Social Experience Shapes Fighting Strategies for Reproductive Success

Reviewer #1 (Public review):

This work addresses an important question in the field of Drosophila aggression and mating- prior social isolation is known to increase aggression in males by increased lunging, which is suppressed by group housing (GH). However, it is also known that single-housed (SH) males, despite their higher attempts to court females, are less successful. Here, Gao et al., developed a modified aggression assay, to address this issue by recording aggression in Drosophila males for 2 hours, over a virgin female which is immobilized by burying its head in the food. They found that while SH males frequently lunge in this assay, GH males switch to higher intensity but very low-frequency tussling. Constitutive neuronal silencing and activation experiments implicate cVA sensing Or67d neurons promoting high-frequency lunging, similar to earlier studies, whereas Or47b neurons promote low-frequency but higher intensity tussling. Using optogenetic activation they found that three pairs of pC1 neurons- pC1SS2 increase tussling. While P1a neurons, previously implicated in promoting aggression and courtship, did not increase tussling in optogenetic activation (in the dark), they could promote aggressive tussling in thermogenetic activation carried out in the presence of visible light. It was further suggested, using a further modified aggression assay that GH males use increased tussling and are able to maintain territorial control, providing them mating advantage over SI males and this may partially overcome the effect of aging in GH males.

Strengths:

Using a series of clever neurogenetic and behavioral approaches, subsets of ORNs and pC1 neurons were implicated in promoting tussling behaviors. The authors devised a new paradigm to assay for territory control which appears better than earlier paradigms that used a food cup (Chen et al, 2002), as this new assay is relatively clutter-free, and can be eventually automated using computer vision approaches. The manuscript is generally well-written, and the claims made are largely supported by the data.

Weaknesses:

I have a few concerns regarding some of the evidence presented and claims made as well as a description of the methodology, which needs to be clarified and extended further.

(1) Typical paradigms for assaying aggression in Drosophila males last for 20-30 minutes in the presence of nutritious food/yeast paste/females or all of these (Chen et al. 2002, Nilsen et al., 2004, Dierick et al. 2007, Dankert et al., 2009, Certel & Kravitz 2012). The paradigm described in Figure 1 A, while important and more amenable for video recording and computational analysis, seems a modification of the assay from Kravitz lab (Chen et al., 2002), which involved using a female over which males fight on a food cup. The modifications include a flat surface with a central food patch and a female with its head buried in the food, (fixed female) and much longer adaptation and recording times respectively (30 minutes, 2 hours), so in that sense, this is not a 'new' paradigm but a modification of an existing paradigm and its description as new should be appropriately toned down. It would also be important to cite these earlier studies appropriately while describing the assay.

(2) Lunging is described as a 'low intensity' aggression (line 111 and associated text), however, it is considered a mid to high-intensity aggressive behavior, as compared to other lower-intensity behaviors such as wing flicks, chase, and fencing. Lunging therefore is lower in intensity 'relative' to higher intensity tussling but not in absolute terms and it should be mentioned clearly.

(3) It is often difficult to distinguish faithfully between boxing and tussling and therefore, these behaviors are often clubbed together as box, tussle by Nielsen et al., 2004 in their Markov chain analysis as well as a more detailed recent study of male aggression (Simon & Heberlein, 2020). Therefore, authors can either reconsider the description of behavior as 'box, tussle' or consider providing a video representation/computational classifier to distinguish between box and tussle behaviors.

(4) Simon & Heberlein, 2020 showed that increased boxing & tussling precede the formation of a dominance hierarchy in males, and lunges are used subsequently to maintain this dominant status. This study should be cited and discussed appropriately while introducing the paradigm.

(5) It would be helpful to provide more methodological details about the assay, for instance, a video can be helpful showing how the males are introduced in the assay chamber, are they simply dropped to the floor when the film is removed after 30 minutes (Figures 1-2)?

(6) The strain of Canton-S (CS) flies used should be mentioned as different strains of CS can have varying levels of aggression, for instance, CS from Martin Heisenberg lab shows very high levels of aggressive lunges. Are the CS lines used in this study isogenized? Are various genetic lines outcrossed into this CS background? In the methods, it is not clear how the white gene levels were controlled for various aggression experiments as it is known to affect aggression (Hoyer et al. 2008).

(7) How important it is to use a fixed female for the assay to induce tussling? Do these females remain active throughout the assay period of 2.5 hours? Is it possible to use decapitated virgin females for the assay? How will that affect male behaviors?

(8) Raster plots in Figure 2 suggest a complete lack of tussling in SH males in the first 60 minutes of the encounter, which is surprising given the longer duration of the assay as compared to earlier studies (Nielsen et al. 2004, Simon & Heberlein, 2020 and others), which are able to pick up tussling in a shorter duration of recording time. Also, the duration for tussling is much longer in this study as compared to shorter tussles shown by earlier studies. Is this due to differences in the paradigm used, strain of flies, or some other factor? While the bar plots in Figure 2D show some tussling in SH males, maybe an analysis of raster plots of various videos can be provided in the main text and included as a supplementary figure to address this.

(9) Neuronal activation experiments suggesting the involvement of pC1SS2 neurons are quite interesting. Further, the role of P1a neurons was demonstrated to be involved in increasing tussling in thermogenetic activation in the presence of light (Figure 4, Supplement 1), which is quite important as the role of vision in optogenetic activation experiments, which required to be carried out in dark, is often not mentioned. However, in the discussion (lines 309-310) it is mentioned that PC1SS2 neurons are 'necessary and sufficient' for inducing tussling. Given that P1a neurons were shown to be involved in promoting tussling, this statement should be toned down.

(10) Are Or47b neurons connected to pC1SS2 or P1a neurons?

(11) The paradigm for territory control is quite interesting and subsequent mating advantage experiments are an important addition to the eventual outcome of the aggressive strategy deployed by the males as per their prior housing conditions. It would be important to comment on the 'fitness outcome' of these encounters. For instance, is there any fitness advantage of using tussling by GH males as compared to lunging by SH males? The authors may consider analyzing the number of eggs laid and eclosed progenies from these encounters to address this.

Reviewer #2 (Public review):

Summary:

Gao et al. investigated the change of aggression strategies by the social experience and its biological significance by using Drosophila. Two modes of inter-male aggression in Drosophila are known: lunging, high-frequency but weak mode, and tussling, low-frequency but more vigorous mode. Previous studies have mainly focused on the lunging. In this paper, the authors developed a new behavioral experiment system for observing tussling behavior and found that tussling is enhanced by group rearing while lunging is suppressed. They then searched for neurons involved in the generation of tussling. Although olfactory receptors named Or67d and Or65a have previously been reported to function in the control of lunging, the authors found that these neurons do not function in the execution of tussling, and another olfactory receptor, Or47b, is required for tussling, as shown by the inhibition of neuronal activity and the gene knockdown experiments. Further optogenetic experiments identified a small number of central neurons pC1[SS2] that induce the tussling specifically. In order to further explore the ecological significance of the aggression mode change in group rearing, a new behavioral experiment was performed to examine territorial control and mating competition. Finally, the authors found that differences in the social experience (group vs. solitary rearing) are important in these biologically significant competitions. These results add a new perspective to the study of aggressive behavior in Drosophila. Furthermore, this study proposes an interesting general model in which the social experience-modified behavioral changes play a role in reproductive success.

Strengths:

A behavioral experiment system that allows stable observation of tussling, which could not be easily analyzed due to its low frequency, would be very useful. The experimental setup itself is relatively simple, just the addition of a female to the platform, so it should be applicable to future research. The finding about the relationship between the social experience and the aggression mode change is quite novel. Although the intensity of aggression changes with the social experience was already reported in several papers (Liu et al., 2011, etc), the fact that the behavioral mode itself changes significantly has rarely been addressed and is extremely interesting. The identification of sensory and central neurons required for the tussling makes appropriate use of the genetic tools and the results are clear. A major strength of the neurobiology in this study is the finding that another group of neurons (Or47b-expressing olfactory neurons and pC1[SS2] neurons), distinct from the group of neurons previously thought to be involved in low-intensity aggression (i.e. lunging), function in the tussling behavior. Further investigation of the detailed circuit analysis is expected to elucidate the neural substrate of the conflict between the two aggression modes.

Weaknesses:

The experimental systems examining the territory control and the reproductive competition in Figure 5 are novel and have advantages in exploring their biological significance. However, at this stage, the authors' claim is weak since they only show the effects of age and social experience on territorial and mating behaviors, but do not experimentally demonstrate the influence of aggression mode change itself. In the Abstract, the authors state that these findings reveal how social experience shapes fighting strategies to optimize reproductive success. This is the most important perspective of the present study, and it would be necessary to show directly that the change of aggression mode by social experience contributes to reproductive success.

In addition, a detailed description of the tussling is lacking. For example, the authors state that the tussling is less frequent but more vigorous than lunging, but while experimental data are presented on the frequency, the intensity seems to be subjective. The intensity is certainly clear from the supplementary video, but it would be necessary to evaluate the intensity itself using some index. Another problem is that there is no clear explanation of how to determine the tussling. A detailed method is required for the reproducibility of the experiment.

Reviewer #3 (Public review):

In this manuscript, Gao et al. presented a series of intriguing data that collectively suggest that tussling, a form of high-intensity fighting among male fruit flies (Drosophila melanogaster) has a unique function and is controlled by a dedicated neural circuit. Based on the results of behavioral assays, they argue that increased tussling among socially experienced males promotes access to resources. They also concluded that tussling is controlled by a class of olfactory sensory neurons and sexually dimorphic central neurons that are distinct from pathways known to control lunges, a common male-type attack behavior.

A major strength of this work is that it is the first attempt to characterize the behavioral function and neural circuit associated with Drosophila tussling. Many animal species use both low-intensity and high-intensity tactics to resolve conflicts. High-intensity tactics are mostly reserved for escalated fights, which are relatively rare. Because of this, tussling in the flies, like high-intensity fights in other animal species, has not been systematically investigated. Previous studies on fly aggressive behavior have often used socially isolated, relatively young flies within a short observation duration. Their discovery that 1) older (14-days-old) flies tend to tussle more often than younger (2-days-old) flies, 2) group-reared flies tend to tussle more often than socially isolated flies, and 3) flies tend to tussle at a later stage (mostly ~15 minutes after the onset of fighting), are the result of their creativity to look outside of conventional experimental settings. These new findings are keys for quantitatively characterizing this interesting yet under-studied behavior.

Precisely because their initial approach was creative, it is regrettable that the authors missed the opportunity to effectively integrate preceding studies in their rationale or conclusions, which sometimes led to premature claims. Also, while each experiment contains an intriguing finding, these are poorly related to each other. This obscures the central conclusion of this work. The perceived weaknesses are discussed in detail below.

Most importantly, the authors' definition of "tussling" is unclear because they did not explain how they quantified lunges and tussling, even though the central focus of the manuscript is behavior. Supplemental movies S1 and S2 appear to include "tussling" bouts in which 2 flies lunge at each other in rapid succession, and supplemental movie S3 appears to include bouts of "holding", in which one fly holds the opponent's wings and shakes vigorously. These cases raise a concern that their behavior classification is arbitrary. Specifically, lunges and tussling should be objectively distinguished because one of their conclusions is that these two actions are controlled by separate neural circuits. It is impossible to evaluate the credibility of their behavioral data without clearly describing a criterion of each behavior.

It is also confusing that the authors completely skipped the characterization of the tussling-controlling neurons they claimed to have identified. These neurons (a subset of so-called pC1 neurons labeled by previously described split-GAL4 line pC1SS2) are central to this manuscript, but the only information the authors have provided is its gross morphology in a low-resolution image (Figure 4D, E) and a statement that "only 3 pairs of pC1SS2 neurons whose function is both necessary and sufficient for inducing tussling in males" (lines 310-311). The evidence that supports this claim isn't provided. The expression pattern of pC1SS2 neurons in males has been only briefly described in reference 46. It is possible that these neurons overlap with previously characterized dsx+ and/or fru+ neurons that are important for male aggressions (measured by lunges), such as in Koganezawa et al., Curr. Biol. 2016 and Chiu et al., Cell 2020. This adds to the concern that lunge and tussling are not as clearly separated as the authors claim.

While their characterizations of tussling behaviors in wild-type males (Figures 1 and 2) are intriguing, the remaining data have little link with each other, making it difficult to understand what their main conclusion is. Figure 3 suggests that one class of olfactory sensory neurons (OSN) that express Or47b is necessary for tussling behavior. While the authors acknowledged that Or47b-expressing OSNs promote male courtship toward females presumably by detecting cuticular compounds, they provided little discussion on how a class of OSN can promote two different types of innate behavior. No evidence of a functional or circuitry relationship between the Or47b pathway and the pC1SS2 neurons was provided. It is unclear how these two components are relevant to each other. Lastly, the rationale of the experiment in Figure 5 and the interpretation of the results is confusing. The authors attributed a higher mating success rate of older, socially experienced males over younger, socially isolated males to their tendency to tussle, but tussling cannot happen when one of the two flies is not engaged. If, for instance, a socially isolated 14-day-old male does not engage in tussling as indicated in Figure 2, how can they tussle with a group-housed 14-day-old male? Because aggressive interactions in Figure 5 were not quantified, it is impossible to conclude that tussling plays a role in copulation advantage among pairs as authors argue (lines 282-288).

Despite these weaknesses, it is important to acknowledge the authors' courage to initiate an investigation into a less characterized, high-intensity fighting behavior. Tussling requires the simultaneous engagement of two flies. Even if there is confusion over the distinction between lunges and tussling, the authors' conclusion that socially experienced flies and socially isolated flies employ distinct fighting strategies is convincing. Questions that require more rigorous studies are 1) whether such differences are encoded by separate circuits, and 2) whether the different fighting strategies are causally responsible for gaining ethologically relevant resources among socially experienced flies. Enhanced transparency of behavioral data will help readers understand the impact of this study. Lastly, the manuscript often mentions previous works and results without citing relevant references. For readers to grasp the context of this work, it is important to provide information about methods, reagents, and other key resources.

Author response:

Public Reviews:

Reviewer #1 (Public review):

This work addresses an important question in the field of Drosophila aggression and mating- prior social isolation is known to increase aggression in males by increased lunging, which is suppressed by group housing (GH). However, it is also known that single-housed (SH) males, despite their higher attempts to court females, are less successful. Here, Gao et al., developed a modified aggression assay, to address this issue by recording aggression in Drosophila males for 2 hours, over a virgin female which is immobilized by burying its head in the food. They found that while SH males frequently lunge in this assay, GH males switch to higher intensity but very low-frequency tussling. Constitutive neuronal silencing and activation experiments implicate cVA sensing Or67d neurons promoting high-frequency lunging, similar to earlier studies, whereas Or47b neurons promote low-frequency but higher intensity tussling. Using optogenetic activation they found that three pairs of pC1 neurons- pC1SS2 increase tussling. While P1a neurons, previously implicated in promoting aggression and courtship, did not increase tussling in optogenetic activation (in the dark), they could promote aggressive tussling in thermogenetic activation carried out in the presence of visible light. It was further suggested, using a further modified aggression assay that GH males use increased tussling and are able to maintain territorial control, providing them mating advantage over SI males and this may partially overcome the effect of aging in GH males.

Strengths:

Using a series of clever neurogenetic and behavioral approaches, subsets of ORNs and pC1 neurons were implicated in promoting tussling behaviors. The authors devised a new paradigm to assay for territory control which appears better than earlier paradigms that used a food cup (Chen et al, 2002), as this new assay is relatively clutter-free, and can be eventually automated using computer vision approaches. The manuscript is generally well-written, and the claims made are largely supported by the data.

Thank you for your precise summary of our study and being very positive on the novelty and significance of the study.

Weaknesses:

I have a few concerns regarding some of the evidence presented and claims made as well as a description of the methodology, which needs to be clarified and extended further.

(1) Typical paradigms for assaying aggression in Drosophila males last for 20-30 minutes in the presence of nutritious food/yeast paste/females or all of these (Chen et al. 2002, Nilsen et al., 2004, Dierick et al. 2007, Dankert et al., 2009, Certel & Kravitz 2012). The paradigm described in Figure 1 A, while important and more amenable for video recording and computational analysis, seems a modification of the assay from Kravitz lab (Chen et al., 2002), which involved using a female over which males fight on a food cup. The modifications include a flat surface with a central food patch and a female with its head buried in the food, (fixed female) and much longer adaptation and recording times respectively (30 minutes, 2 hours), so in that sense, this is not a 'new' paradigm but a modification of an existing paradigm and its description as new should be appropriately toned down. It would also be important to cite these earlier studies appropriately while describing the assay.

We will tone down the description and cite related references.

(2) Lunging is described as a 'low intensity' aggression (line 111 and associated text), however, it is considered a mid to high-intensity aggressive behavior, as compared to other lower-intensity behaviors such as wing flicks, chase, and fencing. Lunging therefore is lower in intensity 'relative' to higher intensity tussling but not in absolute terms and it should be mentioned clearly.

Ww will textually address this issue.

(3) It is often difficult to distinguish faithfully between boxing and tussling and therefore, these behaviors are often clubbed together as box, tussle by Nielsen et al., 2004 in their Markov chain analysis as well as a more detailed recent study of male aggression (Simon & Heberlein, 2020). Therefore, authors can either reconsider the description of behavior as 'box, tussle' or consider providing a video representation/computational classifier to distinguish between box and tussle behaviors.

We will textually address this issue.

(4) Simon & Heberlein, 2020 showed that increased boxing & tussling precede the formation of a dominance hierarchy in males, and lunges are used subsequently to maintain this dominant status. This study should be cited and discussed appropriately while introducing the paradigm.

We will cite this paper and discuss on this issue.

(5) It would be helpful to provide more methodological details about the assay, for instance, a video can be helpful showing how the males are introduced in the assay chamber, are they simply dropped to the floor when the film is removed after 30 minutes (Figures 1-2)?

We will provide more methodological details.

(6) The strain of Canton-S (CS) flies used should be mentioned as different strains of CS can have varying levels of aggression, for instance, CS from Martin Heisenberg lab shows very high levels of aggressive lunges. Are the CS lines used in this study isogenized? Are various genetic lines outcrossed into this CS background? In the methods, it is not clear how the white gene levels were controlled for various aggression experiments as it is known to affect aggression (Hoyer et al. 2008).

We will textually address this issue.

(7) How important it is to use a fixed female for the assay to induce tussling? Do these females remain active throughout the assay period of 2.5 hours? Is it possible to use decapitated virgin females for the assay? How will that affect male behaviors?

We will textually address this issue and provide additional videos.

(8) Raster plots in Figure 2 suggest a complete lack of tussling in SH males in the first 60 minutes of the encounter, which is surprising given the longer duration of the assay as compared to earlier studies (Nielsen et al. 2004, Simon & Heberlein, 2020 and others), which are able to pick up tussling in a shorter duration of recording time. Also, the duration for tussling is much longer in this study as compared to shorter tussles shown by earlier studies. Is this due to differences in the paradigm used, strain of flies, or some other factor? While the bar plots in Figure 2D show some tussling in SH males, maybe an analysis of raster plots of various videos can be provided in the main text and included as a supplementary figure to address this.

We will textually address the first question and provide more detailed analysis for the second question.

(9) Neuronal activation experiments suggesting the involvement of pC1SS2 neurons are quite interesting. Further, the role of P1a neurons was demonstrated to be involved in increasing tussling in thermogenetic activation in the presence of light (Figure 4, Supplement 1), which is quite important as the role of vision in optogenetic activation experiments, which required to be carried out in dark, is often not mentioned. However, in the discussion (lines 309-310) it is mentioned that PC1SS2 neurons are 'necessary and sufficient' for inducing tussling. Given that P1a neurons were shown to be involved in promoting tussling, this statement should be toned down.

We will tone down this statement.

(10) Are Or47b neurons connected to pC1SS2 or P1a neurons?

We conducted pathway analysis in the FlyWire electron microscopy database to investigate the connection between Or47b neurons and pC1 neurons. The results indicate that at least three intermediate neurons are required to establish a connection from Or47b neurons to pC1 neurons. Although the FlyWire database currently only contains neuronal data from female brains, they provide a reference for circuit connect in males. Using the currently available upstream and downstream tracing tools (e.g., retro-/trans-Tango), it is not possible to establish a direct connection between the two. Identifying the intermediate neurons involved in this connection is beyond this study. We will discuss on this concern in our revised manuscript.

(11) The paradigm for territory control is quite interesting and subsequent mating advantage experiments are an important addition to the eventual outcome of the aggressive strategy deployed by the males as per their prior housing conditions. It would be important to comment on the 'fitness outcome' of these encounters. For instance, is there any fitness advantage of using tussling by GH males as compared to lunging by SH males? The authors may consider analyzing the number of eggs laid and eclosed progenies from these encounters to address this.

We will discuss on this concern.

Reviewer #2 (Public review):

Summary:

Gao et al. investigated the change of aggression strategies by the social experience and its biological significance by using Drosophila. Two modes of inter-male aggression in Drosophila are known: lunging, high-frequency but weak mode, and tussling, low-frequency but more vigorous mode. Previous studies have mainly focused on the lunging. In this paper, the authors developed a new behavioral experiment system for observing tussling behavior and found that tussling is enhanced by group rearing while lunging is suppressed. They then searched for neurons involved in the generation of tussling. Although olfactory receptors named Or67d and Or65a have previously been reported to function in the control of lunging, the authors found that these neurons do not function in the execution of tussling, and another olfactory receptor, Or47b, is required for tussling, as shown by the inhibition of neuronal activity and the gene knockdown experiments. Further optogenetic experiments identified a small number of central neurons pC1[SS2] that induce the tussling specifically. In order to further explore the ecological significance of the aggression mode change in group rearing, a new behavioral experiment was performed to examine territorial control and mating competition. Finally, the authors found that differences in the social experience (group vs. solitary rearing) are important in these biologically significant competitions. These results add a new perspective to the study of aggressive behavior in Drosophila. Furthermore, this study proposes an interesting general model in which the social experience-modified behavioral changes play a role in reproductive success.

Strengths:

A behavioral experiment system that allows stable observation of tussling, which could not be easily analyzed due to its low frequency, would be very useful. The experimental setup itself is relatively simple, just the addition of a female to the platform, so it should be applicable to future research. The finding about the relationship between the social experience and the aggression mode change is quite novel. Although the intensity of aggression changes with the social experience was already reported in several papers (Liu et al., 2011, etc.), the fact that the behavioral mode itself changes significantly has rarely been addressed and is extremely interesting. The identification of sensory and central neurons required for the tussling makes appropriate use of the genetic tools and the results are clear. A major strength of the neurobiology in this study is the finding that another group of neurons (Or47b-expressing olfactory neurons and pC1[SS2] neurons), distinct from the group of neurons previously thought to be involved in low-intensity aggression (i.e. lunging), function in the tussling behavior. Further investigation of the detailed circuit analysis is expected to elucidate the neural substrate of the conflict between the two aggression modes.

Thank you for the acknowledgment of the novelty and significance of the study, and your suggestions for improving the manuscript.

Weaknesses:

The experimental systems examining the territory control and the reproductive competition in Figure 5 are novel and have advantages in exploring their biological significance. However, at this stage, the authors' claim is weak since they only show the effects of age and social experience on territorial and mating behaviors, but do not experimentally demonstrate the influence of aggression mode change itself. In the Abstract, the authors state that these findings reveal how social experience shapes fighting strategies to optimize reproductive success. This is the most important perspective of the present study, and it would be necessary to show directly that the change of aggression mode by social experience contributes to reproductive success.

We will either tone down this statement or provide additional analysis.

In addition, a detailed description of the tussling is lacking. For example, the authors state that the tussling is less frequent but more vigorous than lunging, but while experimental data are presented on the frequency, the intensity seems to be subjective. The intensity is certainly clear from the supplementary video, but it would be necessary to evaluate the intensity itself using some index. Another problem is that there is no clear explanation of how to determine the tussling. A detailed method is required for the reproducibility of the experiment.

We will provide more detailed methods and data analysis regarding tussling behavior.

Reviewer #3 (Public review):

In this manuscript, Gao et al. presented a series of intriguing data that collectively suggest that tussling, a form of high-intensity fighting among male fruit flies (Drosophila melanogaster) has a unique function and is controlled by a dedicated neural circuit. Based on the results of behavioral assays, they argue that increased tussling among socially experienced males promotes access to resources. They also concluded that tussling is controlled by a class of olfactory sensory neurons and sexually dimorphic central neurons that are distinct from pathways known to control lunges, a common male-type attack behavior.

A major strength of this work is that it is the first attempt to characterize the behavioral function and neural circuit associated with Drosophila tussling. Many animal species use both low-intensity and high-intensity tactics to resolve conflicts. High-intensity tactics are mostly reserved for escalated fights, which are relatively rare. Because of this, tussling in the flies, like high-intensity fights in other animal species, has not been systematically investigated. Previous studies on fly aggressive behavior have often used socially isolated, relatively young flies within a short observation duration. Their discovery that 1) older (14-days-old) flies tend to tussle more often than younger (2-days-old) flies, 2) group-reared flies tend to tussle more often than socially isolated flies, and 3) flies tend to tussle at a later stage (mostly ~15 minutes after the onset of fighting), are the result of their creativity to look outside of conventional experimental settings. These new findings are key for quantitatively characterizing this interesting yet under-studied behavior.

Precisely because their initial approach was creative, it is regrettable that the authors missed the opportunity to effectively integrate preceding studies in their rationale or conclusions, which sometimes led to premature claims. Also, while each experiment contains an intriguing finding, these are poorly related to each other. This obscures the central conclusion of this work. The perceived weaknesses are discussed in detail below.

Thank you for the precise summary of the key findings and novelty of the study, and your insightful suggestions.

Most importantly, the authors' definition of "tussling" is unclear because they did not explain how they quantified lunges and tussling, even though the central focus of the manuscript is behavior. Supplemental movies S1 and S2 appear to include "tussling" bouts in which 2 flies lunge at each other in rapid succession, and supplemental movie S3 appears to include bouts of "holding", in which one fly holds the opponent's wings and shakes vigorously. These cases raise a concern that their behavior classification is arbitrary. Specifically, lunges and tussling should be objectively distinguished because one of their conclusions is that these two actions are controlled by separate neural circuits. It is impossible to evaluate the credibility of their behavioral data without clearly describing a criterion of each behavior.

We will add more details in methods.

It is also confusing that the authors completely skipped the characterization of the tussling-controlling neurons they claimed to have identified. These neurons (a subset of so-called pC1 neurons labeled by previously described split-GAL4 line pC1SS2) are central to this manuscript, but the only information the authors have provided is its gross morphology in a low-resolution image (Figure 4D, E) and a statement that "only 3 pairs of pC1SS2 neurons whose function is both necessary and sufficient for inducing tussling in males" (lines 310-311). The evidence that supports this claim isn't provided. The expression pattern of pC1SS2 neurons in males has been only briefly described in reference 46. It is possible that these neurons overlap with previously characterized dsx+ and/or fru+ neurons that are important for male aggressions (measured by lunges), such as in Koganezawa et al., Curr. Biol. 2016 and Chiu et al., Cell 2020. This adds to the concern that lunge and tussling are not as clearly separated as the authors claim.

Reply: we will perform additional morphological and functional experiments on pC1^SS2 neurons, e.g., whether they are fru or dsx positive and comparing them with P1a neurons.

While their characterizations of tussling behaviors in wild-type males (Figures 1 and 2) are intriguing, the remaining data have little link with each other, making it difficult to understand what their main conclusion is. Figure 3 suggests that one class of olfactory sensory neurons (OSN) that express Or47b is necessary for tussling behavior. While the authors acknowledged that Or47b-expressing OSNs promote male courtship toward females presumably by detecting cuticular compounds, they provided little discussion on how a class of OSN can promote two different types of innate behavior. No evidence of a functional or circuitry relationship between the Or47b pathway and the pC1SS2 neurons was provided. It is unclear how these two components are relevant to each other. Lastly, the rationale of the experiment in Figure 5 and the interpretation of the results is confusing. The authors attributed a higher mating success rate of older, socially experienced males over younger, socially isolated males to their tendency to tussle, but tussling cannot happen when one of the two flies is not engaged. If, for instance, a socially isolated 14-day-old male does not engage in tussling as indicated in Figure 2, how can they tussle with a group-housed 14-day-old male? Because aggressive interactions in Figure 5 were not quantified, it is impossible to conclude that tussling plays a role in copulation advantage among pairs as authors argue (lines 282-288).

Regarding why Or47b-expressing OSNs regulate two types of innate behaviors, we will add a discussion in the revised manuscript to explore the possible mechanisms underlying this phenomenon.

Regarding the relationship between Or47b-expressing OSNs and pC1^SS2 neurons, we conducted pathway connection analyses using the FlyWire database. Although the FlyWire database currently only contains neuronal data from female brains, these findings provide a certain degree of reference. The results indicate that at least three intermediate neurons are required to establish the connection between these two neuronal types. We hope the editor and reviewers would agree with us that identifying these intermediate neurons involved in this connection is beyond this study.

Regarding the rationale and conclusions from the experiments in Figure 5, we acknowledge the difficulty in quantifying tussling and lunging behaviors in these experiments. In the revised manuscript, we will tone down the statements about the relationship between fighting strategies and reproductive success. Additionally, we will provide further behavioral experiments to support the association between these two factors.

Despite these weaknesses, it is important to acknowledge the authors' courage to initiate an investigation into a less characterized, high-intensity fighting behavior. Tussling requires the simultaneous engagement of two flies. Even if there is confusion over the distinction between lunges and tussling, the authors' conclusion that socially experienced flies and socially isolated flies employ distinct fighting strategies is convincing. Questions that require more rigorous studies are 1) whether such differences are encoded by separate circuits, and 2) whether the different fighting strategies are causally responsible for gaining ethologically relevant resources among socially experienced flies. Enhanced transparency of behavioral data will help readers understand the impact of this study. Lastly, the manuscript often mentions previous works and results without citing relevant references. For readers to grasp the context of this work, it is important to provide information about methods, reagents, and other key resources.

We will add more details in methods and cite additional references, we will also perform additional experiment on pC1^SS2 function.