Author response:
The following is the authors’ response to the original 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 now toned down the description of the paradigm and cited more 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.
We have modified the description as suggested.
(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.
Indeed, we could not faithfully distinguish boxing and tussling. To address this concern, we now made textual changes in the result section we occasionally observed the high-intensity boxing and tussling behavior in male flies, which are difficult to distinguish and hereafter simply referred to as tussling.
We also added this information in the Materials and Methods section Tussling is often mixed with boxing, in which both flies rear up and strike the opponent with forelegs. Since boxing is often transient and difficult to distinguish from tussling, we referred to the mixed boxing and tussling behavior simply as tussling.
(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 now cited this important study in both the Introduction and Discussion sections.
(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 now provided more detailed description about behavioral assays and how we analyze them. For example All testers were loaded by cold anesthesia. After a 30-minute adaptation, the film was gently removed to allow the two males to fell into the behavioral chamber, and the aggressive behavior was recorded for 2 hours.
(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 used the wtcs flies from Baker lab in Janelia Research Campus, and are not sure where they are originated. We appreciate your concern on the use of wild-type strains as they may show different fighting levels, but this study mainly used wild-type strains to compare behavioral differences between SH and GH males. All flies tested in this study are in w+ background, based on w+ balancers flies but are not backcrossed. We have listed detailed genotypes of all tested flies in Table S1 in the revised manuscript.
(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 used a fixed female to restrict it in the center of food. These females remain active throughout the assay as their legs and abdomens can still move. Such design intends to combine the attractive effects from both female and food. One can also use decapitated females, but in this case, males can push the decapitated female into anywhere in the behavioral chamber. The logic to use fixed females has now been added in the Materials and Methods section of the revised manuscript.
(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.
Indeed, tussling is very low in SH males in our paradigm, which may be due to different genetic backgrounds and behavioral assays. Since tussling behavior is a rare fighting form, it is not surprising to see variation between studies from different labs. Nevertheless, this study compared tussling behaviors in SH and GH males, and our finding that GH males show much more tussling behaviors is convincing. The longer duration of tussling in our paradigm may also be due to the modified behavioral paradigm, which also supports that tussling is a high-level fighting form.
(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.
Thank you for this important comment. We now toned down the statement on pC1SS2 function.
(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 levels of interneurons 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.
(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.
Thank you for this suggestion. We agree with you and other reviewers that increased tussling behaviors correlate with better mating competition, but it is difficult for us to make a direct link between them. Thus, in the revised manuscript, we prefer to tone down this statement but not expanding on this part.
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 agree that our data did not directly show that it is the change of aggression mode that results in territory and reproductive advantages in GH males. To address the concern, we have toned down the statement throughout the manuscript. For example, we made textual changes in the abstract as following
Moreover, shifting from lunging to tussling in socially enriched males is accompanied with better territory control and mating success, mitigating the disadvantages associated with aging. Our findings identify distinct sensory and central neurons for two fighting forms and suggest how social experience shapes fighting strategies to optimize 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.
Thank you for this important suggestion. We now analyzed duration of tussling and lunging, and found that a lunging event is often very short (less than 0.2s), while a tussling event may last from seconds to minutes. This new data is added as Figure 2G. In addition, we also provided more detailed methods regarding to tussling behavior
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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.
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.
Thank you for this very important suggestion. We now provided more detailed description of the two fighting forms in the Materials and Methods section. See below
Lunging is characterized by a male raising its forelegs and quickly striking the opponent, and each lunge typically lasts less than 0.2 seconds through detailed analysis. Tussling is characterized by both males using their forelegs and bodies to tumble over each other, and this behavior may last from seconds to minutes. Tussling is often mixed with boxing, in which both flies rear up and strike the opponent with forelegs. Since boxing is often transient and difficult to distinguish from tussling, we referred to the mixed boxing and tussling behavior simply as tussling. As we manually analyze tussling for 2 hours for each pair of males, it is possible that we may miss some tussling events, especially those quick ones.
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.
Thank you very much for this important question. Indeed, there are many experiments that could do to better understand the function of pC1SS2 neurons, and we only provide the initial characterization of them due to the limited scope of this study. My lab has been focused on studying P1/pC1 function in both male and female flies and will continue to do so.
To partially address your concern, we made the following revisions
(1) We provided higher-resolution images of P1a and pC1SS2 (Figure 4C-4E). While their cell bodies are very close, they project to distinct brain regions, in addition to some shared ones.
(2) By staining these neurons with GFP and co-staining with anti-FruM or anti-DsxM antibodies, we showed that P1a neurons are partially FruM-positive and partially DsxM-positive, while pC1SS2 neurons are DsxM-positive and FruM-negative (Figure 5A-5D).
(3) As pC1SS2 neurons are DsxM-positive and FruM-negative, we also examined how DsxM regulates the development of these neurons. We found that knocking down DsxM expression in pC1SS2 neurons using RNAi significantly affected pC1 development regarding to both cell numbers (Figure 5G) and their projections (Figure 5H).
(4) We further found that DsxM in pC1SS2 neurons is crucial for executing their tussling-promoting function, as optogenetic activation of these neurons with DsxM knockdown failed to induce tussling behavior in the initial activation period, and a much lower level of tussling in the second activation period compared to control males (Figure 5I-5K).
(5) While it is very difficult to identify the upstream and downstream neurons of P1a and pC1SS2 neurons, we made an initial step by utilizing trans-tango and retro-Tango to visualize potential downstream and upstream neurons of P1a and pC1SS2 (Figure 4-figure supplement 2), which certainly needs future investigation.
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.
It has been previously found that Or47b-expressing ORNs respond to fly pheromones common to both sexes, and group-housing enhances their sensitivity. Regarding to how Or47b ORNs promotes two different types of innate behaviors, a simple explanation is that they act on multiple second-order and further downstream neurons to regulate both courtship and aggression, not mentioning that neural circuitries for courtship and aggression are partially shared. We did not include this in the discussion as we would like to focus on aggression modes, and how different ORNs (Or47b and Or67d) mediate distinct aggression modes.
Regarding to the relationship between Or47b ORNs and pC1SS2 neurons, or in general ORNs to P1/pC1, it is interesting and important to explore, but probably in a separate study. We tried to conduct pathway connection analyses from Or47b to pC1 using the FlyWire database, and found that Or47b neurons can act on pC1 neurons via three layers of interneurons. Although the FlyWire database currently only contains neuronal data from female brains, they can provide a certain degree of reference. We hope the editor and reviewers would agree with us that identifying these intermediate neurons involved in their connection is beyond this study.
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).
Indeed, we do not have direct evidence to show it is tussling that makes socially experienced males to dominate over socially isolated males. To address your concern, we have made following revisions
(1) We toned down the statements about the relationship between fighting strategies and reproductive success throughout the manuscript. For example, in the abstract Moreover, shifting from lunging to tussling in socially enriched males is accompanied with better territory control and mating success.
(2) Regarding to whether a SH male can engage in tussling with a GH male, we found that while two SH males rarely perform tussling, paired SH and GH males displayed similar levels of tussling like two GH males, although tussling duration from paired SH and GH males is significantly lower compared to that in two GH males (Figure 6-figure supplement 2).
(3) To support the potential role of tussling in territory control and mating competition, we performed additional experiments to silence Or47b or pC1SS2 neurons that almost abolished tussling, and paired these males with control males. We found that males with Or47b or pC1SS2 neurons silenced cannot compete over control males, further suggesting the involvement of tussling in territory control and mating competition.
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.
Thank you very much for this comment and we almost totally agree.
(1) Our results suggest the involvement of distinct sensory neurons and central neurons for lunging and tussling, but do not exclude the possibility that they may also utilize shared neurons. For example, activation of P1a neurons promotes both lunging and tussling in the presence of light.
(2) We have now toned down the statements about the relationship between fighting strategies and reproductive success throughout the manuscript.
(3) We provided more detailed methods, genotypes of flies to improve transparency of the manuscript.
Reviewer #1 (Recommendations for the authors):
(1) Figure 1 Supplement 1 shows that increased aging has a linear and inverse relationship with the number of lunges, this is in contrast to a previous study from Dierick lab (Chowdhury, 2021), where using Divider assays they showed that aggressive lunges increased up to day 10 and subsequently decreased in 30-day old flies. Given that this study did not use 14-day-old flies, it might be useful to comment on this.
Thank you for this comment. Indeed, Chowdhury et al., suggested a decline of lunging after 10 days, which is not contradictory to our findings that lunging in 14d-old males is lower than that in 7d-old males. It is ideally to perform a time-series experiments to reveal the detailed relationship between ages and aggression (lunging or tussling) levels, but given our initial findings that 14d-old males showed stable tussling behavior, we prefer to use this time point for the rest of this study.
(2) For Figure 3, do various manipulations also affect the duration of tussling and boxing besides frequency and latency?
Thank you for this comment. We only analyzed latency and frequency, but not duration, as data analysis was performed manually rather than automatically on every fly pair for about 2 hours, which is very labor-consuming. We hope you could agree with us that the two parameters (frequency and latency) for tussling are representative for assaying this behavior.
(3) For Figure 3 A-F, the housing status of the males is not clearly mentioned either in the main text or the figure. What is the status of the tussling and lunging status when this housing condition is reversed when Or47b neurons are silenced, or the gene is knocked down? Do these manipulations overcome the effect of housing conditions similar to what is seen in NaChBac-mediated activation experiments?
Figure 3A-F used group-housed males and we have now added such information in the figure legends as well as Table S1.
We appreciate your suggestion on using different housing conditions. As silencing Or47b neurons or knocking down Or47b reduced tussling, it is reasonable to use GH males (as we did in Figure 3A-F) that performed stable tussling behavior, but not SH males that rarely tussle.
(4) The connections between Or47b neurons and pC1SS2 or P1a neurons can be addressed by available connectomic datasets or TransTango/GRASP approaches.
Thank you for this important suggestion. We used the FlyWire electron microscope database to analyze the pathway connections between these two types of neurons. The results indicated that there are at least three levels of interneurons for connecting Or47b and pC1 neurons. Although the FlyWire database currently only contains neuronal data from female brains, they can provide a certain degree of reference for males.
The lack of direct synaptic connection also suggests that it is challenging to resolve the connection between these two neuronal types using methods like trans-Tango/GRASP. To partially address this question, we utilized trans-Tango and retro-Tango techniques to visualize potential downstream and upstream neurons of P1a and pC1SS2 (Figure 4-figure supplement 2). Future investigations are certainly needed for clarifying functional connections between Or47b/Or67d and P1a/pC1SS2 neurons.
(5) Figure 5, 'Winning index' and 'Copulation advance index' while described in Material and Methods, should be referred to in the main text.
We now described these two indices briefly in the main manuscript, and in the Discussion section with more details.
(6) Figure 6 shows comparisons for territorial control and mating outcomes where four different housing and aging conditions are organized in a hierarchical sequence. It is not clear from the data in Figure 5, how this conclusion was arrived at. A supplementary table with various outcomes with statistical analysis would help with this.
We now added a supplementary table (Table S2) with various outcomes with statistical analysis.
Minor Comments
(1) Line 26 says that the courtship levels in SH and GH males are not different, however, unilateral wing extension is higher in SH males as compared to GH males (Pan & Baker, 2014; Inagaki et al., 2014), also it was shown that courtship attempts are higher in D. paulsitorium (Kim & Ehrman, 1998). It would be better to clarify this statement.
Indeed, it is found in some cases that SH males court more vigorously than GH males. We have added more references on this matter in the introduction.
(2) Figure 4, correct 'Tussing' to 'Tussling' or 'Box, Tussling' as appropriate.
Corrected.
(3) Duistermars, 2018 should be cited while discussing the role of vision in aggression (Figure 4). [A Brain Module for Scalable Control of Complex, Multi-motor Threat Displays]
We now cited this reference and added more discussion in the revised manuscript.
(4) Reviews on Drosophila aggression and social isolation can be cited in the introduction/discussion to incorporate recent literature e.g., Palavicino-Maggio, 2022 [The Neuromodulatory Basis of Aggression Lessons From the Humble Fruit Fly]; Yadav et al., 2024[Lessons from lonely flies Molecular and neuronal mechanisms underlying social isolation], etc.
We now cited these references in both the introduction and discussion sections.
(5) The concentration of apple juice agar should be mentioned in the methods.
We added this and other necessary information for materials in the Materials and Methods section of the study.
(6) Source of the LifeSongX software and, if available, a Github link would be helpful to include in the materials and methods section.
We now provided the source of the LifesongY software (website https//sourceforge.net/projects/lifesongy/), which is a Windows version of LifesongX (Bernstein, Adam S.et al., 1992).
Reviewer #2 (Recommendations for the authors):
(1) Major comment 1
As pointed out in the public review, the weakness of this study is that the relationship between the aggression strategy and reproductive success is an inference that is not based on experimental facts; I understand that the frequency of tussling is not so high, but at least tussling-like behavior can be observed in the territory control experiment shown in Video 3. Wouldn't it be possible to re-analyse data and examine the correlation between aggressive behavior and territory control? Even if the analysis of tussling itself in this setup is difficult, for example, additional experiments using Or47b knock-out fly or pC1[SS2]-inactivated fly could provide stronger support.
Indeed, we can only make a correlation between the type of aggressive behavior and territory control. We now toned down this statement throughout the manuscript. For example, in the abstract, we changed our conclusions as following
Moreover, shifting from lunging to tussling in socially enriched males is accompanied with better territory control and mating success. Our findings identify distinct sensory and central neurons for two fighting forms and suggest how social experience shapes fighting strategies to optimize reproductive success.
To further address the concern, we now performed additional experiments to silence Or47b or pC1SS2 neurons that almost abolished tussling, and paired these males with control males. We found that males with Or47b or pC1SS2 neurons silenced cannot compete over control males (Figure 6-figure supplement 3), further suggesting the involvement of tussling in territory control and mating competition.
In relation to the above, some of the text in the Abstract should be changed.Line 28 These findings "reveal" how social experience shapes fighting strategies to optimise reproductive success.
"suggest" is more accurate at this stage.
Changed as suggested.
(2) Major comment 2
The tussling is the central subject of this paper. However, neither the main text nor Materials and Methods section provides a clear explanation of how this aggression mode was detected. Did the authors determine this behavior manually? Or was it automatically detected by some kind of image analysis? In either case, the criteria and method for detecting the tussling should be clearly described.
The behavioral data analysis in this study was performed manually. We now provided more detailed description of the two fighting forms in the Materials and Methods section. See below
Lunging is characterized by a male raising its forelegs and quickly striking the opponent, and each lunge typically lasts less than 0.2 seconds through detailed analysis. Tussling is characterized by both males using their forelegs and bodies to tumble over each other, and this behavior may last from seconds to minutes. Tussling is often mixed with boxing, in which both flies rear up and strike the opponent with forelegs. Since boxing is often transient and difficult to distinguish from tussling, we referred to the mixed boxing and tussling behavior simply as tussling. As we manually analyze tussling for 2 hours for each pair of males, it is possible that we may miss some tussling events, especially those quick ones.
For the experimental groups where tussling cannot be observed, the latency is regarded as 120 min, but this is a value depending on the observation time. While it is reasonable to use the latency to evaluate the behavior such as the lunging that is observed at relatively early times, care should be taken when using it to evaluate the tussling. Since similar trends to those obtained for the latency are observed for Number of tussles and % of males performing tussling, it may be better to focus on these two indices.
We initially intended to provide all three statistical metrics. However, we found that using the "% of males performing tussling" would require a significantly larger sample size for subsequent statistical analysis (using chi-square tests), greatly increasing the workload. At the same time, we believe that the trend observed with "% of males performing tussling" is consistent with the other two indices, and the percentage information can also be derived from the individual sample scatter data of the other two metrics. Therefore, we opted to use "latency" and "numbers" as the statistical metrics, despite the caveat as you mentioned.
The authors repeatedly mention that tussling is less frequent but more vigorous. The low frequency can be understood from the data in Fig. 1 and Fig. 2, but there are no measured data on the intensity. As the authors mention in line 125, each tussling event appears to be sustained for a relatively long period, as can be seen from the ethogram in Fig. 2. For example, it would be possible to evaluate the intensity by measuring the duration of the tussling event.
Thank you for your valuable suggestion. We now analyzed duration of tussling and lunging, and found that a lunging event is often very short (less than 0.2s), while a tussling event may last from seconds to minutes, further supporting their relative intensities. This new data is added as Figure 2G.
(3) Minor comments
a) Line 117 How many flies were placed in one vial for group-rearing (GH)? Were males and females grouped together? Please specify in the Materials and Methods section.
We have added this information in the Materials and Methods section. In brief, 30-40 virgin males were collected after eclosion and group-housed in each food vial.
b) Line 174 The trans-Tango is basically a postsynaptic cell labeling technique. It is unlikely that the labeling intensity changes depending on neuronal activity. Do the authors want to say in this text the high activity of Or47b-expressing neurons under GH conditions? Or are they trying to show that the expression level of the Or47b gene, which is supposedly monitored by the expression of GAL4, is increased by GH conditions? The authors should clarify which is the case.
Although the primary function of the trans-Tango technique is to label downstream neurons, the original literature indicates that the signal strength in downstream neurons depends on the use of upstream neurons evidenced by age-dependent trans-Tango signals. Therefore, the trans-Tango technique can indirectly reflect the usage of upstream neurons. Our findings that GH males showed broader Or47b trans-Tango signals than SH males can indirectly suggest that group-housing experience acts on Or47b neurons. We made textually changes to clarify this.
c) Line 178 Which fly line labels the mushroom body; R19B03-GAL4?
Yes, we now provided the detailed genotypes for all tested flies in the Table S1.
d) Line 184 It was reported in Koganezawa et al., 2016 that some dsx-expressing pC1 neurons are involved in aggressive behavior. The authors should also refer to this paper as they include tussling in the observed aggressive behavior.
Thank you for this comment, and we now cited this reference in the revised manuscript.
e) Line 339 I think you misspelled fruM RNAi.
Thank you for pointing this out. fruMi refers to microRNAi targeting fruM, and we have now clearly stated this information in the main text.
f) Line 681 Is tussling time (%) the total duration of tussling occurrences during the observation time? Or is it the percentage of individuals observed tussling during the observation time? This needs to be clarified.
It is the former one. We now clearly stated this definition in the Materials and Methods section
Reviewer #3 (Recommendations for the authors):
For authors to support their conclusion that enhanced tussling among socially experienced flies allows them to better retain resources, it is necessary to quantify aggressive behaviors (mainly tussling and lunging) in Figure 5.
We agree that we can only make a correlation between enhanced tussling behavior and mating competition. We now toned down this statement throughout the manuscript. For example, in the abstract, we changed our conclusions as following Moreover, shifting from lunging to tussling in socially enriched males is accompanied with better territory control and mating success. Our findings identify distinct sensory and central neurons for two fighting forms and suggest how social experience shapes fighting strategies to optimize reproductive success.
To further address the concern, we now performed additional experiments to silence Or47b or pC1SS2 neurons that almost abolished tussling, and paired these males with control males. We found that males with Or47b or pC1SS2 neurons silenced cannot compete over control males (Figure 6-figure supplement 3), further suggesting the involvement of tussling in territory control and mating competition.
In contrast to the authors' data in Figure 4, movies in ref 36 clearly show instances of 2 flies exchanging lunges after the optogenetic activation of P1a neurons, like the examples shown in supplementary movies S1-S3. It is a clear discrepancy that requires discussion (and raises a concern about the lack of transparency about behavioral quantification).
In our study, optogenetic activation of P1a neurons failed to induce obvious tussling behavior, and temperature-dependent activation of P1a neurons can only induce tussling in the presence of light. These data are different from Hoopfer et al., (2015), but are generally consistent with a new study (Sten et al., Cell, 2025), in which pC1SS2 neurons but not P1a neurons promote aggression. Such discrepancy has now been discussed in the revised manuscript.
The authors often fail to cite relevant references while discussing previous results, which compromises the scholarship of the manuscript. Examples include (but are not limited to)
(1) Line 85-86 Simon and Heberlein, J. Exp. Biol. 223 jeb232439 (2020) suggested that tussling is an important factor for flies to establish a dominance hierarchy.
Reference added.
(2) Line 142-143 Cuticular compounds such as palmitoleic acid are characterized to be the ligands of Or47b by ref #18.
Reference added.
(3) Line 185-187 pC1SS1 and pC1SS2 are first characterized by ref #46. Expression data of this paper also implies that pC1SS1 and pC1SS2 label different neurons in the male brain.
We have now added this reference at the appropriate place in the revised manuscript. In addition, we have clarified that these two drivers exhibit sexually dimorphic expression patterns in the brain.
(4) Line 196-199 Cite ref #36, which describes the behavior induced by the optogenetic activation of P1a neurons.
Reference added.
(5) Line 233-235 The authors' observation that control males do not form a clear dominance directly contradicts previous observations by others (Nilsen et al., PNAS 10112342 (2002); Yurkovic et al., PNAS 10317519 (2006); also see Trannoy et al., PNAS 1134818 (2016) and Simon and Heberlein above). The authors must at least discuss why their results are different.
There is a misunderstanding here. We clearly state that there is a ‘winner takes all’ phenomenon. However, for wild-type males of the same age and housing condition, we calculated the winning index as (num. of wins by unmarked males – num. of wins by marked males)/10 encounters * 100%, which is roughly zero due to the randomness of marking.
(6) Line 251-254 The authors' observation that aged males are less competitive than younger males contradicts the conclusion in ref #18. Discussion is required.
We have now added a discussion on this matter. In brief, Lin et al., showed that 7d-old males are more competitive than 2d-old males, which is probably due to different levels of sexual maturity of males, but not a matter of age like our study that used up to 21d-old males.
(7) Line 274-275 It is unclear which "previous studies" "have found that social isolation generally enhances aggression but decreases mating competition in animal models". Cite relevant references.
Reference added.
(8) Line 309-310 The evidence supporting the statement that "there are only three pairs of pC1SS2 neurons". If there is a reference, cite it. If it is based on the authors' observation, data is required.
We have now provided additional data on the number of pC1SS2 neurons in Figure 5G of the revised manuscript.
