Brain-gonad axes harmonize male mating drive and reproductive strategy via steroid hormones

Domain: biology
Published: 2026-01-15
Editorial outcome: major_revision

Abstract
The interplay between the nervous and reproductive systems, encapsulated by the brain-gonad axis, is pivotal for reproduction. However, it remains largely unclear how hormones released from reproductive system actively regulate male sexual behaviors and mating strategies. Here we find that sexual priming induces an elevated sexual drive in both male mice and Drosophila, triggered by the surge of circulating steroid hormones 20-hydroxyecdysone (20E) in flies and testosterone in mice, respectively. In Drosophila, 20E is released from the male reproductive system upon sexual priming, which is controlled by a descending brain-gonad pathway composed of P1 neurons, Dsk neurons, and Crz/CrzR neurons. In return, 20E activates P1 neurons to elevate and sustain male flies’ mating drive. The enhanced mating drive not only fosters consequent mating success but also makes full use of the reproductive materials prepared during the pre-ejaculatory emission stage, e.g., seminal proteins which would otherwise be potentially harmful to males’ gonads. Collectively, our study offers insights into how hormones regulate males’ reproductive strategies.

Introduction
Male animals’ mating motivation is actively regulated by their mate’s availability/quality and sexual experience. During courtship, males integrate female signals and environmental cues to sustain the appropriate level of mating drive 1–3. In mice, key brain regions like the MPOA (medial preoptic area) and BNST (bed nucleus of the stria terminalis) control males’ mating drive. On the other hand, amygdala circuits responding to environmental and social factors negatively regulate mating drive 4–7. In Drosophila, male-specific P1 neurons integrate female stimuli to identify the target mate and trigger vigorous courtship rituals 8. Meanwhile, males adjust their mating drive by monitoring their internal states such as nutrition and prior sexual experience 9–11.

Mating drive drops with ejaculation. Failure to transition from sexual arousal to sexual satiety may lead to an elevated sexual drive state, characterized by intensified mating drive 11. This elevated sexual drive state is an analogy to food hunger to some extent. Notably, mice in starvation exhibit a tolerance for averse tastes 12. It’s thus an intriguing question whether and how males in elevated mating drive adjust their mating drive and pickiness in choosing their mates.

The interplay between the nervous and reproductive systems has been extensively investigated for development 13. This brain-gonad axis regulates gonadal steroidogenesis and the development of gametes, such as oogenesis and spermatogenesis, through the release of gonadotropins from the brain 14. Gonadal steroid hormones, in return, participate in the maturation of the brain and the emergence of sexually dimorphic behavior 15. However, the bi-directional communication of the brain-gonad axis in governing sexual behaviors during adulthood remains elusive.

Hormones likely play a key role in regulating males’ mating behavior, as their levels fluctuate during sexual arousal. For example, testosterone levels increase in their plasm after men watch erotic movies or male mice interact with female mice 16,17. Similarly, the hormone 20-hydroxyecdysone (20E) in Drosophila increases in the male hemolymph with intense courtship behavior 18. In addition, testosterone receptor ARs (Androgen receptors) are widely expressed in sexual-related brain areas of mice 19–21. 20E receptors, EcR and DopEcR, are both found in the fly neurons expressing fruitless (fru)+ and doublesex (dsx)+ 22, two transcription factors developmentally shaping the sexual neural circuits that control sexual behaviors 23. Thus, the expression pattern of hormone receptors in the male brain suggests that hormones may regulate sexual behavior by modulating the activity of sex-related neural circuits.

Here, we found that both male mice and flies transform into an elevated mating drive state and increase their mating drive after experiencing sexual priming, e.g., sexual arousal without ejaculation. During the priming of male flies, 20E released from the reproductive system is triggered by the brain-gonad axis, including P1 neurons and their downstream Dsk and Crz neurons. In return, 20E activates P1 neurons to promote mating drive. Meanwhile, males adjust their allocation of reproductive materials after priming to avoid the waste of prepared reproductive materials. Our findings shed light on the dynamic regulation of mating drive and reproductive strategies by hormones.

Results
Male animals prefer females with high fecundity
Male animals deliberately choose their mates, and they tend to court females with high fecundity 24. We first aimed to quantify males’ mate preference in both flies and mice. For flies, w1118 and CS males were allowed to court with either optimal (5~7 d old, with highest fecundity) or aged (35–45 d old, with lower fecundity) virgin females (Fig. 1A). During the first 5 min of the courtship assay, males showed higher mating drive to the optimal females compared with aged females (Fig. 1B). Similarly, wild type CS males exhibited higher mating drive to the optimal females than aged females (Fig. 1C). These results imply that young females are optimal mates for males as they may have higher fecundity than sub-optimal aged females.

In parallel, we found male mice showed decreased mating intensity (Fig. 1D–F) and towards aged female mice (25–29 weeks) compared with optimal female mice (8–10 weeks). This “mate taste” cross taxa underscores males’ ability to assess and select mates based on female’s reproductive potentials.

Males’ mate preference is regulated by sexual priming
Male animals should be able to adjust their mating strategies to adapt to various environments and mate availability. We wondered whether “mate taste” is regulated by sex experience, such as sexual arousal. A male fly was sexually primed by grouping and vigorously courting with a genitalia-sealed virgin female for 10 min (Fig. 1G). Then, they were allowed to court different female mates. We found that w1118 male flies after priming showed the elevated mating drive to sub-optimal mates (aged females) (Fig. 1H). Similarly, primed CS males performed higher mating drive to the aged females (Fig. 1I). Notably, the courtship index in primed flies to the optimal females was not further increased, suggesting that these optimal mates already elicit plateaued mating intensity that cannot be revealed by the one-on-one courtship assay.

We then performed a mating competition assay for two males to a single optimal female and found that primed males showed higher competitiveness compared to a control male (Supplementary Fig. S1A, B). The results imply that priming causes an increase in males’ mating drive, and primed males no longer show a significantly different courtship intensity towards optimal or aged females.

Similarly, male mice were primed by pairing them with estrus females until they exhibited sexual behaviors (sniffing, mounting, and five times intromissions) without ejaculation (Fig. 1J). Primed male mice showed higher mating intensity to aged female mice (Fig. 1K, L), demonstrating an elevated mating drive on sub-optimal mates. Interestingly, although the mating intensity index to optimal females did not further increase by priming, the reduced latency to mounting behavior argued that primed mice still exhibit a higher mating drive to optimal females (Supplementary Fig. S1E).

The results above suggest that the sex experience of sexual priming without ejaculation induces a stronger mating drive, thus dampening the males’ choosiness to sub-optimal mates. It’s worth noting that in both primed male flies and mice, neither the courtship latency index nor the mating intensity towards the optimal females appears to increase. This may due to the one-on-one mating assays failed to reveal the increase to mating drive to optimal females. In this case, males engage in the elevated mating drive state with the modificatory “sexual taste”.

Steroid hormones boost males’ mating drive
What’s the molecular and neural basis of the priming-induced mating drive elevation? In Drosophila, the 20-hydroxyecdysone (20E) is the major steroid hormone 25. The levels of 20E in the hemolymph were reported to surge during intense courtship behavior 18. We here demonstrated that the levels of 20E in the hemolymph were higher in primed males (Fig. 2A), suggesting that 20E may play a role in the priming induced sexual drive increase. Then, we pinpointed the source of 20E by looking at the expression pattern of the gene dare, which encodes the Drosophila homolog of vertebrate adrenodoxin reductase (AR) and is essential for the synthesis of steroid hormones 26. The dare-GAL4 expression was found across many tissues, such as the intestine and male reproductive system, including the testes (TE), seminal vesicle (SV), anterior ejaculatory duct (AED), and the epithelium of the EB (Supplementary Fig. S2A). Its expression in the nervous system is marginal (Supplementary Fig. S2A). Furthermore, ELISA analysis confirmed the presence of 20E in the gut and the male reproductive system but not in the nervous system of naive males (Fig. 2A and Supplementary Fig. S2B). In contrast, only the male reproductive system showed a decrease in 20E levels after 10 min of priming (Fig. 2A and Supplementary Fig. S2B), indicating that 20E was released from the male reproductive organs during priming.

To directly test whether 20E enhanced males’ mating drive and made them less picky, we fed male flies with 20E (3 mM) for 5 h (Fig. 2C) and found that both CS and w1118 males fed with 20E showed a higher mating drive to the sub-optimal mates (Fig. 2D, E) and stronger courtship competitiveness to optimal females (Supplementary Fig. S1C, D). We thus concluded that sexual priming and 20E feeding had a similar effect in boosting male flies’ sexual drive.

The steroid hormone testosterone levels in the serum of mice and humans are also increased along with sexual arousal/priming 16,17. We also observed that the serum testosterone levels of the mice were increased after they were primed with the estrus female (sniffing, mounting, and five times intromissions without ejaculation) (Fig. 2B). To test whether the increased testosterone modulates male mice’s mating intensity, we subcutaneously injected 500 ng testosterone in male mice (Fig. 2F). Thirty minute after injection, male mice performed more vigorous and consistent mating behavior to aged females comparable to optimal mates (Fig. 2G, H). Moreover, male mice injected with testosterone showed a reduced mounting latency to both optimal and aged female mice (Supplementary Fig. S1E), similar to the male mice which were sexually primed (Supplementary Fig. S1E). By contrast, vehicle control of corn oil injection alone does not enhance mating motivation (Supplementary Fig. S6). Taken together, we found that steroid hormones of both rodents and insects are released from their reproductive system and accumulated in the hemolymph or plasm during sexual arousal/priming, converting males into elevated mating drive states.

20E activates P1 neurons to regulate male flies’ mating drive
How do steroid hormones elevate the mating drive for male animals? In Drosophila, male-specific P1 neurons control males’ mating drive 8. We thus asked whether 20E activates P1 neurons to enhance mating drive. Two 20E receptors were identified in flies, with EcR as a nuclear complex and DopEcR as a G-protein coupled receptor (GPCR) 27. We then conducted RNAi knockdown for ECR or DopECR in P1 neurons (labeled by R71G01-GAL4) (Fig. 3A). Knocking down ECR but not DopECR in P1 neurons even resulted in decreased mating drive towards optimal mates (Fig. 3B), indicating that 20E and ECR may be required for the developmental and normal function of P1 neurons. In contrast, knocking down dopECR in P1 neurons blocked the priming-induced higher mating drive to aged females (Fig. 3C) and reduced the priming-induced stronger courtship competitiveness to optimal females (Supplementary Fig. S2C). Thus, we reasoned that 20E released during priming activates P1 neurons through DopECR to elicit a higher mating drive to sub-optimal mates.

We then used GCaMP7s to monitor P1 neurons’ activity and found that these neurons were activated by 10 min priming of males (Supplementary Fig. S2D). Importantly, knockdown of DopECR in P1 neurons abolished the priming-induced activation (Supplementary Fig. S2E). We thus proposed that P1 neurons’ activity may be upregulated by 20E. However, this ex vivo experimental setting may not be able to fully capture the activity of P1 neurons. To further test the hypothesis that 20E can activate P1 neurons via dopECR, we performed the following experiments. Firstly, to confirm that DopECR is indeed expressed in P1 neurons, we used an intersectional strategy to label the overlapping neurons between DopECR-GAL4 and FruLexA or R71G01-LexA and found that the intersectional neurons included the male-specific P1 neurons (Fig. 3D). Secondly, To gain direct evidence that 20E activates P1 neurons, we performed ex vivo calcium imaging on P1 neurons. The calcium level of P1 neurons exhibited a dosage-dependent increase with the concentration of 20E from 10−1 nM to 10−3 nM, a range comparable with the physiological condition (Fig. 3E and Supplementary Fig. S2F) 18. In contrast, when we knocked down DopECR using RNAi in P1 neurons, 20E could no longer activate P1 neurons (Fig. 3F and Supplementary Fig. S2G). Thus, we demonstrated that 20E is released by the male reproductive system during priming, activating P1 neurons through its receptor dopECR. Thus, we have demonstrated that 20E released from the reproductive system can act on the brain to regulate mating drive.

A brain-gonad axis controls 20E released from the male reproductive system
We next asked how the 20E release from the male reproductive system was controlled during the sexual priming. As an integrative center in mating behavior, P1 neurons are actively involved in the priming stage and are likely to play an important role in regulating the activity of the male reproductive system. Here, we found that thermo-genetically activating P1 neurons with dTPRA1 for 10 min could induce 20E release from the male reproductive system without any sexual behavior (Fig. 4A), suggesting that P1 neurons control the secretion of 20E from the male reproductive system during priming.

We then investigated the neural pathway that links P1 neurons and the male reproductive system in the flies. Four male-specific Crz neurons and their downstream CrzR neurons located in the ventral nerve cord (VNC) are responsible for male ejaculation 28. CrzR neurons projected to the male reproductive system with elaborate arborizations 28, suggesting that Crz and CrzR neurons may trigger 20E emission during sexual arousal. To test this notion, we blocked Crz neurons using the inwardly rectifying potassium channel kir2.1 and measured the levels of 20E in the male reproductive system after priming. In this case, we found that priming-induced 20E emission was blocked (Fig. 4B). To rule out the involvement of the Crz neurons in the brain, we specifically targeted brain Crz neurons by suppressing Crz-GAL4 in the VNC with tsh-GAL80 (Fig. 4C). Blocking brain-specific Crz neurons did not interfere with 20E emission during courtship (Fig. 4D), suggesting that Crz neurons in the VNC exclusively control 20E emission. To further confirm the functional connection between P1 neurons and Crz neurons, we performed Ca2+ imaging on VNC Crz neurons with the calcium sensor GCaMP7s. The Ca2+ level in Crz neurons was largely increased when we used ATP to activate P1 neurons bearing P2X2 (Fig. 4F), suggesting that VNC Crz neurons are downstream of P1 neurons.

A neuropeptide pathway mediates the descending control of 20E emission
How is the activation from the P1 neuron during priming relayed to VNC Crz neurons? Drosulfakinin (DSK) neurons, which receive direct synaptic transmission from P1 neurons, are known to act downstream of P1 neurons to regulate mating behaviors 29,30. Two putative receptors of Dsk peptides, CCKLR-17D1 and CCKLR-17D3 have been identified. We found that selectively knocking down CCKLR-17D3 but not CCKLR-17D1 with RNAi in Crz neurons significantly suppressed 20E emission during courtship behavior (Fig. 5A), indicating that DSK and its receptor CCKLR-17D3 are indispensable for 20E emission. We then examined the CCKLR-17D3 expression in Crz neurons with a knock-in GAL4 into the CCKLR-17D3 locus (17D3 GAL4) 29 and the Crz antibody. Two out of the four male-specific Crz neurons in the abdominal ganglion (AbG) of the VNC expressed CCKLR-17D3 (Fig. 3B), consistent with the single-cell RNA sequencing results from FCA (FLY CELL ATLAS). Furthermore, stimulation of DSK neurons with ATP elicited a Ca2+ response in all four Crz neurons in the AbG (Fig. 3C). While only two Crz neurons express CCKLR-17D3 and are the direct downstream of DSK neurons, mutual activation between the four Crz neurons could trigger the synchronized activation of all four Crz neurons 31. Thus, the DSK/CCKLR pathway bridges P1 neurons and Crz neurons to control the emission of 20E. However, we did not rule out the possibility that the activation of the DSK/CCKLR pathway could result in the biosynthesis of 20E in EB. This scenario is likely, considering that the CCKLR may elicit a downstream cascade to boost the biosynthesis of 20E. Up to this point, we have identified the two-directional communication along the brain-gonad axis during sexual priming. During sexual priming, this axis controls the release of 20E from the male reproductive system. As feedback, 20E activates P1 neurons to promote the males’ mating drive, as reflected by the fact that males became less picky about mates.

Males’ sexual arousal is correlated with their reproductive strategy
What’s the biological significance of the priming-induced mating drive elevation? Male animals display lower mating intensity toward aged females, probably due to the lower fecundity of those mates. It is conceivable that the mating drive levels of males are determined by the expectation for the reproductive outcome of their mates. It has been well demonstrated that aged females laid significantly fewer eggs and produced markedly fewer progeny than optimal mates 32. Thus, we speculated that the increased mating drive of males to sub-optimal mates (aged females) may result from an increased anticipation of the reproductive outcome when mated with aged females. Indeed, we found that aged females mated with males who experienced 10 min priming laid more eggs on the first day after mating than those mated with naïve males (Fig. 6A and Supplementary Fig. S3A). Specifically, it primarily affects the initial phase of egg-laying after mating. This result suggest that multiple factors may influence female fecundity, and the increased mating plug from males induced by priming is just one of them. Nevertheless, as females lay the most eggs in the first day after mating and are more likely to mate with other males later on, this initial increase may be important for the number of this males’ offspring. Additionally, the larger mating plug may be beneficial for the males in a competitive environment where tandem matings usually occur.

To investigate how primed males enhanced the reproductive outcomes of the sub-optimal mates, we quantified the different reproductive strategies of males between optimal and sub-optimal mates. We first examined the number of sperms transferred to the females during mating. By visualizing sperms using protamine-GFP, we observed that both optimal and aged females processed a similar number of sperms in their uterus immediately after ejaculation (Fig. 6B and Supplementary Fig. S3B), indicating that males transferred a similar number of sperms regardless of the states of their mates. Interestingly, aged females stored fewer sperms in their seminal receptacle compared with optimal females 8 h after mating, which may account for the reduced egg number (Fig. 6C and Supplementary Fig. S3C).

Males with priming invest more mating plug in sub-optimal mates
Despite around a thousand sperms being transferred by the males during mating, as few as 30-40% of them are stored in mated females 33. The mating plug from the ejaculates acts as a barrier to prevent sperm loss and facilitates sperm storage in mated females 34,35. Mating plugs undergo a phase transition in the female reproductive tract to form a barrier against sperm loss 35. In addition, mating plugs enhances sperm competitiveness by subsequent competing males 36,37. The mating plug’s comprehensive functionality prompts questions about its impact on sperm movement and its activation of reproductive tract stretch receptors.

Given that the sperm number transferred to the aged females was not reduced, we reasoned that a defective mating plug may cause sperm loss after mating in the aged females. The mating plug can be visualized with PEBme, a major protein component with auto-fluorescence under UV light 34. We observed that the mating plug in the uterus of aged females is small after ejaculation (Fig. 6D), suggesting that males reduce their allocation of the mating plug to the aged females. Mated females expel both the mating plug and the majority of sperm from their uterus several hours after mating 33. We found that the aged females ejected the mating plug faster than the optimal females (Fig. 6E), likely due to the small size of the mating plug. Thus, males make different reproductive investment strategies between optimal and sub-optimal mates by adjusting mating plug allocation.

We then found that males after priming 10 min allocated more mating plug to the aged females (Fig. 6F), indicating that males increase their reproductive investment to aged females when they are at high mating drive state.

In parallel, we observed a smaller size mating plug in aged female mice after mating with a naïve male (Fig. 6G). Quantifying mating plug weight, the mating plug in aged female mice is lighter than in optimal female mice (Fig. 6H). In addition, the weight of the mating plug is not correlated with the weight of the female or the male, or the female receptivity index (Fig. S3D–F). Then, we observed that male mice experienced priming increased their mating plug allocation to aged females (Fig. 6G, H). Taken together, we can conclude that males with priming invest more mating plug in sub-optimal mates across taxa, making males less picky.

Ejaculatory bulb emission contributes to mating plug allocation
To study how the male mating drive affects mating plug allocation, we sought to elucidate the neural basis underlying mating plug emission during sexual priming. The mating plug is mainly composed of proteins secreted by the ejaculatory bulb (EB) of the male reproductive system. We proposed that sexual priming triggers the emission of EB, e.g., proteins secreted to the lumen, through the brain-gonad axis, and the increase of mating plug allocation on sub-optimal mates avoids the waste of prepared reproductive materials. To test it, we dissected the male flies and examined the EB size after they were primed for 10 min (Fig. 7A). The EB size of the primed males was larger than the naive males (Fig. 7B), due to the accumulation of mating plug proteins in the EB lumen during courtship. These results suggest that mating plug proteins are secreted to the EB lumen before copulation, a process resembling the emission of mammals, e.g., the preparation of the contents in the seminal vesicles 38. Thus, primed males increase their mating plug allocation to aged females to avoid wasting prepared reproductive materials.

On the other hand, a prepared mating plug stored in males for a long time may be potentially harmful for males’ reproductive organs. To test this notion, males who experienced priming were single-housed, and their EB was examined. We observed significant protein precipitation and aggregation in EB 11 days after priming, compared with 1 day (Supplementary Fig. S4A, B). The major component of the mating plug is PEBme, which is transferred from a liquid state in males to a gel-like state in mated females 39. We then observed that purified PEBme precipitates and aggregates in vitro with increasing concentrations ranging from 5 μM to 60 μM (Supplementary Fig. S4C), indicating that PEBme may form aggregation when stored in EB for a long time. We thus proposed that high concentration PEBme may form precipitation and aggregation inside the lumen of males’ EB, which would block emission and ejaculation. By expelling the prepared mating plug, primed male flies and mice may be able to maintain the normal function of the EB.

The brain-gonad axis coordinates male reproductive investment
We then investigated whether the P1-DSK-Crz pathway also controls EB emission. Males with P1 neurons blocked failed to expand their EB after they were primed with optimal females (Supplementary Fig. S5A). Furthermore, activating P1 neurons with dTPRA1 for 10 min could induce EB enlargement without any sexual experience (Fig. 7C). These results indicate that P1 neurons control the EB emission to modulate males’ reproductive material allocation.

We found that CrzR neurons projected to the male reproductive system with elaborate arborizations (Supplementary Fig. S5B), suggesting that Crz neurons may also trigger EB enlargement during sexual priming. We found that silencing Crz neurons in the VNC blocked EB enlargement during priming (Fig. 7D). To exclude the effect of Crz neurons in EB enlargement, we used tsh-gal80 specifically labeled Crz neurons in the brain and we found these neurons are not involved in EB enlargement during courtship behavior (Fig. 7C). Meanwhile, CCKLR-17D3 but not CCKLR-17D1 in Crz neurons regulates males’ mating plug allocation (Fig. 7F). The reduced mating plug size of ΔCCKLR-17D3 mutant males could be rescued by expressing CCKLR-17D3 with Crz-GAL4 (Supplementary Fig. S5C). Thus, the brain-gonad axis controls EB enlargement during sexual priming via the P1-DSK-Crz pathway. This pathway conveys prior sexual experience into a stronger mating drive to boost reproductive output and probably also to avoid the waste of the prepared mating plug materials.

Methods
We confirm that our research complies with all relevant ethical regulations. All experimental procedures in this study were performed in accordance with the Tsinghua University guidelines and animal protocols, which were approved by Tsinghua University Institutional Animal Care and Use Committee (IACUC).

Fly experiments
Animals. Fly strains utilized in this study were procured from the Bloomington Drosophila Stock Center (BDSC) or other reputable laboratories. Flies were maintained on a standard medium under controlled environmental conditions, including a temperature of 25°C, 60% relative humidity, and a 12 h light–dark cycle. Exceptions were made for thermo-activation and thermo-inactivation experiments, during which flies were subjected to an 18°C environment. w1118 was used as a wild-type control and aged female (35–45 d old). All assays were completed with 3–7 days flies except the RNAi experiment and rescue experiment, which used flies aged 10–16 days. The sub-optimal female refers to the aged female if not otherwise mentioned. All male flies were raised single-housed. For a comprehensive list of fly strains used in this study, please refer to the Supplementary Materials (see KRT).

Courtship and copulation assays
Courtship assays were conducted using cylindrical courtship chambers with dimensions of 10 mm in diameter and 3 mm in height. These chambers provided a controlled environment for observing courtship behaviors. The experiments were carried out at a temperature of 25°C with 60% relative humidity. Unless otherwise noted, a male fly was paired with a w1118 virgin female fly in each chamber. The courtship assays were videotaped to allow for detailed analysis and manual scoring of courtship behaviors. The courtship index was manually scored as the fraction of time during which the male fly exhibited courtship behaviors. These behaviors included orientation, following, single-wing vibration, attempted copulation, and copulation. The scoring was performed within a 5 min window, initiated once the male fly commenced courtship activities.

Priming assays
Before the assays, the female flies were anesthetized using CO2 to facilitate handling. The genitalia of the female flies were carefully sealed using nail polish. This procedure aimed to prevent copulation during the primed-courtship assays. Following genital sealing, the female flies were allowed a recovery period of 2 h and this recovery time took place in individual food tubes. After the recovery period, the prepared female flies were utilized in 10 min priming assay. During these assays, individual male flies were introduced to the chambers.

Courtship competition assay
Courtship competition assay was carried out in a chamber with dimensions of 10 mm in diameter and 3 mm in height. All w1118 female flies were collected within 2 h after eclosion and housed in groups. Male flies were collected and singly housed right after eclosion. Female flies aged for 4–6 days were briefly anesthetized on ice and introduced into the recording chamber to recover for at least 2 h. Two competing male flies aged for 4-13 days were introduced into the chamber simultaneously. To identify each genotype, the flies in either the control group or experimental group were marked with a dot on their wings.

20E ELISA
The hemolymph samples for each experiment were collected from 20 adult flies. 20E are extracted by adding 3 volumes of chilled methanol (75% of the total volume) and vortexing for 30 s. The solution was centrifuged at 9391 × g for 10 min at 4°C. Supernatant was carefully aspirated without disturbing the pellet, and the pellet was dried down completely using a centrifugal concentrator at 30°C for 2-3 hrs. The dried powder/pellet can be stored at −20°C until assayed or can be dissolved in a minimum of 125 μL ELISA buffer to immediate quantification. A 50 μL sample was used to detect the levels of 20E using an enzyme immunoassay kit (ACE Enzyme Immunoassay).

The central neural system, gut, and reproductive system samples were collected from 10 adult flies. The dissection was performed in a dissection buffer (PBST: 0.015% Triton X-100 in 1x PBS. The collected samples were ground in methanol at 4°C to extract 20-hydroxyecdysone (20E), a hormone. The grinding process was facilitated by sonication, performed five times for five seconds each at a frequency of 20 KHz. Following sonication, the samples were centrifuged at 10,000 × g for 20 min at 4°C. The resulting supernatant, containing the extracted 20E, was carefully collected. The collected supernatant was subjected to evaporation until complete dryness. The dried pellet was reconstituted in 125 μL of ELISA buffer. This step aimed to ensure the solubility of the extracted 20E for subsequent analysis. The pellet was dissolved in 125 μL ELISA buffer. A 50 μL sample was used to detect the levels of 20E using an enzyme immunoassay kit (ACE Enzyme Immunoassay).

20E administration
For 20E supply, flies first underwent a starvation period lasting 4 to 6 h. A 25 mM stock solution of 20E in ethanol was prepared for use. The final concentration used for administration was 3 mM. This concentration was achieved by diluting the 25 mM stock solution appropriately. The ethanol served as a solvent for 20E. Following the starvation period, flies were provided with the 3 mM 20E solution for 5 h.

Fecundity (egg-laying) assays
Each female fly was paired with a male in a food vial. The mating pairs were allowed to interact for a duration of 2 h. Mated females were subsequently identified by the presence of a mating plug. Confirmed mated females were then transferred onto a grape juice plate. The grape juice medium was prepared by dissolving 0.25% agarose in grape juice, resulting in a final concentration of 15% (v/v) agarose. This grape juice plate served as the substrate for egg laying. The mated females were given 24 h for egg laying on the grape juice plate. After this period, the total number of eggs laid by five mated females was counted. This count provided a measure of reproductive output within the given time frame.

Each female fly was paired with a male in a food vial for a duration of 2 h. Mated females were identified by the presence of a mating plug. Mated females were then transferred onto a new food vial. This food vial served as the substrate for egg laying. The food vial was changed every day. The number of eggs laid by each mated female was counted for 10 days.

Sperm counts
Female flies were allowed to mate with protamine-GFP males. The use of protamine-GFP males facilitated the visualization and identification of sperm cells through green fluorescent protein (GFP) labeling. Post-mating, the female reproductive tract and seminal receptacle were carefully dissected in a dissection buffer (PBST: 0.015% Triton X-100 in 1x PBS). The dissected female reproductive tract and seminal receptacle were mounted between two pieces of cover glass with two spacers of 0.18 mm. Confocal image stacks were acquired to visualize and manually count GFP-positive sperm heads. Two specific time points were considered for analysis: immediately after ejaculation to assess the number of sperm transported to the uterus, and 8 h post-mating to examine sperm storage in the seminal receptacle. It was noted that confocal imaging might not capture every sperm, particularly in deeper optical Z-sections, due to the thickness of the uterus. As a result, the sperm counts obtained from this procedure were consistent between samples but may be underestimated compared to previous reports.

Mating plug size and retention time measurement
To assess the size of the mating plug in the female uterus, the following steps were taken. Female reproductive tracts were dissected 2 h after ejaculation in a dissection buffer (PBST: 0.015% Triton X-100 in 1x PBS). The dissected female reproductive tracts were mounted between two pieces of cover glass with two spacers of 0.18 mm. Confocal images were acquired at 405 nm/440 nm excitation/emission to detect the auto-fluorescence of PEBme in the mating plug. This choice of wavelength allowed for the visualization of specific components within the mating plug. ImageJ, a widely used image analysis tool, was employed to analyze the projected area of fluorescence particles. This analysis provided quantitative data on the size of the mating plug.

For mating plug retention time measurement, UV light was used to detect the presence of mating plugs every half hour after ejaculation.

EB size measurement
EB also includes PEBme and auto-fluorescence, the secretions transferred to the female are stored in the lumen of EB. We used the same method described in the mating plug size measurement to dissect EB and analyze the projected area of the lumen in EB.

Thermo-genetics
Flies utilized in heat activation or inactivation experiments were raised in a controlled incubator set to a temperature of 18°C. All experiments, specifically heat activation or inactivation studies, were conducted at 30°C. This elevated temperature was chosen to induce specific physiological responses or conditions in the experimental flies.

Tissue dissection, staining, and imaging
Brains and VNC of 5–7 d old flies were dissected in dissection buffer (PBST: 0.015% tritonX-100 in 1x PBS) and fixed in 4% PFA at room temperature on a shaker for 30 min, then washed for 4 × 15 min in wash buffer (0.3% triton in 1x PBS). After this, the tissues were blocked in block buffer (1x heat-inactivated normal goat serum with 0.3% triton in 1x PBS) for 30 min at room temperature. The samples were then incubated with the primary antibody at 4°C overnight. The primary antibodies used were: Rabbit-GFP (1:500 dilution), Mouse-GFP (1:500 dilution), Chicken-GFP (1:500 dilution), and Mouse-nc82 (1:50 dilution). On the second day, tissues were washed for 4 × 15 min and then incubated in secondary antibodies. The secondary antibodies were all from Invitrogen and used at 1:200 dilution: Alexa Fluor 555 anti-Rabbit (1:200 dilution), Alexa Fluor 488 anti-Mouse (1:200 dilution), Alexa Fluor 647 anti-Mouse (1:200 dilution). After incubation, tissues were washed for 4 × 15 min. VNC and brains were mounted on a slide for imaging. An Olympus FV1000 microscope with 10X air lens or 20X air lens was used for confocal imaging.

Calcium imaging
The male flies were raised single-housed for 7 to 11 days, and the entire brain and VNC were dissected in a recording chamber filled with the AHL (103 mM NaCl, 3 mM KCl, 5 mM TES, 10 mM trehalose, 10 mM glucose, 26 mM NaHCO3, 1 mM NaHPO4, and 4 mM MgCl2, pH 7.25, 310 mOsm). 2 mM CaCl2 was added to the saline before use. Calcium imaging was performed using an Olympus BX51WI microscope with a 40× water immersion objective, an Andor Zyla camera, and a Uniblitz shutter. The Ca2+ indicator GCaMP7s was used to measure the Ca2+ signal. GCaMP7s was excited with 488 nm light, and the fluorescent signals were collected at 1 Hz. For P2X2 stimulation, 10 μl of 0.2 M ATP solution or AHL was loaded into the recording chamber to reach the final concentration of 1 mM. ROIs were manually selected with ImageJ. The fluorescent change was calculated as %Peak ΔF/F0 = (Fpeak − F0)/F0 × 100, where F0 corresponds to the average intensity of 20 frames of background-subtracted baseline fluorescence before ATP application and F corresponds to the highest fluorescence after ATP application.

For monitoring the activity of P1 neurons, brains were prepared as described above. Images were acquired on an Olympus FV1000 confocal microscope using a 20X Air lens. GCaMP7s signals were excited with 488 nm light, and green fluorescent signals were collected. All operation was automatically controlled by the FV10-ASW 3.0 software. Selected regions of interest (ROIs) of P1 neurons using the tdTomato channel and the GCaMP7s channel. ROI sizes were kept roughly the same between samples. The average fluorescence in each ROI was calculated with ImageJ. Normalized fluorescence was calculated as GCaMP7s/tdTomato.

For 20E stimulation, 20E dissolved in DMSO or AHL was loaded into the recording chamber to reach the final concentration of 10−1 nM to 10−3 nM. ROIs of P1 neurons were manually selected with ImageJ. The fluorescent change was calculated as %Peak ΔF/F0 = (Fpeak − F0)/F0 × 100, where F0 corresponds to the average intensity of 20 frames of background-subtracted baseline fluorescence before 20E application, and F corresponds to the highest fluorescence after 20E application.

PEBme expression and purification
The coding sequence of PEBme was amplified from Drosophila melanogaster and cloned into an expression vector pET-28b with 6×His tag. BL21 cells were transformed with the expression plasmids and plated on Ampicillin LB plates. Single clones were selected, sub-cultured in median volume flasks, and transferred to larger ones in LB medium with agitation on an incubated shaker (180 rpm at 37°C) until they reached an OD600 of 0.8. Then 0.5 M IPTG was added to induce protein expression, and culture continued at 220 rpm for 4 h.

Bacteria were centrifuged at 1503 × g and lysed in Native Lysis Buffer (150 mM NaCl, 20 mM Tris-HCl, pH = 7.5, 1 mM PMSF, Protein Inhibitor Cocktail 1:1000) and sonicated for 30 min (10 s on/10 s off, 180 W). After centrifugation at 20,000 × g for 40 min, the pellet was collected and resuspended in Denaturing Lysis Buffer (6 M Urea, 150 mM NaCl, 20 mM Tris-HCl, pH = 7.5, 1 mM PMSF, Protein Inhibitor Cocktail 1:1000). Purification of His-tagged PEBme was performed by Ni-affinity chromatography. After centrifugation at 20,000 × g, the supernatant was collected. Purification of His-tagged PEBme was then performed by Ni-affinity chromatography. The purified proteins were further concentrated to 300 μM using centrifugal filters.

In vitro phase transition assays
The PEBme was diluted in a pH 7.5 buffer solution, with concentrations ranging from 5 μM to 60 μM. The phase transition products were added to glass-bottom 384-well plates and settled at room temperature for 30 min. Images were captured using an Olympus IXplore spinning disk confocal microscope.

Mice experiments
Animals. C57BL/6J mice were used for all studies. All care and experimental procedures involving mice were conducted following the approval of the Laboratory Animal Resources Center of Tsinghua University and Beijing Vital River Laboratory Animal Technology Co., Ltd. Ethical considerations were prioritized to ensure the well-being and humane treatment of the animals. Male and female mice of 8 weeks old were used in the experiments. Aged female mice between 25 to 27 weeks were used. Mice were housed in a controlled environment with a 12-h light and 12 h darkness cycle. The temperature was maintained between 20–22°C, and humidity levels were kept within the range of 30–70%. Standard mouse chow (Experimental mouse breeding feed with 6% fat content. Other ingredients are similar to those of 5K52 used by JAX) and water (Reverse Osmosis water, sterile) were provided ad libitum unless otherwise specified. Male mice were single-housed in their home cage, while female mice were group-housed, accommodating up to five individuals. Mice were euthanized by carbon dioxide inhalation in accordance with institutional guidelines.

Mice behavioral experiments and data analysis
All behavioral experiments were conducted 1 h after the onset of the dark cycle in a new cage. Before tests, mice were habituated in the behavior recording room for 1-2 h. Behavioral experiments were video-recorded using a Logitech camera for 30 min at a frame rate of 30 per second.

Male-female mating assays
Male mice were single-housed for at least 1 week. Female mice were estrous females, typically housed in groups, and were selected for high receptivity. Before the experiment, female mice were introduced into a new cage, and male mice were introduced into the cage after approximately one hour.

Sexual priming behavior assays
Individually housed male mice were primed by an estrous female before the assay. In the priming-mating assay, males were allowed to interact with females for up to 5 times intromission, and were separated before ejaculation. Intromissions longer than 15 s were interrupted to prevent early ejaculation. Behaviors of the primed male mice with another estrous female were recorded.

Testosterone injection mating behavior assays
Each male mouse was subcutaneously injected with 500 ng testosterone (dissolved in ethanol and diluted with corn oil) in the back, with a 30 min rest. Behaviors of the testosterone-injected mice when exposed to estrous female mice were recorded.

Data analysis
We used BORIS to analyze male mice mating behavior, including sniffing, mounting, intromission, and ejaculation. The onset of about of sniffing was defined as the moment when the male’s nose touched any part of the female’s body and culminated when the male stopped making contact with the female. The onset of about of mounting was defined as the moment when the male climbed over a female and began rapid, shallow lower-body thrusting, and terminated when the male was no longer over the female. During about of mounting, the male could gain intromission, which was evident from the male’s thrusting movements becoming deeper and slower. The ejaculation was marked by a specific series of events in which an intromitted male began to shake for a few seconds, then fell on his side, then became temporarily immobilized even when the female moved away, and then stood up again to clean himself.

To measure a male’s latency to mounting, we calculated the time period between male entry and the first attempt to mounting.

To measure a male’s mating intensity, we calculated the fraction of time that a male spent sniffing, mounting, and intromission behavior in the ejaculation latency.

To measure a male’s female receptivity index, we calculated the fraction of time that a male spent intromission behavior in mounting behavior.

Weighing mice mating plug
After each mating session, which lasted for 3–8 h, female mice were selected for further analysis. Female mice were anesthetized using avertin at a dosage of 0.15 mL per gram of body weight. Avertin, a commonly used anesthetic, was administered to ensure the mice were under anesthesia during the mating plug retrieval process. The mating plug was obtained from the vagina of anesthetized female mice. To facilitate the retrieval, a blunt wire was used to gently remove the mating plug from the reproductive tract. The retrieved mating plug was weighed using a precise scale. After weighing, the mating plug was stored at 4°C. This storage condition helps maintain the integrity of the mating plug for further analysis.

Testosterone ELISA
The testosterone from each mouse was extracted from 200 μl of serum. Firstly, 5 times the sample volume of diethyl ether was added and thoroughly mixed with a vortex. After allowing the layers to separate, 90% of the ether (upper layer) was transferred into a clean test tube using a pipette. This extraction process was repeated three more times. The combined ether extracts were then evaporated under a gentle stream of nitrogen. The resulting extract was dissolved in 500 μl of ELISA buffer. A 50 μL sample was used to measure the levels of testosterone using an enzyme immunoassay kit (ACE Enzyme Immunoassay).

Quantification and statistical analysis
GraphPad Prism 8 software was employed for statistical analysis. Error bars in all figures represent the Standard Error of the Mean (SEM). For all analyses, statistical notations are as follows: *p<0.05. **p<0.01, ***p<0.001, ****p<0.0001. No sample size estimation, inclusion and exclusion of any data or subjects were conducted in this study.

Normality Tests, Small Sample (n<8): Shapiro-Wilk test was utilized for normality testing. Large Sample (n≥8): D’Agostino & Pearson test was used for normality testing.

Comparison Tests, Parametric Datasets (Two Groups): Two-tailed unpaired t test was employed to evaluate significance. Nonparametric Datasets (Two Groups): The Mann-Whitney test was used for evaluating significance. Parametric Datasets (Three Groups): One-way ANOVA was employed for evaluating significance. Correlation Test: A correlation test was performed to assess the correlated degree between the weight of the mating plug and the weight of the female, male, or female receptivity index.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The submitted research article provides a mouse/ftuit-fly comparative analysis on the involvment od sex steroids (testosterone in mice and 20-hydroxyecdysone in fruit-fly) on male mating drive and reproductive strategy. A circuitry involving P1 neurons, 20-hydroxyecdysone, Dsk neurons, and Crz/CrzR neurons in the regulation of mating drive has been reported flies.\nThis approach is really good and original; presented results are well interpteted; the article is scienfically sound and methodologically well prresented.\nNevertheless, result section is quite difficult to follow due to the mixed order of figure panels and a large set of data presented as supplementary.\nSuggestion: Within the main text Figure panels have to be described in alphabetical order and not jumping from panel A to panel F ect. Too many information are presented as supplementary data.The authors surely made this choice to present a compative analysis between fruit-fly/mouse, but for the readers it not easy to jump from a panel to an other and to the very large amount of data presented as supplementary.", "reviewer_3": "Li et al. argue that sexual priming makes male flies and mice more willing to court older females. They propose a neuroendocrine pathway that controls this priming in flies, running from P1 neurons to Dsk neurons in the brain, to Crz neurons of the VNC, to 20E producing cells in the male reproductive system, which then signal back to P1 neurons in the brain. The authors assert that increased sexual behavior following priming avoids waste of reproductive resources prepared during priming and keeps the male reproductive tract functional by preventing potential obstruction due to precipitation of unutilized reproductive fluids. There are some interesting and believable findings in this paper, but they are intermingled with many weaker results and contorted logic, making the overall pathway and major conclusions difficult to believe.\nFigure 1: In this figure the authors work to establish the effects of sexual priming on subsequent selectivity for a mating partner. There is some immediate confusion because the text indicates that one male fly is asked to choose between a young and old fly, but the methods section says that males are placed individually with either a young or old fly, and the figures generally seem to be more in line with the 1-on-1 assays. In any case, the effect is somewhat surprising and appears generally reproducible—though I believe that the small effect size and large inter-animal variability lead to problems in the authors’ mechanistic work.\nIn mice, priming has long been established, but to my knowledge priming (usually scored as a reduced latency to mating behaviors) has not been shown to specifically increase the sexual behavior toward older females. The data in this paper do not convince me that this is true. The mating intensity index in mice seems arbitrarily derived: sniffing, mounting, and intromission are given equal weight and the ejaculation latency does not get factored in. It is therefore not clear to what extent their index reflects actual changes in the male’s sexual behavior toward older female mice. In fact, the index is construed such that it cannot detect the standard, well-known priming effect, because the “mating intensity index” towards “optimal” females does not change with priming.\nFor example, when comparing the ethograms in Fig. 1H and K, the latency to ejaculation looks substantially reduced for primed compared to naïve males both in the optimal and aged group. Despite the mating intensity index being the same (0.4) for naïve male+optimal female and primed male+optimal female (Fig. 1I, L) the latency to ejaculation (which seems like a good readout of the intensity of a mating, too) is a lot shorter in the ethograms shown, suggesting an increase in mating intensity caused by priming even towards optimal females, which is what is classically thought of as priming and is not reflected in the mating intensity index.\nFigure 2: Here the authors find decreased ecdysone (flies) and testosterone (mice) following priming. The data look strong but the interpretation of the fly data is strange and forced. The authors argue that ecdysone levels are decreased in primed animals because it is being released…so it is actually increased. Much more convincing data would be required to make this point, including seeing ecdysone levels being increased in the hemolymph, as is shown for testosterone in mice. The authors then experimentally increase ecdysone and testosterone and report selective increases in sexual behavior towards aged females. This result is more believable in flies than in mice, for reasons outlined above.\nFigure 3: Now the authors switch to a mechanistic investigation in flies, arguing that the DopEcr receptor transduces the ecdysone signal in P1 courtship neurons. Here the small magnitude and high variance of the behavioral priming effect becomes a big issue. The authors find no statistical difference between primed and unprimed flies in which DopEcr has been knocked down from P1 neurons. But the non-primed courtship is slightly higher in this genotype and the primed control moves down ~10% to achieve the statistical effect. Given the spread of the data, this effect could easily be due to chance. In any case, these experiments show that this readout of priming would require many, many more flies to produce a convincing effect (because of small effect size and extreme variability (0.0 to 0.7 unprimed; 0.15 to 0.8 primed with a sample size of only 10-12).\nI have mixed feelings about the imaging in this figure. I do not believe the baseline activity measurements at all, given the very, very modest effect compared to the spread of the data. A lack of effect would not be surprising because P1 is not thought to be basally active and these are explant preparations. I was more convinced by the response to experimenter-supplied ecdysone, and the requirement for DopEcr for this effect. But, if this were true, why would the high ecdysone titer (argued via low ecdysone in the reproductive tract) not cause an increase in courtship toward optimal targets as well as aged ones? Instead of addressing these issues as they arise, the authors accept their results at face value and the story moves along, occasionally presenting interesting results, but always surrounded by experiments with less solid conclusions and forced logic.\nAt the end of several thorough readings, I attempted to take stock of what I believed. Here is the complete list:\n1. Less courtship by male flies of older females.\n2. Exogenously supplied ecdysone can stimulate P1 neurons in an explant preparation.\n3. Ecdysone levels in the male reproductive tract are decreased by P1 activation.\n4. P1 activation causes an increase in ejaculatory bulb volume.\nThese are interesting findings for researchers in the field, but they do not constitute a coherent story.\nMinor points:\nFig. 6A: Assessing female fecundity for just 24h is not sufficient to claim these females are less fertile, since female flies lay eggs in response to mating for about 10-14 days.\nProblematic wording:\n“Sexual hunger” “optimal”, “enhanced libido”, and “sexual desire” all imply more knowledge of the workings of the male fly’s mind than the authors can reasonably assert.\n316-319: “To avoid the waste of the prepared mating plug materials, males increase their mating plug investment in aged females. This elevated reproductive investment boosts reproductive output and is the reason why males are no longer picky about mates” In addition to being very forceful about a conclusion that can only be speculative, this statement seems at odds with the fact that mating plug aggregation/precipitation is found only after 11 days.\n48-50: “However, the involvement of the brain-gonad axis in directly governing sexual behaviors during adulthood remains elusive” The authors must mean something more specific than is conveyed by this statement. It is obvious that both the brain and gonads are involved in sexual behaviors.\nConclusion:\nThis is a wide-ranging paper with some interesting findings obscured by confusing wording, lack of experimental detail, and over-interpretations. The conclusions are often out of line with the results in the paper and therefore I cannot support its publication in Nature Communications."}, "author_response": "Response to reviewers’ comments\nWe sincerely appreciate the positive and supportive evaluations provided by both reviewers of our\nwork, as well as their valuable suggestions for improving our manuscript. In response to their\ncomments, we have conducted numerous additional experiments and made corresponding\nmodifications to the figures and manuscript. With the incorporation of the new data, we have both\nconfirmed and expanded upon the conclusions drawn in the original submission. We are confident\nthat we have effectively addressed the concerns raised by the reviewers and have substantially\nenhanced the quality of the paper. The modifications and additions have been highlighted\nthroughout the manuscript. We hope the reviewers will now find our revised paper suitable for\npublication in Nature Communications.\nReply to reviewers:\nReviewer #1 (Remarks to the Author):\nThe submitted research article provides a mouse/ftuit-fly comparative analysis on the involvment\nod sex steroids (testosterone in mice and 20-hydroxyecdysone in fruit-fly) on male mating drive\nand reproductive strategy. A circuitry involving P1 neurons, 20-hydroxyecdysone, Dsk neurons,\nand Crz/CrzR neurons in the regulation of mating drive has been reported flies.\nThis approach is really good and original; presented results are well interpteted; the article is\nscienfically sound and methodologically well prresented.\nNevertheless, result section is quite difficult to follow due to the mixed order of figure panels and\na large set of data presented as supplementary. Suggestion: Within the main text Figure panels\nhave to be described in alphabetical order and not jumping from panel A to panel F ect.\nThanks for the suggestion. We have rearranged the order of the figure panels to improve the\nreadability of the paper.\nToo many information are presented as supplementary data. The authors surely made this choice\nto present a comparative analysis between fruit-fly/mouse, but for the readers it not easy to jump\nfrom a panel to another and to the very large amount of data presented as supplementary.\nWe arranged the data in the main and supplementary figures according to the following principles:\n1. When illustrating similar results, the data for w1118 is placed in the main figures and the data for\nCS is placed in the supplementary figures. 2. The main experiments supporting the conclusion that\npriming can lead to changes in male mate pickiness is placed in the main figures. While additional\nevidence such as priming increases male competitiveness are placed in supplementary. We have\nmodified the text so that the results are easier to follow.\nReviewer 3:\nLi et al. argue that sexual priming makes male flies and mice more willing to court older females.\nThey propose a neuroendocrine pathway that controls this priming in flies, running from P1\nneurons to Dsk neurons in the brain, to Crz neurons of the VNC, to 20E producing cells in the\nmale reproductive system, which then signal back to P1 neurons in the brain. The authors assert\nthat increased sexual behavior following priming avoids waste of reproductive resources prepared\nduring priming and keeps the male reproductive tract functional by preventing potential\nobstruction due to precipitation of unutilized reproductive fluids. There are some interesting and\nbelievable findings in this paper, but they are intermingled with many weaker results and contorted\nlogic, making the overall pathway and major conclusions difficult to believe.\nFigure 1:\n1, In this figure the authors work to establish the effects of sexual priming on subsequent selectivity\nfor a mating partner. There is some immediate confusion because the text indicates that one male\nfly is asked to choose between a young and old fly, but the methods section says that males are\nplaced individually with either a young or old fly, and the figures generally seem to be more in\nline with the 1-on-1 assays. In any case, the effect is somewhat surprising and appears generally\nreproducible—though I believe that the small effect size and large inter-animal variability lead to\nproblems in the authors’ mechanistic work.\nWe are sorry for the confusion. All of the courtship experiments were one-on-one assays. We have\nchanged the sentence “For flies, w1118 and CS males were allowed to choose freely between virgin\nfemales at two different stages, optimal (5~7d old, with highest fecundity) and aged (35-45d old,\nwith lower fecundity).” to “For flies, w1118 and CS males were allowed to court with either optimal\n(5~7d old, with highest fecundity) or aged (35-45d old, with lower fecundity) virgin females.”\nFurthermore, we performed courtship competition assay and found that sexual priming could\nincrease the males’ mating drive to optimal females as indicated by a higher competitiveness to\nnaïve males, although this increase was not evident in the courtship index assay (Figure S1D).\n2. In mice, priming has long been established, but to my knowledge priming (usually scored as a\nreduced latency to mating behaviors) has not been shown to specifically increase the sexual\nbehavior toward older females. The data in this paper do not convince me that this is true.\nThis’s a very helpful suggestion. We analyzed the time from sexual behavior initiation to mounting\nbehavior and found this latency is shorter in primed males compared to optimal female (Figure\nS1H). Together with the data that priming induces stronger mating behavior towards older females,\nwe think sexual priming generally elevates male mice’ mating drive to all females.\n3, The mating intensity index in mice seems arbitrarily derived: sniffing, mounting, and\nintromission are given equal weight and the ejaculation latency does not get factored in. It is\ntherefore not clear to what extent their index reflects actual changes in the male’s sexual behavior\ntoward older female mice.\nIn the Methods, we noted that “To measure a male’s mating intensity, we calculated the fraction\nof time that a male spent on sniffing, mounting, and intromission behavior in the ejaculation\nlatency.” We agree that priming behavior is usually scored as a reduced latency to mating\nbehaviors. We thus analyzed the latency to mounting behavior of the male mice, as shown in point\n2 above.\n4, In fact, the index is construed such that it cannot detect the standard, well-known priming effect,\nbecause the “mating intensity index” towards “optimal” females does not change with priming.\nBased on the literature search of priming on mice, we found that many of them used different\nmethods for mating intensity analysis, and they also used different priming assays [1, 2]. In this\nstudy, we used the similar method to calculate the mating intensity of mice and flies to facilitate\ncross-species comparison. In flies, this method for calculating mating intensity has been widely\nreported in a line of previous studies[3, 4]. In addition, we found that the mounting latency is\nshorter in primed males to optimal female (Figure S1H) as shown in point 2, although the mating\nintensity index did not change based on the current method.\n5, For example, when comparing the ethograms in Fig. 1H and K, the latency to ejaculation looks\nsubstantially reduced for primed compared to naïve males both in the optimal and aged group.\nDespite the mating intensity index being the same (0.4) for naïve male+optimal female and primed\nmale+optimal female (Fig. 1I, L) the latency to ejaculation (which seems like a good readout of\nthe intensity of a mating, too) is a lot shorter in the ethograms shown, suggesting an increase in\nmating intensity caused by priming even towards optimal females, which is what is classically\nthought of as priming and is not reflected in the mating intensity index.\nThanks for the valuable suggestion. The reduced latency to ejaculation indeed suggests that\npriming have a positive effect on the mating intensity of the male mice to optimal females,\nalthough it not evident from mating intensity index. In the new Figure S1H, we show that the\nmounting latency is reduced in primed male mice to optimal females. A similar change can be seen\nin males with testosterone injection. These results are consistent with the reduced latency to\nejaculation. In the current study, we did not further analyze the ejaculation latency changed by\npriming, because we focus on the mechanisms of priming induced mating drive change in the pre-\nejaculatory stage.\nFigure 2:\n1, Here the authors find decreased ecdysone (flies) and testosterone (mice) following priming. The\ndata look strong but the interpretation of the fly data is strange and forced. The authors argue that\necdysone levels are decreased in primed animals because it is being released…so it is actually\nincreased.\nThis is a critical question. We speculate that 20E released by the reproductive system corresponds\nto the increase of 20E level in hemolymph of flies, which is supported by the following evidence\nand experiments. First, the gene dare encodes the Drosophila homolog of vertebrate adrenodoxin\nreductase (AR) and is essential for 20E synthesis. We detected dare-GAL4 expression in many\ntissues such as the intestine and male reproductive system (Fig S2A), suggesting that these tissues\nare capable of secreting 20E. Secondly, ELISA analysis confirmed the presence of 20E in the gut\nand the male reproductive system but not in the nervous system of sexually naive males (Fig 2A\nand Fig S2B). In contrast, only the male reproductive system but not the gut showed a decrease in\n20E levels after 10 minutes of priming (Fig 2A and Fig S2B). Thus, these experiments indicate\nthat 20E was released from the male reproductive organs to the circulating hemolymph during\npriming.\nWe have also improved the experimental method of 20E extraction from the reproductive system.\nAs noted in the method, we used ultrasound to break down the tissues to make this result more\nreliable.\n2, Much more convincing data would be required to make this point, including seeing ecdysone\nlevels being increased in the hemolymph, as is shown for testosterone in mice. The authors then\nexperimentally increase ecdysone and testosterone and report selective increases in sexual\nbehavior towards aged females. This result is more believable in flies than in mice, for reasons\noutlined above.\nWe agree that detecting ecdysone levels directly in the hemolymph of flies would be more\nconvincing. We have thus conducted this measurement. The hemolymph from 20 flies was\nextracted and the 20E ELISA kit was used to detect the 20E level. We demonstrated that the levels\nof 20E circulating in the hemolymph were higher in primed males (Fig 2A), suggesting that 20E\nmay play a role in the priming phase.\nFigure 3:\n1, Now the authors switch to a mechanistic investigation in flies, arguing that the DopEcr receptor\ntransduces the ecdysone signal in P1 courtship neurons. Here the small magnitude and high\nvariance of the behavioral priming effect becomes a big issue. The authors find no statistical\ndifference between primed and unprimed flies in which DopEcr has been knocked down from P1\nneurons. But the non-primed courtship is slightly higher in this genotype and the primed control\nmoves down ~10% to achieve the statistical effect. Given the spread of the data, this effect could\neasily be due to chance. In any case, these experiments show that this readout of priming would\nrequire many, many more flies to produce a convincing effect (because of small effect size and\nextreme variability (0.0 to 0.7 unprimed; 0.15 to 0.8 primed with a sample size of only 10-12).\nThanks for the suggestion. We agree that increasing the sample size helps to produce a more\nconvincing conclusion. We increased the sample sizes for these experiments and the new results\nare now in Figure 3C. The sample sizes are now 17-19 for this experiment. D'Agostino & Pearson\ntest indicated that the data show the normal distribution. Two-tailed unpaired t-test was employed\nto evaluate the statistical significance. P value between A and B column is 0.0315. P value between\nC and D column is 0.0215. P value between E and F column is 0.7313 (all are shown in the Figure\nLegend).\n2. I have mixed feelings about the imaging in this figure. I do not believe the baseline activity\nmeasurements at all, given the very, very modest effect compared to the spread of the data. A lack\nof effect would not be surprising because P1 is not thought to be basally active and these are\nexplant preparations.\nThere are limitations of the explant preparations, although this method has been used by multiple\nstudies [5, 6]. However, this method can be useful to measure the prolonged state transition of P1\nneurons. Moreover, our conclusions are further justified with additional experiments, such as co-\nlabeling experiments, and behavior experiments.\nAdditionally, the activity of P1 neurons was measured as a ratio between GCaMP7s and tdTomato,\nfurther enhancing the credibility of the data. We also increased the sample sizes to 22-26 for these\nexperiments in Fig 3D and E. D'Agostino & Pearson test indicated that the data show normal\ndistribution. Two-tailed unpaired t-test was employed to evaluate the statistical significance of P1\nactivity between naïve and primed males. P value in R71G01>GCaMP7s is 0.0050. P value in\nR71G01> GCaMP7s;DopECR-RNAi is 0.3880.\n3, I was more convinced by the response to experimenter-supplied ecdysone, and the requirement\nfor DopEcr for this effect. But, if this were true, why would the high ecdysone titer (argued via low\necdysone in the reproductive tract) not cause an increase in courtship toward optimal targets as\nwell as aged ones?\nThanks for the comments. This point is related to Figure 1 point 1&2. The courtship index may\nnot fully reflect the activity of P1 neurons, as males’ exhibit very strong courtship to both young\nand older virgin females. Thus, we have performed courtship competition assay to demonstrated\nthat priming by 20E could indeed increase the males’ mating drive as they had a higher\ncompetitiveness in the assay (Figure S1F).\nMinor points:\nFig. 6A: Assessing female fecundity for just 24h is not sufficient to claim these females are less\nfertile, since female flies lay eggs in response to mating for about 10-14 days.\nThanks for the comments. We added the 10 days fecundity (egg-laying) assay (FIG S3A). The\nnumber of eggs laid by each mated female was counted for 10 days. 1 to 7 days after mating, the\ndifference numbers of eggs laid by optimal and aged females can be seen. Because of the dramatic\ndecline in egg-laying number in both groups during ageing, there is no differences can be seen\nafter 8 to 10 days. Moreover, we found that priming caused an increase in egg-laying of aged\nfemale, but the effect of priming only lasted for one day.\nProblematic wording:\n“Sexual hunger” “optimal”, “enhanced libido”, and “sexual desire” all imply more knowledge of\nthe workings of the male fly’s mind than the authors can reasonably assert.\nWe agree with these comments. We change all “Sexual hunger” state to “elevated sexual drive”\nstate, all “enhanced libido” to “enhanced mating drive” and “sexual desire” to “mating intensity”.\nThe optimal means the optimal fecundity. We used the optimal female to describe virgin and\nyoung (3-7 days) females which have a highest fecundity such that they are the “optimal” mates\nfor males.\n316-319: “To avoid the waste of the prepared mating plug materials, males increase their mating\nplug investment in aged females. This elevated reproductive investment boosts reproductive output\nand is the reason why males are no longer picky about mates” In addition to being very forceful\nabout a conclusion that can only be speculative, this statement seems at odds with the fact that\nmating plug aggregation/precipitation is found only after 11 days.\nWe have modified the sentence to “This pathway conveys prior sexual experience into stronger\nmating drive to boost reproductive output and probably also to make use the prepared mating plug\nmaterials.”\n48-50: “However, the involvement of the brain-gonad axis in directly governing sexual behaviors\nduring adulthood remains elusive” The authors must mean something more specific than is\nconveyed by this statement. It is obvious that both the brain and gonads are involved in sexual\nbehaviors.\nWe have changed the sentence to “However, the bi-directional communication of the brain-gonad\naxis in governing sexual behaviors during adulthood remains elusive.”\nReference:\n1. Zhang, S.X., et al., Hypothalamic dopamine neurons motivate mating through persistent\ncAMP signalling. Nature, 2021. 597(7875): p. 245-249.\n2. Zhou, X., et al., Hyperexcited limbic neurons represent sexual satiety and reduce mating\nmotivation. Science, 2023. 379(6634): p. 820-825.\n3. Zhang, S.X., et al., Hormonal control of motivational circuitry orchestrates the transition\nto sexuality in <i>Drosophila</i>. Science Advances, 2021. 7(25): p. eabg6926.\n4. Keleman, K., et al., Dopamine neurons modulate pheromone responses in Drosophila\ncourtship learning. Nature, 2012. 489(7414): p. 145-9.\n5. Zhang, S.X., D. Rogulja, and M.A. Crickmore, Dopaminergic Circuitry Underlying\nMating Drive. Neuron, 2016. 91(1): p. 168-81.\n6. Oh, Y., et al., A glucose-sensing neuron pair regulates insulin and glucagon in Drosophila.\nNature, 2019. 574(7779): p. 559-564."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors carried out all the suggested changes; they rerganized result section and also supplementary materials.", "reviewer_3": "The responses of the authors satisfy only one of my many concerns. This was my concern that a decrease of ecdysone in the reproductive organs meant higher circulating ecdysone. The authors now directly measure circulating ecdysone and find that it is increased after priming (Figure 2A), making me somewhat more confident in this conclusion.\nIn other cases the authors have simply restated their earlier experiments/interpretations (Figure 1, most of Figure 2, Figure 3 imaging). Or they have increased the sample size, which does not ameliorate my concern about small effect sizes (Figure 3 behavior). And in one case, a concern I raised about fecundity turned out to be true (no real change with priming). The authors decide to keep their misleading data in Figure 6A, while providing the more complete data that contradicts their hypothesis in the supplement (Figure S3A). And finally, when their own data don’t match their story (if priming increases ecdysone and ecdysone increases P1 and P1 increases courtship, why doesn’t priming increase courtship from ~0.5 in figure 1I?) the authors perform an experiment that doesn’t address the question.\nI understand that the authors may not agree with my criticisms, but I found it frustrating that in only one case were the concerns even directly addressed with new, useful information (data or explanation) and in no cases were the conclusions altered when additional justification was not provided (or when more thorough experiments led to a contradicting conclusion)."}, "author_response": "Point-by-point response to reviewer #3:\n1.The responses of the authors satisfy only one of my many concerns. This was my\nconcern that a decrease of ecdysone in the reproductive organs meant higher\ncirculating ecdysone. The authors now directly measure circulating ecdysone and find\nthat it is increased after priming (Figure 2A), making me somewhat more confident\nin this conclusion.\n2.In other cases the authors have simply restated their earlier\nexperiments/interpretations (Figure 1, most of Figure 2, Figure 3 imaging).\nThank you for the comments.\nRegarding Drosophila courtship related experiments in Figure 1 and 2, we performed\nthe following experiments. We added behavioral data from wild-type CS flies and\nreached the same conclusion as w1118 flies: 1. Male CS flies naturally pursue young\nfemales with higher courtship index than old females (Fig 1C). 2. Sexually primed CS\nmales show the similar courtship index to young and old females (Fig 1I). 3. 20E fed\nCS male flies showed similar courtship index to young and old females (Fig 2E).\nSimilarly, we found that there is a plateau effect of courtship index to optimal females,\nas we did not see an increase of courtship index of primed males to these females.\nTherefore, we further conducted competitive experiments of CS flies and found that\npriming or 20E feeding can indeed increase their mating drive which is not revealed\nby courtship index (Fig S1A-D). These data consistent with our previous findings on\nw1118 flies and suggested that sexual priming can increase mating drive independent of\ngenetic background.\nRegarding mice related experiments, we further analyzed the behavior of the mice as\nsuggested by reviewer3 to more clearly explain the effects of priming. We found that\nalthough the mating intensity index to optimal females did not further increase by\npriming or testosterone injecting, male mice primed or injected with testosterone\nshowed the reduced mounting latency to both optimal and aged female mice (Fig\nS1E). These results tell us mice primed or injected with testosterone still exhibit a\nhigher mating drive to both optimal and aged females.\nFor the imaging results in Figure 3, although a line of studies have used this\nexperimental explant method [1, 2], we also have the similar concerns with the\nreviewer that ex vitro measurements can fully reflect the neuronal activity in vivo, so\nwe moved this part of the data to supplementary data (Fig S2D and E). Thus, the\nconclusion that 20E is involved in priming by activating P1 neurons through DopECR\nis verified by courtship behavior experiments, courtship competition experiments,\nco-labeling experiments between P1 neurons and DopECR neurons, and calcium\nimaging experiments in 20E-activated P1 neurons in our paper. These data suggest\nthat P1 neurons play a role in sexual priming in a DopECR dependent manner.\n3.Or they have increased the sample size, which does not ameliorate my concern about\nsmall effect sizes (Figure 3 behavior).\nThank again for this comment. Firstly, we have further increased the sample sizes in\nFigure 3 behavior to improve the credibility of our results. The current sample sizes\nfor this experiment range from 30 to 36 and each group of data shows a clear normal\ndistribution. A two-tailed unpaired t-test has confirmed that there is a statistically\nsignificant difference between the data sets. Secondly, we added male fly courtship\ncompetition experiments to demonstrate that 20E and its receptor DopECR also affect\nthe enhancement of courtship competition triggered by sexual priming (Fig S2C).\n4.And in one case, a concern I raised about fecundity turned out to be true (no real\nchange with priming). The authors decide to keep their misleading data in Figure\n6A, while providing the more complete data that contradicts their hypothesis in the\nsupplement (Figure S3A).\nOur findings indicate that priming can lead to an increase of fecundity in females, but\nthis effect is limited to the first day post-mating. Specifically, it primarily affects the\ninitial phase of egg-laying after mating. This result suggests that multiple factors may\ninfluence female fecundity, and the increased mating plug from males induced by\npriming is just one of them. Nevertheless, as females lay the most eggs in the first day\nafter mating and are more likely to mate with other males later on, this initial increase\nmay be important for the number of this males’ offspring. Additionally, the larger\nmating plug may be beneficial for the males in the competitive environment where\ntandem matings usually occur. We have elaborated on this part in the Discussion.\n5.And finally, when their own data don’t match their story (if priming increases\necdysone and ecdysone increases P1 and P1 increases courtship, why doesn’t priming\nincrease courtship from ~0.5 in figure 1I?) the authors perform an experiment that\ndoesn’t address the question.\nWe think this is from the plateau effect of the courtship index induced by optimal\nfemales. We found that the mean value of the maximum courtship index was 0.5 for\nw1118 males, and 0.7 for CS males. After a series of literature review, we found that\nmany articles reported that the courtship intensity of w1118 male was lower than that of\nCS male, which was most likely caused by mutation of the White gene[3]. In addition,\ndifferent experimental courtship chambers and statistical methods also affect the value\nof courtship index. Meanwhile, when activating P1 neurons and fru+ neurons\ncontaining P1 neurons with dTrpA1, the male courtship index did not reach 100% [4,\n5], suggesting a maximum courtship index exists.\nIn our study, we observed that the male courtship index for optimal female courtship\nis already relatively high, thereby masking the potential positive impact of priming in\nthe experimental settings with courtship index. To further investigate this the role of\npriming on sexual drive, we conducted competitive experiments which revealed that\nprimed males exhibited higher mating competitiveness (Fig S1A-D). This finding\nsupports the conclusion that primed males exhibit an elevated mating drive.\nReferences:\n1. Zhang, S.X., D. Rogulja, and M.A. Crickmore, Dopaminergic Circuitry Underlying\nMating Drive. Neuron, 2016. 91(1): p. 168-81.\n2. Oh, Y., et al., A glucose-sensing neuron pair regulates insulin and glucagon\nin Drosophila. Nature, 2019. 574(7779): p. 559-564.\n3. Xiao, C., S. Qiu, and R.M. Robertson, The white gene controls copulation success\nin\nsuccess\nin Drosophila melanogaster. Scientific Reports, 2017. 7(1): p. 7712.\n4. Pan, Y., G.W. Meissner, and B.S. Baker, Joint control of Drosophila male\ncourtship behavior by motion cues and activation of male-specific P1 neurons.\nProc Natl Acad Sci U S A, 2012. 109(25): p. 10065-70.\n5. Wu, S., et al., Drosulfakinin signaling in fruitless circuitry antagonizes P1\nneurons to regulate sexual arousal in Drosophila. Nat Commun, 2019. 10(1): p.\n4770."}, {"round": 3, "reviewer_comments": {"reviewer_4": "This manuscript reports observations that sexual priming renders males less sexually discriminating of female quality in both the fly and rodent models. The authors then showed that priming also increases circulating levels of 20E in the fly and testosterone in the rodent. Interestingly, feeding 20E or injecting testosterone without sexual priming appears to increase sexual arousal. The authors then used molecular genetic analysis and optical imaging experiments to show that 20E activates P1 command neurons via DopEcR. Similarly, DSK neurons function downstream of P1 neurons to connect with CRZ neurons, which have previously been shown to control ejaculation duration. Aged females mated with primed had higher fecundity than those mated with non-primed males. In addition, primed males produce the larger mating plug and greater EB emission compared to non-primed males. This work reports an unknown role for P1 neurons and their downstream neurons in the transient elevation of 20E during sexual priming. However, it is unclear if and how some of these neurons stimulate the transient 20E release. In the later part of manuscript, the authors reported a role of priming and P1 and its downstream neurons in EB release. This part is more or less descriptive. The relationship between priming and EB emission is not fully explored. The relationship between 20E and increased EB emission remains unexplored. I found several data lacking proper control (I have listed a few). The manuscript has typos and confusing phrases that need some attention. Image quality and resolutions are not adequate. I offer the following comments, which I hope will help improve manuscript.\nThis study showed that sexual priming increased the amount of 20E in the hemolymph by 3-4 pg/fly, while simultaneously reducing the amount of 20E in the male reproductive system (MRS) by a similar amount. Thus, the authors used the degree of reduction in MRS 20E levels as an indicator of humoral 20E levels in males with altered P1-DSK-CRZ pathways. However, since several peptide neurons are known to regulate 20E biosynthesis, it is possible that some of these peptide neurons (i.e., DSK or CRZ neurons) affect 20E biosynthesis rather than 20E secretion. This is of particular concern because CRZ neurons directly arborize MRS. In the absence of direct evidence that CRZ neurons increase 20E secretion in the MRS, it is a leap of faith to assume that the P1-DSK-CRZ pathway regulates 20E secretion during sexual priming.\nThe authors have shown that the P1-DSK-CRZ pathway induces EB emission during sexual priming. Are 20E secretion and EB emission separate events? If not, is one a consequence of the other? Is EB the source of 20E? This needs a clarification and a plausible explanation.\nDoes 20E or priming make males indiscriminate toward optimal vs. aged females? The authors seem to imply this, but the data presented do not really show this. Does the choice experiment in which males discriminate between optimal and aged females work in this setting?\nWhat is the source of dare-Gal4 (no information in the M&M)? It is also important to show that dare-Gal4 positive cells in the male reproductive system (MRS) are the source of 20E secreted by the MRS. Are dare-positive cells arborized by CRZ neurons?\nDo DSK neurons in the brain project to the VNC to arborize where CRZ neurons are located? Is it possible to visualize synaptic connections between DSK neurons in the brain receiving inputs from P1 and CRZ neurons receiving inputs from DSK neurons?\nCan CRZ activation alone lead to 20E secretion, increased sex drive, and the (larger) EB emission?\nLine 292. \" We proposed that sexual priming triggers the emission of EB through the brain-gonad axis, and the increase of mating plug allocation on sub-optimal mates avoids the waste of prepared reproductive materials.\" Including this, the later parts of this manuscript are not clear about cause and effect. For example, does 20E lead to the greater investment (the greater amount of EB emission)? Or does greater investment lead to more 20E secretion and lead to increased sexual drive? Do authors propose a positive relationship between EB emission and 20E secretion? If so, how does it happen?\nThe relationship between the amount of EB released and the amount of ejaculate transferred to the female during copulation (i.e., the size of the mating plug) is not entirely clear. Does a male transfer the entire EB content to the female during a single copulation? If so, primed males with the greater amount of EB content will transfer the greater amount of ejaculate to their mates, regardless of the quality of their mate. Otherwise, does a male transfer some of the EB content and adjust the amount of EB content transferred based on the quality of his mate? I suggest to examine EB content after ejaculation to confirm that both primed and non-primed males have similarly depleted levels of EB content regardless of female quality.\nFig 6C: Fig. 6A shows that aged females mated with primed males lay more eggs than those mated with unprimed males. Therefore, it is required to compare the number of sperm in the seminal receptacle of aged females mated with males with and without priming.\nIt appears that aged females mated with primed males have larger mating plug. Are the authors claiming that the larger mating plug causes the females to expel the seminal receptacle more slowly and store more sperm as a consequence. If so, what mechanism does the author suggest?\nMinor Comments and Typos\n1. Confocal images do not have sufficient resolution\n- Fig 2: Confocal images with higher resolution.\n- Fig. S2A: The resolution of the images is too low to distinguish cells. In addition, the brain image is not confocal and it is difficult to tell which cells express GFP. The intestine also appears to express dare-Gal4, but it is not clear which area of the intestine is positive for dare-Gal4. It is also not clear which cells are the source of circulating 20E after priming.\n- Fig. 3D: Increasing the resolution of images to identify specific cells/neurons. What is the number of DopECR positive P1 cells that are positive for both DopECR and R71G01?\n- Fig. 4C. Show both CRZ-Gal4 images with and without tsh-Gal80 for comparison\n- Fig 5C: Stinger-GFP should be in the nucleus while anti-CRZ should be in the cytoplasm. However, both GFP and anti-CRZ appear in almost exactly the same area, which looks odd. The high resolution image is needed to resolve this issue.\n2. Additional controls are needed:\n- Fig 2D, E, F: Vehicle control is missing\n- Fig S2C: RNAi, Gal4 control is missing\n3. Line 223: \"Consistent with single cell RNA sequencing results from FCA (Fly Cell Atlas)\". Show relevant scRNA results and provide references.\n4. 10 min priming is sufficient to increase the courtship index measured for 10 min. Compared to the priming experiment, 5 hours of 20E feeding is simply too long. In addition, it is unclear how much 20E is circulating when males are fed 3 mM 20E for 5 hours. Any reference to support this experimental protocol?\n5. Figure S2F needs to include experimental protocol indicating the timing of 20E treatment and imaging.\n6. Figure 6F. Why not show the exact data points for this graph.\n7. Line 261. “by preventing subsequent competing males from mating” ? Incomplete sentence.\n8. Line 277. \" This increase eventually leads males less picky about mates.” This statement is confusing because the logic behind it is unclear.\n9. Does ‘the emission of EB’ mean the accumulation of the ejaculation-ready materials within the EB. To me, it sounds like the expulsion of materials from EB. To help readers understand, the authors need to clearly explain the meaning of ‘the emission of EB’ and its relationship with mating plug allocation earlier in the text.\n10. Line 83: \"Comparing the mating latency of primed or unprimed males to aged females in mouse experiments\" Does \"the emission of EB\" mean the accumulation of ejaculatory materials within the EB? To me, it sounds like the expulsion of materials within the EB. To help readers understand, the authors need to clearly explain the meaning of \"the emission of EB\" and its relationship to the allocation of mating plugs earlier in the text. Do ‘the emission of EB’ and ‘EB emission’ mean the same thing?.\n11. Line 297. A process similar to mammalian emission. Again, explain what \"the emission\" means in this context. Can it be more specific?\n12. The authors need to provide evidence for the statement in lines 312-314.\nTypos\nLine 82: then   than\nLine 85: cross tax   across taxa\nLines 168-169: “However… the activity of P1 neurons” is not needed.\nLine 277: “This increase … about mate” is out of context.\nFig 2D. X-axis label. ‘optiaml’   optimal\nCCKLR-17D3-GAL4(X); sp/cyo (Yufeng Pan). Is this correct genotype? CCKLR-17D3GAL4"}, "author_response": "Reply to reviewer #4\nREVIEWERS' COMMENTS\nReviewer #4 (Remarks to the Author):\nThis manuscript reports observations that sexual priming renders males less sexually\ndiscriminating of female quality in both the fly and rodent models. The authors then showed that\npriming also increases circulating levels of 20E in the fly and testosterone in the rodent.\nInterestingly, feeding 20E or injecting testosterone without sexual priming appears to increase\nsexual arousal. The authors then used molecular genetic analysis and optical imaging\nexperiments to show that 20E activates P1 command neurons via DopEcR. Similarly, DSK\nneurons function downstream of P1 neurons to connect with CRZ neurons, which have\npreviously been shown to control ejaculation duration. Aged females mated with primed had\nhigher fecundity than those mated with non-primed males. In addition, primed males produce the\nlarger mating plug and greater EB emission compared to non-primed males. This work reports\nan unknown role for P1 neurons and their downstream neurons in the transient elevation of 20E\nduring sexual priming. However, it is unclear if and how some of these neurons stimulate the\ntransient 20E release. In the later part of manuscript, the authors reported a role of priming and\nP1 and its downstream neurons in EB release. This part is more or less descriptive. The\nrelationship between priming and EB emission is not fully explored. The relationship between\n20E and increased EB emission remains unexplored. I found several data lacking proper control\n(I have listed a few). The manuscript has typos and confusing phrases that need some attention.\nImage quality and resolutions are not adequate. I offer the following comments, which I hope\nwill help improve manuscript.\nThis study showed that sexual priming increased the amount of 20E in the hemolymph by 3-4\npg/fly, while simultaneously reducing the amount of 20E in the male reproductive system (MRS)\nby a similar amount. Thus, the authors used the degree of reduction in MRS 20E levels as an\nindicator of humoral 20E levels in males with altered P1-DSK-CRZ pathways. However, since\nseveral peptide neurons are known to regulate 20E biosynthesis, it is possible that some of these\npeptide neurons (i.e., DSK or CRZ neurons) affect 20E biosynthesis rather than 20E secretion.\nThis is of particular concern because CRZ neurons directly arborize MRS. In the absence of\ndirect evidence that CRZ neurons increase 20E secretion in the MRS, it is a leap of faith to\nassume that the P1-DSK-CRZ pathway regulates 20E secretion during sexual priming.\nWe agree with the reviewer that the it’s possible that the P1-DSK-CRZ pathway may regulate\nthe biosynthesis of 20E, thus elevating the circulating level of 20E. At the current stage, we are\nnot able to distinguish the two possibilities. We have discussed this issue in the revised\nmanuscript.\nThe authors have shown that the P1-DSK-CRZ pathway induces EB emission during sexual\npriming. Are 20E secretion and EB emission separate events? If not, is one a consequence of the\nother? Is EB the source of 20E? This needs a clarification and a plausible explanation.\n20E secretion and EB emission are two separate events. However, the two events might be\nsynergistically triggered by the P1-DSK-CRZ pathway. The expression of the gene dare\nindicates that 20E is synthesized in different parts of the male reproductive organs, especially in\nEB and ejaculatory duct, suggesting that 20E could be released from not only EB but other part\nof the male reproductive organs.\nDoes 20E or priming make males indiscriminate toward optimal vs. aged females? The authors\nseem to imply this, but the data presented do not really show this. Does the choice experiment in\nwhich males discriminate between optimal and aged females work in this setting?\nIn Figure 1G-I, primed male showed a similar court intensity to optimal and aged females,\nsuggesting that priming make male flies less discriminative to the age of the female flies.\nWhat is the source of dare-Gal4 (no information in the M&M)? It is also important to show that\ndare-Gal4 positive cells in the male reproductive system (MRS) are the source of 20E secreted\nby the MRS. Are dare-positive cells arborized by CRZ neurons?\nThe dare-Gal4 was from Bloomington Drosophila Stock Center 79316. Based on Fig S5B, Crz\nneurons seem to extensively innervate EB, although it’s not clear whether dare-positive cells are\ndirectly arborized by CRZ neurons.\nDo DSK neurons in the brain project to the VNC to arborize where CRZ neurons are located? Is\nit possible to visualize synaptic connections between DSK neurons in the brain receiving inputs\nfrom P1 and CRZ neurons receiving inputs from DSK neurons?\nWu et at (https://www.nature.com/articles/s41467-019-12758-6) showed that DSK neurons have\nrich arbors in the VNC abg where CRZ neurons are located. In Fig 5D, we showed that the\nactivation of DSK neurons triggered calcium response in CRZ neurons, suggesting that they are\nfunctionally connected.\nCan CRZ activation alone lead to 20E secretion, increased sex drive, and the (larger) EB\nemission?\nWe were unable to test this as the activation of CRZ neurons triggers ejaculation.\nLine 292. \" We proposed that sexual priming triggers the emission of EB through the brain-\ngonad axis, and the increase of mating plug allocation on sub-optimal mates avoids the waste of\nprepared reproductive materials.\" Including this, the later parts of this manuscript are not clear\nabout cause and effect. For example, does 20E lead to the greater investment (the\ngreater amount of EB emission)? Or does greater investment lead to more 20E secretion and lead to\nincreased sexual drive? Do authors propose a positive relationship between EB emission and\n20E secretion? If so, how does it happen?\n20E secretion and EB emission are two separate events. However, it’s possible that these two\nevents may be linked biochemically as they happen in the same organ. In the current study, we\nfocus on the mechanism that the two events may be synergistically triggered by the P1-DSK-\nCRZ pathway.\nThe relationship between the amount of EB released and the amount of ejaculate transferred to\nthe female during copulation (i.e., the size of the mating plug) is not entirely clear. Does a male\ntransfer the entire EB content to the female during a single copulation? If so, primed males with\nthe greater amount of EB content will transfer the greater amount of ejaculate to their mates,\nregardless of the quality of their mate. Otherwise, does a male transfer some of the EB content\nand adjust the amount of EB content transferred based on the quality of his mate? I suggest to\nexamine EB content after ejaculation to confirm that both primed and non-primed males have\nsimilarly depleted levels of EB content regardless of female quality.\nThanks for the suggestion. We will examine these possibilities in future work.\nFig 6C: Fig. 6A shows that aged females mated with primed males lay more eggs than those\nmated with unprimed males. Therefore, it is required to compare the number of sperm in the\nseminal receptacle of aged females mated with males with and without priming.\nIn Figure 6F, we showed that the mating plug is larger in aged females mated with primed males.\nAs males transfer as many sperms to optimal and aged females, the different eggs laying in\nFigure 6C suggest that more sperms are preserved in aged females mated with primed males due\nto large mating plug.\nIt appears that aged females mated with primed males have larger mating plug. Are the authors\nclaiming that the larger mating plug causes the females to expel the seminal receptacle more\nslowly and store more sperm as a consequence. If so, what mechanism does the author suggest?\nMating plug serves as a physical barrier. The mating plug physically obstructs the reproductive\ntract, making it mechanically difficult for the female to expel the ejaculate. This larger plug acts\nas a more effective physical barrier.\nMinor Comments and Typos\n1. Confocal images do not have sufficient resolution\n- Fig 2: Confocal images with higher resolution.\n- Fig. S2A: The resolution of the images is too low to distinguish cells. In addition, the brain\nimage is not confocal and it is difficult to tell which cells express GFP. The intestine also\nappears to express dare-Gal4, but it is not clear which area of the intestine is positive for dare-\nGal4. It is also not clear which cells are the source of circulating 20E after priming.\n- Fig. 3D: Increasing the resolution of images to identify specific cells/neurons. What is the\nnumber of DopECR positive P1 cells that are positive for both DopECR and R71G01?\n- Fig. 4C. Show both CRZ-Gal4 images with and without tsh-Gal80 for comparison\n- Fig 5C: Stinger-GFP should be in the nucleus while anti-CRZ should be in the cytoplasm.\nHowever, both GFP and anti-CRZ appear in almost exactly the same area, which looks odd. The\nhigh resolution image is needed to resolve this issue.\nRevised.\n2. Additional controls are needed:\n- Fig 2D, E, F: Vehicle control is missing\nRevised. The control experiments are not in Fig S6.\n- Fig S2C: RNAi, Gal4 control is missing\nThis result is a supplement of Figure 3B, C. The control expriments are included in the main\nfigure.\n3. Line 223: \"Consistent with single cell RNA sequencing results from FCA (Fly Cell Atlas)\".\nShow relevant scRNA results and provide references.\nRevised.\n4. 10 min priming is sufficient to increase the courtship index measured for 10 min. Compared to\nthe priming experiment, 5 hours of 20E feeding is simply too long. In addition, it is unclear how\nmuch 20E is circulating when males are fed 3 mM 20E for 5 hours. Any reference to support this\nexperimental protocol?\nRevised.\n5. Figure S2F needs to include experimental protocol indicating the timing of 20E treatment and\nimaging.\nRevised.\n6. Figure 6F. Why not show the exact data points for this graph.\nRevised.\n7. Line 261. “by preventing subsequent competing males from mating” ? Incomplete sentence.\nRevised.\n8. Line 277. \" This increase eventually leads males less picky about mates.” This statement is\nconfusing because the logic behind it is unclear.\nRevised.\n9. Does ‘the emission of EB’ mean the accumulation of the ejaculation-ready materials within\nthe EB. To me, it sounds like the expulsion of materials from EB. To help readers understand, the\nauthors need to clearly explain the meaning of ‘the emission of EB’ and its relationship with\nmating plug allocation earlier in the text.\nEmission of EB indicated that the contents of the mating plug are release from the EB cells to the\nEB lumen. We have clarified this in the text.\n10. Line 83: \"Comparing the mating latency of primed or unprimed males to aged females in\nmouse experiments\" Does \"the emission of EB\" mean the accumulation of ejaculatory materials\nwithin the EB? To me, it sounds like the expulsion of materials within the EB. To help readers\nunderstand, the authors need to clearly explain the meaning of \"the emission of EB\" and its\nrelationship to the allocation of mating plugs earlier in the text. Do ‘the emission of EB’ and ‘EB\nemission’ mean the same thing?.\nRevised.\n11. Line 297. A process similar to mammalian emission. Again, explain what \"the emission\"\nmeans in this context. Can it be more specific?\nRevised.\n12. The authors need to provide evidence for the statement in lines 312-314.\nRevised.\nTypos\nLine 82: then  than\nLine 85: cross tax  across taxa\nLines 168-169: “However… the activity of P1 neurons” is not needed.\nLine 277: “This increase … about mate” is out of context.\nFig 2D. X-axis label. ‘optiaml’  optimal\nCCKLR-17D3-GAL4(X); sp/cyo (Yufeng Pan). Is this correct genotype? CCKLR-17D3GAL4\nAll corrected."}]

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