Multisensory inputs control the regulation of time investment for mating by sexual experience in male Drosophila melanogaster

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   Review Commons

   Nov 9, 2022

   Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

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   Reply to the reviewers

   Manuscript number: RC-2022-01680

   Corresponding author(s): Woo Jae, Kim

   1. __ General Statements__ The goal of this study is to provide the groundwork for future studies of genetically controlled neuronal regulation of ‘interval timing’ through the provision of a behavioral paradigm. Interval timing, or the sense of time in the seconds to hours range, is important in foraging, decision making, and learning in humans via activation of cortico-striatal circuits. Interval timing requires completely distinct brain processes from millisecond or circadian timing. In summary, interval timing allows us to subjectively sense the passage of physical time, allowing us to …

   Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Reply to the reviewers

   Manuscript number: RC-2022-01680

   Corresponding author(s): Woo Jae, Kim

   1. __ General Statements__ The goal of this study is to provide the groundwork for future studies of genetically controlled neuronal regulation of ‘interval timing’ through the provision of a behavioral paradigm. Interval timing, or the sense of time in the seconds to hours range, is important in foraging, decision making, and learning in humans via activation of cortico-striatal circuits. Interval timing requires completely distinct brain processes from millisecond or circadian timing. In summary, interval timing allows us to subjectively sense the passage of physical time, allowing us to integrate action sequences, thoughts, and behavior, detect developing trends, and predict future consequences.

   Many researchers have tried to figure out how animals, including humans, can estimate time intervals with such precision. However, most investigations have been conducted in the realm of psychology rather than biology thus far. Because the study of interval timing was limited in its ability to intervene in the human brain, many psychologists concentrated on developing convincing theoretical models to explain the known occurrence of interval timing.

   To overcome the limits of studying interval timing in terms of genetic control, we have reported that the time investment strategy for mating in* Drosophila* males can be a suitable behavioral platform to genetically dissect the principle of brain circuit mechanism for interval timing. For example, we previously reported that males prolong their mating when they have previously been exposed to rivals (Kim, Jan & Jan, "Contribution of visual and circadian neural circuits to memory for prolonged mating induced by rivals" Nature Neuroscience, 2012), and this behavior is regulated by visual stimuli, clock genes, and neuropeptide signaling in a subset of neurons (Kim, Jan & Jan, “A PDF/NPF Neuropeptide Signaling Circuitry of Male Drosophila melanogaster Controls Rival-Induced Prolonged Mating” Neuron, 2013).

   Throughout their lives, all animals must make decisions in order to optimize their utility function. Male reproductive success is determined by how many sperms successfully fertilize an egg with a restricted number of investment resources. To optimize male reproductive fitness, a time investment strategy has been devised. As a consequence, we believe that the flexible responses of mating duration to different environmental contexts in Drosophila males might be an excellent model to investigate neural circuits for interval timing.

   One of the most well-known features of human interval timing is the association of different sensory inputs with perception of time intervals, which influences our estimate of time intervals. Therefore, the first step toward comprehending the neural regulation of interval timing is to dissect the role that numerous sensory inputs play in determining the time duration. In this article, we discuss a different time-investment strategy adopted by males, called "Shorter-Mating-Duration" (SMD). According to our findings, male Drosophila with more sexual experience had shorter mating duration. During our investigation into the sensory inputs for SMD behavior, we found a small number of cells that express sugar receptors and pheromone receptors (ppk25 and ppk29) and thus transmit the multisensory information from females in order to generate memories of sexual experiences, which will determine the final decision of mating duration.

   Our discovery of sensory integration mechanisms associated with complex behavioral trait in male Drosophila at the brain circuit and genetic network levels will be a huge step forward in our knowledge of interval timing behavior.

   __ Description of the planned revisions__

   REVIEWER #1

   1. • Overall I think this would be difficult for a general audience as the rationale and explanation of experiments needs to be clearer. * Answer: During the revision process, we will make our text more legible for wide audiences.

   REVIEWER #2

   1. • 'The knockdown of LUSH, an odorant-binding protein' Lush is expressed in trichoid sensilla in olfactory organs , from the beginning, they exclude the role of olfaction and later one they said 'suggesting that the expression of the pheromone sensing proteins LUSH and Snmp1 in Gr5a-positive gustatory neurons is critical for generating SMD behavior.' ? Therefore, I recommend If available, please provide a reference for the statement in the Methods section that the Orco1 line was "validated via electrophysiology", or include the electrophysiology data itself in this manuscript as supplementary figure. Ideally, positive behavioral controls for this line would also be included in the manuscript. * Answer: We value the reviewer's concern. LUSH has been discovered as an odorant-binding protein; nevertheless, current research suggests that LUSH may be involved in the sensing of additional pheromones to cVA, implying the presence of a lush-independent cVA detection mechanism [1]. Billeter et al. demonstrated in their paper that LUSH detects a female stimulatory chemical and modifies male mating latency (Fig. 2 of Billeter at al.). As Billeter et al. stated, our present understanding of pheromonal recognition in Drosophila is insufficient, and we concur. As a result, we attempted to validate the expression of Snmp1 in the male leg by experiments (Fig. 7I-J) performing sncRNA seq analysis on the Fly SCope dataset, as shown in Fig. 12. As demonstrated in Fig.12, Snmp1 and LUSH is higly expressed fly leg and wing system. Future study will look at the roles of Snmp1 and LUSH in female pheromone sensing, as well as PPK receptors.

   Following the reviewer's advice, we will repeat the electrophysiologically validated Orco2 mutant phenotype with proper control and attach it when we submit the complete revision to the journal.

   • What is this (GustDx6)? I suggest using Poxn mutant line. *

   Answer: We value the reviewer's recommendation. We believe we have previously demonstrated that the Gr5a-mediated gustatory pathway is essential for the generation of sensory input for SMD behavior, but we will test the Poxn mutant and Poxn-RNAi to replace the GustDx6 mutant result.

   __ Description of the revisions that have already been incorporated in the transferred manuscript__

   __REVIEWER #1 __

   • My copy of this ms does not have page numbers or line numbers, this makes it extremely difficult to identify where I am making queries/ suggestions. I don't know whether this is a decision of the journal or authors, but please change this in the future.* Answer: We put page numbers and line numbers.

   • A general point, there is simply too much in this ms. It covers too much ground and so doesn't give proper descriptions, discuss the consequences of the data fully or integrate properly with existing literature. Quantity does not equal impact. *

   Answer: We appreciate the reviewer's insight. We have previously separated this document from our original preprint [2] in response to a prior reviewer's advice; we believe we have included too much data, which may confuse readers. As a result, we will delete all of the mechanosensory/thermosensory receptor screening data from our present paper and write a second manuscript on sensory integration for the production of SMD behavior. We also removed the most of sncRNA seq data analysis except Fig.12 which confirms our finding in a single diagram.

   • Results paragraph 1 says that white mutant background had no effect "unlike that of LMD behavior as reported previously", ignoring that there has been a contrary report that extension of mating duration after exposure to a rival does not involve visual cues and so is not affected by the white mutation (Bretman et al 2011 Curr Biol). *

   Answer: We recognize that there is a conflicting report concerning white mutation on LMD behavior, however because we are now reporting SMD rather than LMD behavior, we deleted the statement comparing white mutant results to earlier reports, as shown below;

   “thus suggesting that the effect of the *white *mutant genetic background was not evident.” (line 97)

   • A general point in the methodology, it's not very helpful just to say "as in a previous study" without giving at least a brief idea of what that was (e.g. the explanation of egg counting procedures).

   A "sperm depletion" assay is described in the results that I cannot find any methodology for. *

   Answer: We thank the reviewer for allowing us to clarify our lacking methodologies for a better comprehension of our manuscript.

   We included the egg counting procedure to the EXPERIMENTAL PROCEDURES section to further illustrate our approach of egg laying assay as below;

   “In short, wild type females mated with naïve or experienced males were transferred to a fresh new vial and allowed to lay eggs for 24 hr at 25°C. After 24 hr of egg laying, number of eggs were counted under the stereomicroscope. After we count the number of eggs, we kept vials in 25°C incubator and counted the total number of progenies ecolsed from them.” (line 956-960)

   We included “Sperm Depletion from Males” section in EXPERIMENTAL PROCEDURES as below;

   “To deplete sperm from males, 40 virgin Defexel6234 females which lacks SPR and shows multiple mating with males (Yang 2009) were placed in a vial containing four CS males for indicated time (2 h, 4 h, 8 h, and 24 h).” (line 880)

   • Was the "excessive mating" with SPR females actually observed, or inferred from previous work? Needs to be clear. In what way do virgins expressing fruitless behave like mated females? It is so unclear how all the evidence in this paragraph leads to the conclusion that both cues from females and successful copulation. Especially as in the next paragraph experience with feminized females (with which the focal males cannot copulate) elicits the response.

   It might be helpful to combine the results into a table, so it is easy to see under which conditions males reduce mating duration. *

   Answer: We modified the sentence describing SPR mutant female experiment and added references as below;

   “Sexual experiences with sex peptide receptor (SPR) mutant females which exhibit a delayed post-mating response and multiple mating with males [3] had no additional effect on SMD (Fig. 2I).” (line 135)

   We clarify in which extent, *fru>UAS-mSP *virgin females behave like mated females as below;

   “Virgin females behave like mated females by expressing a membrane-bound version of male sex-peptide in fruitless-positive neurons, hence rejecting the male's copulation attempt.” (line 136)

   In the instance of feminized males, we assume that these feminine males can give adequate signals for inducing SMD and eliminated the term "successful copulation" since we are unsure if males can copulate these feminized males or not, despite the fact that males can mount and mate with them (Fig. 2O-P).

   Tables S1 and S2 describe the conditions, genotypes, and descriptions of an experiments illustrated in Fig. 2. We believe that these tables may assist general audiences in comprehending our experimental design.

   • Why are no statistics reported in the results? Identifying sig diffs on figures is not sufficient. I'm very sceptical that "mating duration of males showed normal distribution" for all comparisons, but then it's also difficult to identify which were analysed in this way (if statistics were properly reported this would not be an issue). *

   Answer: We described our statistical analysis with mating duration previously [4–7] and followed the statistical analysis of copulation duration assay reported by Crickmore et al., published in CELL (2013) and NEURON (2020) [8,9]. To further validate our statistical analysis, we added estimation statistics which focuses on the effect size of one's experiment/intervention, as opposed to significance testing [10]. We already described our statistical analysis in EXPERIMENTAL PROCEDURES section in details. We also described our statistical analysis for mating duration will be same in all other figures in the Fig.1 legend.

   We appreciate the reviewer's recommendation that the normal distribution of our mating duration data be validated. As a consequence, we performed the normailty test with Graphpad prism and added the histogram and QQ plot results to Fig. S1M and N.__ Table S3__ also contains the results of the normality and lognormality tests.

   • Gr5a/ Gr66a mediate acceptance/ avoidance of what? Why would you hypothesise these in particular to be involved? *

   Answer: We accidentally left out the citation for that phrase and updated it with Wang et al.'s CELL (2004) paper. Wang et al. wrote in their article about taste representations in the Drosophila brain, “Our behavioral studies reveal that Gr5a cells recognize sugars and mediate acceptance/attractive behaviors whereas Gr66a cells recognize bitter compounds and mediate avoidance…. This suggests that Gr5a cells may be “acceptance” cells rather than “sweet” cells…. Our expression and behavioral studies reveal that Gr5a marks cells that recognize sugars and mediate taste acceptance, whereas Gr66a marks cells that recognize bitter compounds and mediate avoidance.” [11]

   As a result, we hypothesize that Gr5a and Gr66a-positive cells influence acceptance or avoidance of "taste." We also changed certain sentences to make them clearer, as seen below;

   “Of the various gustatory receptors, Gr5a marks cells that recognize sugars and mediate taste acceptance, whereas Gr66a marks cells that recognizes bitter compounds and mediates avoidance.” (line 173)

   • As Orco was not found to affect the behaviour, why test Or67d? *

   Answer: We appreciate the reviewer bringing this to our attention. We omitted the Or67d result from the present manuscript to simplify it and make it easier for readers to grasp.

   • "Mate guarding" suddenly appears in the modelling section. Can a difference of a couple of minutes in a mating duration of 15-20min really be considered mate guarding? A similar variation in response to rival males is not considered mate guarding, but is linked to adjustments in ejaculate expenditure (admittedly not in a very straight forward way). Surely in a system like this the benefits arise more from how many females the male can mate with in a given time? How does this model relate to any of the previous models of mate guarding?

   In this section the work of Linklater et al 2007 is important, they showed progeny declined over successive matings, and related this to exhaustion of Acps rather than sperm. I would urge the authors to consider that what they observe does not necessarily have an adaptive explanation. *

   Answer: We have defined “mate guarding” in the text now. The costs and benefits of mate guarding have been extensively studied in insects and demonstrated to shape the optimal mating duration of males. In our experiment, we cannot specify whether the shortened mating duration was caused by the adjustments in ejaculate expenditure or a shorted stay after the ejaculation. Instead, our model has a general assumption that the costs of mate guarding increase linearly at the same rate in both pre- and post-ejaculation periods, which is highlighted in the model text.

   There exist many models for the optimal mating duration (earlier models include Grafen and Ridley, 1983. A model of mate guarding. J. Theor. Biol. 102: 549 – 567 [12]). While our model was not built upon a novel theoretical approach (it was built based on the classical Charnov’s marginal value theorem equation), our model was developed specifically for generating testable predictions for the observed SMD behaviors.

   We have rephrased the text as follow;

   “This model assumes that (i) the shortened (or prolonged) mating duration is controlled by males and shaped by a trade-off between the benefit of mate guarding (remaining with the female both before and after the sperm ejaculation) and opportunistic costs (e.g. searching for another mate).” (line 970)”

   • I can't find a data accessibility statement. *

   Answer: We added it in the manuscript.

   • That said, a current grand challenge in understanding behaviour is discovering the mechanisms that enable individuals to respond plastically to changing environments. This speaks directly to that challenge. However, this behavioural observation is not novel, as claimed. Generally the idea of refractoriness is widely known, and specifically the reduction in mating duration over successive matings in D. melanogaster was shown by Linklater et al 2007 Evolution. Moreover, the time between exposure to females has been shown to be important. Linklater et al 2007 gave males mating attempts in quick succession and observed the decrease in mating duration, whereas given recovery time of 3 days, males either mate equally as long, or even longer across their life course (Bretman et al 2011 Proc B, Bretman et al 2013 Evolution). These papers should be discussed, and more broadly the work understood in the light of previous knowledge. The behaviour does not need to be novel for this manuscript to make a significant contribution to the field. *

   Answer: We believe the reviewer highlighted relevant past research that examined the influence of female experiences on mating duration. We agree with the reviewer that SMD behavior does not have to be original in order to contribute significantly to the field. As a result, we examined past reports and updated the introduction as follows;

   “It has been reported that previous sexual experience with females influences the mating duration of male D. melanogaster [15,16,34]; however, the neural circuits and physiology underlying this behavior have not been deeply investigated. Here, we report the sensory integration mechanisms by which sexually experienced males exhibit plastic behavior by limiting their investment in copulation time; we refer to this behavior as "shorter mating duration (SMD)."” (line 85)

   • Both in the introduction and discussion the extended mating duration in response to rivals is raised. A great deal of work has been done on this plasticity and yet the way this is written implies just two papers from these authors (whilst referencing others elsewhere). *

   Answer: We agree with the reviewer. In the introductory and discussion sections, we cited as many key publications explaining the plastic responses of male mating duration as we could.

   __REVIEWER #2

   __

   1. • Summary: The submitted manuscript reports that Drosophila melanogaster males use information derived from their previous sexual experiences from multiple sensory inputs to optimize their investment in mating. They refer to this plasticity as 'shorter-mating duration (SMD)'. SMD requires sexually dimorphic taste neurons. They identified several neurons in the male foreleg and midleg that express specific sugar, pheromone and mechanosensory receptors. Unfortunately, several aspects of the study design and methods used are inappropriate. Although the statistical approaches used are appropriate, the results are questionable. The discussion and conclusions are therefore too speculative in my view and overstretch the implications of the results as presented. Below I explain each one of these concerns about the study design, methods and results in detail as follows.* Answer: We appreciate the reviewer's assessment, especially the statement that our statistical approaches were appropriate. We will revise our manuscript in response to the reviewer's suggestions.

   • The conclusions (as the authors point out) hinge on small (often extremely small) effect sizes. This is not an insurmountable problem, so long as the assays are robust across trials. Unfortunately, they are not-the variation in the baseline for control replicates is often as large as, or larger than, the effects from which the conclusions are derived. Given the extreme experimental challenges of small effect size combined with large intertrial variability, it is notable that the authors do not report any likely false negative or false positive data, as would be frequently expected under these conditions. One explanation for the reproducibility of statistical effect seen across many experiments despite these experimental hurdles is manipulation of sample size. The authors acknowledge the extreme variability in sample size offer seemingly harmless explanations, but a closer look shows how problematic this practice is. For example, see Figure 1 (I, J, L) there is a big different between naive and experience males? *

   Answer: We value the reviewer's feedback. Several research have been conducted to investigate the mating duration of male fruit fly. For example, our lab [2,13–15] and others [13–30] have regularly reported that previous rival exposure increases male fruit fly mating duration. Bretman A et al. utilized 49-59 males in their studies to compare the variations in mating duration between circumstances. Crickmore et al. also reported the effect of mating duration differences caused by genetic or experimental modification [8]. They utilized 10-18 male flies in their study to compare the variations in mating duration across circumstances, as shown in Figs. 1G (n=15-18) and 2A (n=10-27). All of these findings indicate that our mating duration sample size is sufficient to examine the effect size variations between the naive and experienced conditions. To confirm our statistical analysis further, we incorporated estimate statistics, which focus on the effect size of one's experiment/intervention rather than significance tests [10]. We have already detailed our statistical analyses under the EXPERIMENTAL PROCEDURES section. We conducted hundreds of mating duration assays using this configuration and confirmed that all of our results are reproducible in a blind test. As a result, we believe our mating duration assay has been validated by other groups' findings, several analytic tools, and numerous blind tests conducted by us. We appreciate the reviewers' concerns, but our data meets the reproducibility requirements.

   • I am not sure if you keep using the same control with different experiments (that is okay if those exp is done in the same time) as in figure 1 B, I,J,K,L.But I don't think you did Fig 1B in the same time with Fig 1I, J, K,L. *

   Answer: We appreciate the reviewer's feedback. Yes, all of our tests comparing the differences in mating duration between naive and experienced conditions were conducted under the same conditions and at the same time. We replaced Fig.1B with new data (n=49-51) obtained lately in a new lab in China. As previously stated, SMD behavior could be reproduced by the same Canton S genotype in different locations by different experimenters.

   • It will be clear if you mention in the text how much reduction in percent happened in copulation duration when the males had previous sexual experience? *

   Answer: We appreciate the reviewer’s suggestion and added in the manuscript as follow;

   “We found that the mating duration of various wild-type and *w1118 *naïve males are significantly longer (wild type 15.7~15.8%, w1118 12.4%) than that of sexually experienced males (Fig. 1B-D, Fig. S1A)” (line 99)

   • 'Drosophila simulans, the sibling species of D. melanogaster also exhibits SMD, thus suggesting that SMD is conserved between close species of D. melanogaster (Fig. S1B).'. If you want come with this conclusion, you need to test D. erecta, D. sechelia and D. yakuba. *

   Answer: We appreciate the reviewer's feedback. We removed the D. simulans data because it is not required for the conclusion of this manuscript. In future research, we will look on the conservation of SMD behavior between species.

   • The authors mention that Gr66a is salt. This is not 100% correct. GR66a is expressed in many bitter sensing neurons and is required for the physiological and behavioral responses to many bitter compounds. check this reference DOI:https://doi.org/10.1016/j.cub.2019.11.005. *

   Answer: We made the following changes and cited the article reviewer's suggestion.

   “Of the various gustatory receptors, Gr5a marks cells that recognize sugars and mediate taste acceptance, whereas Gr66a marks cells that recognizes bitter compounds and mediates avoidance (Wang et al, 2004; Dweck & Carlson, 2020).” (line 180)

   • Drosophila melanogaster mating duration is between 21- 23 mins. I never saw copulation duration in normal condition (control) 10-15 mins as in figure fig 2E, Fig 7 C,E,F, Fig 8 E and fig 12 G . To the best of my knowledge, of all of the papers on copulation duration, the only one that ascribes a shortened duration to manipulations of the female is Rideout...Goodwin Nature Neuroscience 2010, who argue that this shortening results from markedly increased female activity/agitation during mating, leading the male to terminate early. *

   Answer: We appreciate the reviewer's feedback. Copulation duration in Drosophila melanogaster male is extremely variable and has been reported to be approximately 20 minutes. However, as other groups documented, male copulation duration can range from 10-15 minutes depending on sperm completion (Fig. 1a-c of Bretman A et al.) [30] and genetic background (Fig. 1C, Fig. 2E, Fig. 5D, and Fig. 7A and E of Crickmore et al) [8]. And, as previously stated, males dominate copulation duration [8,30], not females, and we always utilized the same genotype of females for mating duration experiment. As a result, we believe that these rather short mating duration outcomes are the product of a distinct genetic background. Because we employed the same genotype of males while altering the female experience condition, we believe our mating duration results are all equivalent and comparable.

   • In some experiments, the authors test very few number of replicates which is not convinced me to their conclusion as example Fig 2F and Fig 12 E. Why you test 100, 103 replicates in this exp fig 10 F? How you compare 47 replicates against 9 replicates in fig S10 I? *

   Answer: We appreciate the reviewer's input. As we previously stated in response to Reviewer Question 2, the n number of males exhibited in Figs. 2F and 12E is statistically significant. To corroborate findings with replication, we examined 100, 103 duplicates of Fig. 10F, which represents pyx-RNAi screening results. The results of Fig. S10I are screening data, and we cannot rule out the possibility that TrpA1 knockdown in Gr5a neurons affects the mating success of sexually experienced males. We only placed it there because it was screening results and the differences between naive and experienced conditions were substantial despite the small sample size. However, we deleted Fig. 10F and Fig. S10I data from the current paper in response to Reviewer #1's advice, thus it will not be an issue for the manuscript's conclusion.

   • 'Next, to decipher whether DEG/NaC channel-expressing pheromone sensing neurons require the function of OBP, we expressed lush-RNAi using ppk23-, ppk25- and ppk29-GAL4 drivers to knockdown LUSH in each channel-expressing neuron. The knockdown of LUSH in ppk25- and ppk29-GAL4 labeled cells, but not in ppk23-GAL4 labeled cells, led to a disturbance in SMD behavior, thus suggesting that LUSH functions in ppk25- and ppk29-positive neurons to detect pheromones and elicit SMD behavior (Fig. 9G-I). The knockdown of SNMP1 in ppk29-GAL4- labeled neurons also inhibited SMD behavior (Fig. 9J), thus suggesting that SNMP1 also functions in ppk29-positive neurons to induce SMD behavior.' What about ppk25? **

   Answer: As indicated by the reviewer, we included ppk25-GAL4/snmp1-RNAi data in Fig. S9I, indicating that snmp1 expression in ppk25-positive cells is similarly implicated in SMD behavior.

   • There are no page or line numbers throughout the ms! *

   Answer: We included page and line numbers.

   • The use of subheadings in the results section makes reading much easier.*

   Answer: We added subheadings in the results section.

   • 'We found that the mating duration of various wild-type and w 1118 naïve males are significantly longer than that of sexually experienced males (Fig. 1B-D, Fig. S1A)' . I think you should change various wild type to CS and WT Berlin as in legend and figure 1B,C .*

   Answer: The revised sentence is as follows:

   “We found that the mating duration of Canton S, WT-Berlin, Oregon-R, and *w1118 *naïve males are significantly longer (wild type 15.7~15.8%, w1118 12.4%) than that of sexually experienced males (Fig. 1B-D, Fig. S1A)” (line 102)

   • Suggested exp , Fig S1E-H , they might test 2,6, 12 hours males separation from females to test exactly when this behavior change over time. *

   Answer: We value the reviewer's recommendation. As seen in Fig. S4B of Kim et al., we have previously conducted experiments for examining the memory circuit of SMD [6]. Briefly, the male with a shorter mating duration recovers completely after 12 to 24 hours of isolation from females. As we are currently preparing the memory section of the SMD study, this information will be included in a future manuscript.

   • General comment in figures, you could remove the common y axis as example in figure 1 B,C,D , difference between means and mating duration. *

   Answer: We welcome the reviewer's idea, however in this situation we believe that the y axis of each data set is independent from one another and will thus retain the originals. We feel this would be more useful for the general audiences.

   • You might move the number of replicates to the legend. *

   Answer: We appreciate the reviewer's idea, however we feel that adding more information to the graphic will aid the general audience in comprehending our statistics.

   • Latin name should be italic as example Drosophila simulans.*

   Answer: We fixed it.

   __ Description of analyses that authors prefer not to carry out__

   N/A

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   20. Rouse J, Bretman A. Exposure time to rivals and sensory cues affect how quickly males respond to changes in sperm competition threat. Anim Behav. 2016;122: 1–8. doi:10.1016/j.anbehav.2016.09.011
   21. Bretman A, Fricke C, Hetherington P, Stone R, Chapman T. Exposure to rivals and plastic responses to sperm competition in Drosophila melanogaster. Behav Ecol. 2010;21: 317–321. doi:10.1093/beheco/arp189
   22. Rouse J, Watkinson K, Bretman A. Flexible memory controls sperm competition responses in male Drosophila melanogaster. Proc Royal Soc B Biological Sci. 2018;285: 20180619. doi:10.1098/rspb.2018.0619
   23. Maguire CP, Lizé A, Price TAR. Assessment of Rival Males through the Use of Multiple Sensory Cues in the Fruitfly Drosophila pseudoobscura. Plos One. 2015;10: e0123058. doi:10.1371/journal.pone.0123058
   24. Bretman A, Westmancoat JD, Gage MJG, Chapman T. COSTS AND BENEFITS OF LIFETIME EXPOSURE TO MATING RIVALS IN MALE DROSOPHILA MELANOGASTER. Evolution. 2013;67: 2413–2422. doi:10.1111/evo.12125
   25. Bretman A, Fricke C, Westmancoat JD, Chapman T. Effect of competitive cues on reproductive morphology and behavioral plasticity in male fruitflies. Behav Ecol. 2016;27: 452–461. doi:10.1093/beheco/arv170
   26. Price TAR, Lizé A, Marcello M, Bretman A. Experience of mating rivals causes males to modulate sperm transfer in the fly Drosophila pseudoobscura. J Insect Physiol. 2012;58: 1669–1675. doi:10.1016/j.jinsphys.2012.10.008
   27. Bretman A, Westmancoat JD, Gage MJG, Chapman T. Males Use Multiple, Redundant Cues to Detect Mating Rivals. Curr Biol. 2011;21: 617–622. doi:10.1016/j.cub.2011.03.008
   28. Fowler EK, Leigh S, Rostant WG, Thomas A, Bretman A, Chapman T. Memory of social experience affects female fecundity via perception of fly deposits. Bmc Biol. 2022;20: 244. doi:10.1186/s12915-022-01438-5
   29. Dore AA, Rostant WG, Bretman A, Chapman T. Plastic male mating behavior evolves in response to the competitive environment*. Evolution. 2021;75: 101–115. doi:10.1111/evo.14089
   30. Bretman A, Fricke C, Chapman T. Plastic responses of male Drosophila melanogaster to the level of sperm competition increase male reproductive fitness. Proc Royal Soc B Biological Sci. 2009;276: 1705–1711. doi:10.1098/rspb.2008.1878

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   Referee #2

   Evidence, reproducibility and clarity

   Summary:

   The submitted manuscript reports that Drosophila melanogaster males use information derived from their previous sexual experiences from multiple sensory inputs to optimize their investment in mating. They refer to this plasticity as 'shorter-mating duration (SMD)'. SMD requires sexually dimorphic taste neurons. They identified several neurons in the male foreleg and midleg that express specific sugar, pheromone and mechanosensory receptors. Unfortunately, several aspects of the study design and methods used are inappropriate. Although the statistical approaches used are appropriate, the results are questionable. The …

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   Referee #2

   Evidence, reproducibility and clarity

   Summary:

   The submitted manuscript reports that Drosophila melanogaster males use information derived from their previous sexual experiences from multiple sensory inputs to optimize their investment in mating. They refer to this plasticity as 'shorter-mating duration (SMD)'. SMD requires sexually dimorphic taste neurons. They identified several neurons in the male foreleg and midleg that express specific sugar, pheromone and mechanosensory receptors. Unfortunately, several aspects of the study design and methods used are inappropriate. Although the statistical approaches used are appropriate, the results are questionable. The discussion and conclusions are therefore too speculative in my view and overstretch the implications of the results as presented. Below I explain each one of these concerns about the study design, methods and results in detail as follows.

   Major comments:

   1. The conclusions (as the authors point out) hinge on small (often extremely small) effect sizes. This is not an insurmountable problem, so long as the assays are robust across trials. Unfortunately, they are not-the variation in the baseline for control replicates is often as large as, or larger than, the effects from which the conclusions are derived. Given the extreme experimental challenges of small effect size combined with large intertrial variability, it is notable that the authors do not report any likely false negative or false positive data, as would be frequently expected under these conditions. One explanation for the reproducibility of statistical effect seen across many experiments despite these experimental hurdles is manipulation of sample size. The authors acknowledge the extreme variability in sample size offer seemingly harmless explanations, but a closer look shows how problematic this practice is. For example, see Figure 1 (I, J, L) there is a big different between naive and experience males?
   2. I am not sure if you keep using the same control with different experiments (that is okay if those exp is done in the same time) as in figure 1 B, I,J,K,L.But I don't think you did Fig 1B in the same time with Fig 1I, J, K,L.
   3. It will be clear if you mention in the text how much reduction in percent happened in copulation duration when the males had previous sexual experience?
   4. 'Drosophila simulans, the sibling species of D. melanogaster also exhibits SMD, thus suggesting that SMD is conserved between close species of D. melanogaster (Fig. S1B).'. If you want come with this conclusion, you need to test D. erecta, D. sechelia and D. yakuba.
   5. The authors mention that Gr66a is salt. This is not 100% correct. GR66a is expressed in many bitter sensing neurons and is required for the physiological and behavioral responses to many bitter compounds. check this reference DOI:https://doi.org/10.1016/j.cub.2019.11.005.
   6. Drosophila melanogaster mating duration is between 21- 23 mins. I never saw copulation duration in normal condition (control) 10-15 mins as in figure fig 2E, Fig 7 C,E,F, Fig 8 E and fig 12 G . To the best of my knowledge, of all of the papers on copulation duration, the only one that ascribes a shortened duration to manipulations of the female is Rideout...Goodwin Nature Neuroscience 2010, who argue that this shortening results from markedly increased female activity/agitation during mating, leading the male to terminate early.
   7. In some experiments, the authors test very few number of replicates which is not convinced me to their conclusion as example Fig 2F and Fig 12 E
   8. Why you test 100, 103 replicates in this exp fig 10 F?
   9. How you compare 47 replicates against 9 replicates in fig S10 I?
   10. 'The knockdown of LUSH, an odorant-binding protein' Lush is expressed in trichoid sensilla in olfactory organs , from the beginning, they exclude the role of olfaction and later one they said 'suggesting that the expression of the pheromone sensing proteins LUSH and Snmp1 in Gr5a-positive gustatory neurons is critical for generating SMD behavior.' ? Therefore, I recommend If available, please provide a reference for the statement in the Methods section that the Orco1 line was "validated via electrophysiology", or include the electrophysiology data itself in this manuscript as supplementary figure. Ideally, positive behavioral controls for this line would also be included in the manuscript.
   11. 'Next, to decipher whether DEG/NaC channel-expressing pheromone sensing neurons require the function of OBP, we expressed lush-RNAi using ppk23-, ppk25- and ppk29-GAL4 drivers to knockdown LUSH in each channel-expressing neuron. The knockdown of LUSH in ppk25- and ppk29-GAL4 labeled cells, but not in ppk23-GAL4 labeled cells, led to a disturbance in SMD behavior, thus suggesting that LUSH functions in ppk25- and ppk29-positive neurons to detect pheromones and elicit SMD behavior (Fig. 9G-I). The knockdown of SNMP1 in ppk29-GAL4- labeled neurons also inhibited SMD behavior (Fig. 9J), thus suggesting that SNMP1 also functions in ppk29-positive neurons to induce SMD behavior.' What about ppk25?

   Minor comments:

   1. There are no page or line numbers throughout the ms!
   2. The use of subheadings in the results section makes reading much easier.
   3. 'We found that the mating duration of various wild-type and w 1118 naïve males are significantly longer than that of sexually experienced males (Fig. 1B-D, Fig. S1A)' . I think you should change various wild type to CS and WT Berlin as in legend and figure 1B,C .
   4. Suggested exp , Fig S1E-H , they might test 2,6, 12 hours males separation from females to test exactly when this behavior change over time.
   5. What is this (GustDx6)? I suggest using Poxn mutant line.
   6. General comment in figures, you could remove the common y axis as example in figure 1 B,C,D , difference between means and mating duration.
   7. You might move the number of replicates to the legend.
   8. Latin name should be italic as example Drosophila simulans.

   Referees cross-commenting

   I found the comments of the other reviewer reasonable and fair. I agree that the time for fixing all these comments is about six months.

   Significance

   The idea of the work is interesting, but the design of experiments in some places is inappropriate (see above). The discussion mainly depends on doi: 10.1016/j.neuron.2013.09.034. I study chemoreception and sexual communication in Drosophila and insect vectors of human disease.

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   Referee #1

   Evidence, reproducibility and clarity

   My copy of this ms does not have page numbers or line numbers, this makes it extremely difficult to identify where I am making queries/ suggestions. I don't know whether this is a decision of the journal or authors, but please change this in the future.

   Overall I think this would be difficult for a general audience as the rationale and explanation of experiments needs to be clearer.

   Results paragraph 1 says that white mutant background had no effect "unlike that of LMD behavior as reported previously", ignoring that there has been a contrary report that extension of mating duration after exposure to a rival does not involve visual …

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   Referee #1

   Evidence, reproducibility and clarity

   My copy of this ms does not have page numbers or line numbers, this makes it extremely difficult to identify where I am making queries/ suggestions. I don't know whether this is a decision of the journal or authors, but please change this in the future.

   Overall I think this would be difficult for a general audience as the rationale and explanation of experiments needs to be clearer.

   Results paragraph 1 says that white mutant background had no effect "unlike that of LMD behavior as reported previously", ignoring that there has been a contrary report that extension of mating duration after exposure to a rival does not involve visual cues and so is not affected by the white mutation (Bretman et al 2011 Curr Biol).

   A general point in the methodology, it's not very helpful just to say "as in a previous study" without giving at least a brief idea of what that was (e.g. the explanation of egg counting procedures).

   A "sperm depletion" assay is described in the results that I cannot find any methodology for.

   Was the "excessive mating" with SPR females actually observed, or inferred from previous work? Needs to be clear. In what way do virgins expressing fruitless behave like mated females? It is so unclear how all the evidence in this paragraph leads to the conclusion that both cues from females and successful copulation. Especially as in the next paragraph experience with feminized females (with which the focal males cannot copulate) elicits the response.

   It might be helpful to combine the results into a table, so it is easy to see under which conditions males reduce mating duration.

   Why are no statistics reported in the results? Identifying sig diffs on figures is not sufficient. I'm very sceptical that "mating duration of males showed normal distribution" for all comparisons, but then it's also difficult to identify which were analysed in this way (if statistics were properly reported this would not be an issue).

   Gr5a/ Gr66a mediate acceptance/ avoidance of what? Why would you hypothesise these in particular to be involved?

   As Orco was not found to affect the behaviour, why test Or67d?

   "Mate guarding" suddenly appears in the modelling section. Can a difference of a couple of minutes in a mating duration of 15-20min really be considered mate guarding? A similar variation in response to rival males is not considered mate guarding, but is linked to adjustments in ejaculate expenditure (admittedly not in a very straight forward way). Surely in a system like this the benefits arise more from how many females the male can mate with in a given time? How does this model relate to any of the previous models of mate guarding?

   In this section the work of Linklater et al 2007 is important, they showed progeny declined over successive matings, and related this to exhaustion of Acps rather than sperm. I would urge the authors to consider that what they observe does not necessarily have an adaptive explanation.

   I can't find a data accessibility statement.

   Significance

   A general point, there is simply too much in this ms. It covers too much ground and so doesn't give proper descriptions, discuss the consequences of the data fully or integrate properly with existing literature. Quantity does not equal impact.

   That said, a current grand challenge in understanding behaviour is discovering the mechanisms that enable individuals to respond plastically to changing environments. This speaks directly to that challenge.

   However, this behavioural observation is not novel, as claimed. Generally the idea of refractoriness is widely known, and specifically the reduction in mating duration over successive matings in D. melanogaster was shown by Linklater et al 2007 Evolution. Moreover, the time between exposure to females has been shown to be important. Linklater et al 2007 gave males mating attempts in quick succession and observed the decrease in mating duration, whereas given recovery time of 3 days, males either mate equally as long, or even longer across their life course (Bretman et al 2011 Proc B, Bretman et al 2013 Evolution). These papers should be discussed, and more broadly the work understood in the light of previous knowledge. The behaviour does not need to be novel for this manuscript to make a significant contribution to the field.

   Both in the introduction and discussion the extended mating duration in response to rivals is raised. A great deal of work has been done on this plasticity and yet the way this is written implies just two papers from these authors (whilst referencing others elsewhere).

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   Version published to 10.1101/2022.09.23.509131 on bioRxiv

   Sep 24, 2022