implicated in a range of social and sexual behaviors in both male and female fruit flies (Dweck et al., 2015; Gaspar et al., 2022; Kohlmeier et al., 2021; Lin et al., 2016; Lone et al., 2015; Lone and Sharma, 2012; Zhuang et al., 2016). Methyl laurate and trans-palmitoleic acid are odorant ligands for Or47b that account for many of these functions, particularly in males (Dweck et al., 2015; Lin et al., 2016). In this study, we provide compelling evidence that 2MC induces cAMP elevation in pC1 neurons and EHP shortening via both the Or47b receptor and Or47b ORNs, suggesting that 2MC functions as an odorant ligand for Or47b. Notably, gas chromatography-mass spectrometry analysis of CHCs from 4-day-old wild-type D. melanogaster revealed the presence of 2MC exclusively in males (Dweck et al., 2015). Surprisingly, however, unlike 2MC, neither methyl laurate nor trans-palmitoleic acid affected EHP. The reason for this paradoxical result remains unclear. A plausible interpretation is that the EHP shortening induced by 2MC may require not only Or47b but also other as yet unidentified ORs. With the establishment of a behavioral and cellular assessment of 2MC activity, the search for additional odorant receptors responsive to 2MC is now feasible. Another important avenue for further research is whether 2MC can also elicit behaviors previously associated with methyl laurate or trans-palmitoleic acid, such as promoting male copulation and courtship (Dweck et al., 2015; Lin et al., 2016).

We observed that both 2MC and 7-T exhibit both cellular and behavioral activity within a specific concentration range (Figure 3—figure supplement 3, Figure 4—figure supplement 1, Figure 5—figure supplement 2). This observation is of particular interest, given the multitude of environmental and biological factors that influence the levels of 2MC and 7-T, potentially affecting the capacity of males to induce MIES. For instance, exposure to low temperatures during development has been linked to increased production of both 2MC and 7-T (Bontonou et al., 2013). Similarly, mutation of the desiccation stress gene CG9186, which encodes a protein associated with lipid droplets, has been found to impact 2MC levels (Werthebach et al., 2019). Furthermore, 2MC levels rise with age in males (Everaerts et al., 2010). Thus, we propose that levels of 2MC, and possibly 7-T, may serve as indicators of male age and resilience to environmental stress in a complex manner.

In mated females, treatment with 2MC or 7-T increases cAMP levels in pC1b, c neurons but not in pC1a neurons. In contrast, pC1a neurons in virgin females are fully responsive to both male pheromones, showing increases in cAMP levels that are similar to those of pC1b,c neurons (Figure 5—figure supplement 3). The absence of cAMP levels in pC1a neurons in mated females likely results from the mating signal (i.e. sex peptide) silencing pC1a neurons. Connectome and electrophysiology data support this interpretation, as SAG neurons, which relay sex peptide signals, have the strongest synaptic connection with the pC1a among five pC1 subtypes (Wang et al., 2020). However, the activity of SAG neurons may also influence pC1c neurons, as they also have substantial synaptic connections with pC1c neurons, as seen in the hemibrain connectome dataset (Scheffer et al., 2020). Future studies are needed to understand the role of SAG neurons in the regulation of EHP.

We found that increased cAMP levels cause pC1b, c neurons in mated females, which are typically unresponsive to male courtship cues like cVA and pulse song, to become responsive and exhibit strong Ca²⁺ transients. Since pC1b, c neurons play a role in generating sexual drive and increasing female receptivity to male courtship, the 2MC- or 7-T-induced increases in cAMP are likely to control the removal of the mating plug and the engagement of mated females in further mating (Figure 7). This hypothesis aligns well with the previous report that mating reduces the sensitivity of Or47b ORNs, which we found to be responsive to 2MC, leading to an increased preference for pheromone-rich males after mating (Kohlmeier et al., 2021). Moreover, the finding that 2MC and 7-T induce cAMP levels in pC1b, c neurons in virgin females suggests that virgin females may also use 2MC and 7-T as odorant cues to assess male quality during their first mating. Indeed, females seem to evaluate male quality by the amount of 7-T, as increased 7-T promotes mating receptivity and shortens mating latency (Grillet et al., 2006).

Physiological factors like the nutritional status of females prior to mating and the nutritional status of their mates have been shown to influence EHP (Kim et al., 2023), and therefore potentially MIES. Hence, it is highly probable that MIES is regulated by additional central neurons such as Dh44-PI neurons that regulate these processes (Lee et al., 2015). However, it remains unclear whether and how Dh44-PI neurons and pC1 neurons interact to modulate EHP and MIES. The observation that double knockdown of Dh44R1 and Dh44R2 has only a marginal effect on MIES suggests that Dh44-PI neurons may also function independently of pC1 neurons, raising the possibility that multiple independent central circuits may contribute to the production of MIES.

Our initial screening of ORNs responsible for MIES revealed the involvement of Or47b ORNs, as well as several other ORNs. In addition to 2MC, which acts through Or47b-expressing ORNs, our findings indicate that 7-T and ppk23 neurons, which are capable of detecting 7-T, also play a role in MIES induction. In D. melanogaster and other related species, food odors typically serve as volatile long-range signals that attract both males and females (Lin et al., 2015; Verschut et al., 2023), suggesting that specific food odors may also influence EHP (Duménil et al., 2016). The involvement of multiple ORNs in the regulation of EHP predicts that pC1 neurons may process multiple odorants, not limited to those associated with mating behavior, including food odors. Future studies will explore the full spectrum of odorants processed by pC1 neurons in the regulation of EHP.

In conclusion, we have identified a circuit that, via the detection of a novel male pheromone, potentially signals male quality and governs the female’s decision to remove the mating plug of her last mate and mate again.

All data generated or analysed during this study are included in the manuscript and supporting files.

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Author details

1. Minsik Yun

   School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju, Republic of Korea

   Contribution

   Conceptualization, Investigation, Writing – original draft

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0011-0942

2. Do-Hyoung Kim

   School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju, Republic of Korea

   Contribution

   Investigation

   Competing interests

   No competing interests declared

3. Tal Soo Ha

   Department of Biomedical Science, College of Natural Science, Daegu University, Gyeongsan, Republic of Korea

   Contribution

   Investigation

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-0015-6075

4. Kang-Min Lee

   School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju, Republic of Korea

   Contribution

   Methodology

   Competing interests

   No competing interests declared

5. Eungyu Park

   School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju, Republic of Korea

   Contribution

   Investigation

   Competing interests

   No competing interests declared

6. Markus Knaden

   1. Department of Evolutionary Neuroethology, Max Planck Institute for Chemical Ecology, Jena, Germany
   2. Next Generation Insect Chemical Ecology, Max Planck Centre, Max Planck Institute for Chemical Ecology, Jena, Germany

   Contribution

   Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-6710-1071

7. Bill S Hansson

   1. Department of Evolutionary Neuroethology, Max Planck Institute for Chemical Ecology, Jena, Germany
   2. Next Generation Insect Chemical Ecology, Max Planck Centre, Max Planck Institute for Chemical Ecology, Jena, Germany

   Contribution

   Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4811-1223

8. Young-Joon Kim

   School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju, Republic of Korea

   Contribution

   Conceptualization, Supervision, Funding acquisition, Writing – original draft, Writing – review and editing

   For correspondence

   kimyj@gist.ac.kr

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-7990-754X

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