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  1. Author Response:

    Reviewer #2 (Public Review):

    The reported study includes an overall well-conducted and well-presented set of experiments. Ample data are reported and a clear and conclusive picture of the findings is portrayed.

    1. The Introduction falls short of providing the background needed for fully appreciating the current findings and their importance. The authors don't present the current understanding regarding the role of 4-vinylanisole in locusts (mostly their own work). Nor do they present the accepted knowledge of the control of sexual maturation in locusts (mostly several decades-old work). Moreover, the importance of reproductive synchrony in the life history of gregarious locusts, including its tentative roles in maintenance of the homogeneity and integrity of the swarm, in ensuring high density conditions for the next generation, and more, is also not adequately presented.

    We appreciate the reviewer’s helpful comments. According to these comments, we have revised the introduction part by enriching the significance of reproductive synchrony in ecological adaption of gregarious locusts and the research progresses on sexual maturation control in locusts. Details were shown as: “Depending on population density, locusts display striking phenotypic plasticity, with a cryptic solitarious phase and an active gregarious phase (Wang and Kang, 2014). Gregarious locusts, compared to solitarious conspecifics, show much higher synchrony in physiological and behavioral events, such as egg hatching and sexual maturation, as well as synchronous feeding and marching behaviors (Norris, 1954, Uvarov, 1977). Reproductive synchrony in gregarious locusts provides benefits for individuals in several aspects, such as more favorable microenvironment, lower risk of predation, efficiently forging, as well we more encounters with mates, therefore ensures high density conditions for the next generation, and is essential for maintenance of locust swarm (Beekman et al., 2008, Maeno et al., 2021). Some sort of vibratory stimulus, maternal microRNAs, and SNARE protein play important roles in the egg-hatching synchrony of gregarious locusts (Chen et al., 2015b, He et al., 2016, Nishide and Tanaka, 2016). It has been revealed that the presence of mature male adults has effectively accelerating effects on synchrony of sexual maturation of immature male and female conspecifics in two locust species, Schistocerca gregaria and Locusta migratoria (Norris, 1952, Loher, 1961, Guo and Xia, 1964, Norris, 1964). The accelerating effects of several prominent volatiles released by gregarious mature males in male maturation have been exampled in the desert locust. Four volatile pheromones (benzaldehyde, veratrole, phenylacetonitrile, and 4-vinylveratrole) have significantly stimulatory effects on sexual maturation of male adults, with phenylacetonitrile having the most pronounced effect. (Mahamat et al., 1993, Assad et al., 1997). However, how conspecific interaction affects female sexual maturation remains unclear and the pheromones those contribute to maturation synchrony of females have not been determined so far”. In the current study, we identify 4-vinylanisole as a key pheromone promoting sexual maturation synchrony through validating the role of five gregarious male-abundant volatiles one by one, instead of following up our previous work on 4-VA. Thus, we have fully elaborated the multifunction of 4-VA as both aggregation pheromone and maturation accelerating pheromone in the formation and maintenance of locust swarm in the discussion part.

    2. Research on pheromonal signaling in locusts have traditionally focused on compounds with a putative role in density-dependent phase-specific behaviors. Hence, it is common to compare the response of crowd-reared vs. solitary locusts to applied chemicals. The challenge, however, is maintaining the density context, while attempting to conduct controlled similar experiments with locusts of the two phases (i.e. keeping the solitary phase locusts isolated, while the gregarious locusts must always be crowded). This is even more challenging when studying reproductive physiology. By the basic nature of the two phases, there can be a multitude of interacting factors (behavioral and/or physiological) affecting the much-desired reproductive synchronization in gregarious locusts, while such synchronization is not expected at all in solitary ones (it may even be claimed to have no fitness-related advantage).

    3. In general, the authors of the current report have dealt well with these challenges, taking extra care to conduct multiple controls and making an effort to specifically test all the possible factors. However, there are several points that raise some uncertainties. For example:

    o If I am not mistaken, females of both phases were included in the study only if already mated by day A+7 (LL355-357). While this is reasonable for gregarious locusts, it may not be suitable for the solitary locusts, imposing an undesired and unequal selection criterion.

    We thank the reviewer’s comments. We don’t think the criterion (mated at PAE 6-7 days) cause significant bias in either gregarious locusts or solitarious locusts. In fact, the limitation of mating before PAE 7 days is used to rule out the effects on oviposition synchrony caused by difference in mating age among individuals. This criterion is only limited during the analysis of the first oviposition date. On the premise of consistent mating time, oviposition consistency in gregarious female adults may largely present the sexual maturation synchrony among individuals (Figure 1A). For subsequent experiments, we mainly concentrate on regulation of sexual maturation using only virgin females in all experiments.

    o In the test of the effects of conspecifics interactions, 10 gregarious locusts provided stimulation to the tested gregarious female, while only one insect was the stimulating factor for the solitary female.

    Actually, we carried out two independent experiments to test the effects of conspecifics interactions. The population densities were kept in solitarious context for comparison of female sexual maturation synchrony between typical gregarious and solitarious phases (Figure 1D). For locust emissions treatments, ten solitarious locusts were used to ensure the stimulations at the same density level (Figure 1F). Both of two experiments suggested that solitarious male adults had no effects on female sexual maturation.

    o It is not clear how were egg pods attributed to specific gregarious females (maintained in groups of 10)

    Thanks for the reviewer’s comments. To monitor the oviposition activities of each individual of gregarious females in a group, locusts were individually marked, and their first oviposition times were determined by collecting egg pods every 4 hours per day after mating. Females those laid new eggs could be easily distinguished by much thinner abdomen with white foam around ovipositor. We have provided the method details in the revised manuscript.

    Overall, since the focus of this study is actually not on the comparison between the phases, it might have been beneficial to the readers if the focus was on the gregarious locusts only, with maybe a couple of experiments conducted on solitary insects and presented separately.

    We understand the reviewer’s concern. Actually, the aim of this study is to explore the mechanism underlying sexual maturation synchrony by comparing phase- and sex-dependent conspecific interactions in locusts. The reproductive synchrony in gregarious might be not highlighted without comparison with solitarious locusts, including both first oviposition time and sexual maturation, although the mechanism studies were mostly performed in gregarious locusts. Moreover, phase-dependent comparison of volatile contents is helpful for us to screen candidate volatiles responsible for the acceleration of sexual maturation synchrony in females.

    4. Assuming that within a locust group there is overall agreement in the age of males and females, there seem to be a not-fully-explained mismatch between the age of max 4-VA release by males (linearly increasing with age) and the age of max effect in females (critical period at A+3-4)

    We appreciate the reviewer’s query. We have provided additional discussions on the “mismatch” of between age-dependent release of 4-VA by males and the age of max effect in females (PAE 3-4 days). Details were shown as: “. We find that the release of 4-VA by gregarious males continuously increased after adult eclosion, with maximal 4-VA release at PAE 8 days. The age of maximal 4-VA production outwardly seems to be unmatched with the sensitive developmental stage to 4-VA of females (PAE 3-4 days). In insects, it is very common for males to mature earlier than females (Alonzo, 2013). In the locust, male adults also display earlier sexual maturation for several days, compared to females. In given locust population, individuals emerge to adults successively in a couple of days, not in completely synchronous period. Therefore, age-dependent increase in 4-VA release in gregarious male adults presents a persistent stimulus for less-developed young female adults, and thus maximizes synchronous maturation of female locusts, which could reduce male competitions for mate selection”.

    5. Similar to the introduction, the discussion section also does not present comprehensive arguments regarding the importance of reproductive synchronization in female locusts. Points that could have been discussed include: females' oviposition disrupting migration, synchronization affecting sexual selection, accelerating intra-sex competition over mates as well as oviposition sites, and more.

    We appreciate the reviewer’s nice suggestions. We have provided additional discussions on this point following these suggestions. Details were shown as: “Reproduction synchrony involves consistence in maturation, mating, and egg laying, among which sexual maturation synchrony serves as the most foundational step for oviposition uniformity (Hassanali et al., 2005). Extremely high energy cost for female reproduction could restrict migration to pre, post, or inter oviposition period in locusts, thus have crucial effects on collective movement of local populations (Min et al., 2004). Given this, a balance of sexual maturation timing among female members presents an essential subject for maintenance of locust swarms. We here demonstrated that young female adults reared with older gregarious male adults show faster and more synchronous sexual maturation in the migratory locust, supporting the accelerate role of crowding in sexual maturation of females (Guo and Xia, 1964, Norris and Richards, 1964,). Together with the accelerating effects on immature male sexual maturation induced by older gregarious male adults reported previously (Torto et al., 1994, Mahamat et al., 2000), young adults of both sexes lived in gregarious conditions prefers more synchronous maturation than individuals reared in solitary. The consistent maturation in both sexes will greatly reduce intra- and inter-sexes competitions for mate selection and thus ensures reproductive synchronous in whole locust populations. We demonstrated that a single minor component (4-VA) of the volatiles abundantly released by gregarious male adults is sufficient to induce the maturation synchrony of female adults. By comparison, four volatiles (benzaldehyde, veratrole, phenylacetonitrile, and 4-vinylveratrole) showed stimulatory effects on male maturation (Mahamat et al., 2000). Thus, there might exist a sex-dependent action modes of maturation-accelerating pheromones: multi-component pheromones for males and single active component for females, possibly due to different selective pressures between two sexes in response to social interaction. Further exploration will be performed to confirm this hypothesis by determining whether 4-VA has maturation-accelerating effects on male adults in the migratory locust in future”.

    Reviewer #3 (Public Review):

    Strengths: Grouping behavior for marching, sexual maturation, swarming, oviposition and egg hatching in gregarious locusts is complex and it's mediated by a combination of cues-olfactory, tactile, and visual cues to ensure synchronous behavior. The authors show that only olfactory cues released by gregarious adult males mediates maturation synchrony of females. This finding is a confirmatory result of a well-established phenomenon for maturation synchrony in both sexes of adult locusts, although in this study, the authors focused on only females. Further, the authors validated their findings using gene editing techniques to show that maturation synchrony was diffused in Or35-/- mutant adult females but not in wild type females exposed to adult male volatiles and the individual component identified as 4-vinylanisole among five male-abundant volatiles as promoting synchronous sexual maturation in only post adult eclosion females (PAE) 3-4 days old. Use of molecular and single sensillum recordings, followed by physiological experiments focused on the interaction between this specific adult pheromone and juvenile hormone to validate the behavioral results found for females add scientific value to the study.

    Weaknesses: Firstly, synchronous and accelerated sexual maturation of young adults by older pheromone-producing ones, is a primer effect driven by males and this facilitates 'integration and cohesion' of both sexes of adults. In my view, the fact that this study focused on only females but not on both sexes, weakens the contribution of the study towards increased understanding of the biology/ecology of locusts.

    We accepted the reviewer’s comment that synchronous and accelerated sexual maturation of young adults by older pheromone-producing ones occurs in both sexes. In fact, early studies have reported that mature males can accelerate sexual maturation of young males through several candidate compounds (Mahamat et al.,1993, Chemoecology; and Mahamat et al., 2000; International Journal of Tropical Insect Science). However, the effects of conspecific interaction on sexual maturation of females are rarely reported. Moreover, distinct volatiles that can accelerate female sexual maturation have not been characterized before this work. Therefore, we focus on female sexual maturation synchrony in the current study. A comparison of regulatory mechanisms underlying sexual maturation synchrony in males and females has been discussed in the revised manuscript.

    There are also weaknesses in the methods, such as focusing on only the five-abundant male volatiles based on heat maps. Basically, the decision as to which components in adult male volatiles may be contributing to sexual maturation should be made by antennae of different ages of PAE females and males to avoid selecting only abundant compounds based on artificial intelligence (AI). Since most studies in this subject area have demonstrated that there is no direct correlation between volatile abundance and detection at the periphery or central nervous systems of an insect, I believe that the authors will agree with me that often some of the minor volatile components tend to contribute more to the chemical ecology of an insect than the more abundant components. Without testing minor components identified in male volatiles as a blend or individually, as additional controls to increase the robustness of the study, I am not convinced that the authors have fully achieved their aim in identifying a male-produced volatile that promotes sexual maturation in females.

    We agree the reviewer’s comments that the activities of volatiles are not always determined by the absolute contents. In fact, in our work, the selection of candidate effective compounds for female sexual maturation did not rely on the absolute content of these volatiles, but mainly based on comparative analysis of their relative contents between gregarious and solitarious male adults, because only volatiles from gregarious male adults could accelerate sexual maturation of females (Figure 1C-F). In the revision process, given that the volatiles released by gregarious males, rather than gregarious females and solitarious males, have the accelerate effects on female sexual maturation, we further performed more comparative analysis of volatile contents among these three groups (G-males, G-females, and S-males). Compared to volatiles released by G-females, and S-males, only five kinds of volatiles display significantly higher emission in G-males (PAN, guaicol, 4-VA, vertrole, and anisole). The roles of five candidate volatiles in female sexual maturation were individually validated by removing the volatile from the stimulation blend one by one. The results showed that only the omission of 4-VA from the blends lost the accelerating effects on sexual maturation synchrony of gregarious females (Figure 2B). Based on these findings, we inferred that 4-VA played major roles in promoting female sexual maturation synchrony.

    JH experiments- My main concern is the lack of proper controls to fully investigate the interactive effect of the male-produced pheromone promoting sexual maturation and juvenile hormone production. JH titers were not measured in females exposed to the other male-abundant compounds including PAN, guaiacol, veratrole and anisole or blend/individual minor components.

    We understand the reviewer’s query. In fact, the potential role of JH pathway was inferred firstly by the RNA-seq analysis of CC-CA, which showed that the expression levels of JH metabolism-related genes were significantly affected by 4-VA treatment at PAE 3-4 days. The measurement of JH titer after 4-VA treatment was further performed to support the involvement of JH in 4-VA-accelerated sexual maturation in female adults. Since other male-abundant compounds have been excluded due to the omission of any of the four volatiles (Figure 2B), we don’t think it is necessary to detect their effects on JH titers in females including PAN, guaiacol, veratrole, or anisole.

    Another notable weakness is the 'JH Rescue Experiment'. The authors did not inhibit JH synthesis in the corpora allata (allalectomized locusts) in treated locusts before injecting the JH-analog methoprene to accelerate maturation and reproduction in females.

    Thanks for the reviewer’s comments. The JH rescue experiments in Figure 4D-F were performed in Or35 female mutants, which showed lower JH levels and sexual maturation rate. Thus, the JH analog was applied to Or35^-/- females to test whether activation of JH pathway could recover sexual maturation rate and Vg expression. To provide additional evidence, we performed addition rescue experiments in WT females by inhibiting JH synthesis using Precocene (PI) before JH treatment. The results showed that PI treatment significantly inhibited sexual maturation rate and Vg expression in 4-VA-exposed WT females, whereas JH treatment post PI application can obviously recovered the sexual maturation rate and Vg expression (Figure 4G-I).

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  2. Evaluation Summary:

    The current study follows up on previous studies from this group, uncovering the role of olfactory signaling in the migratory locust. Specifically, it follows up on a recent report demonstrating that 4-vinylanisole serves as a locust aggregation pheromone. Here, this pheromone is also assigned an instrumental role in control and synchronization of female sexual maturation. This study will be useful for the understanding of swarming behaviour in locusts, and it will also interest those who work on behaviour and its modulation.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)

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  3. Reviewer #1 (Public Review):

    In their previous work, the authors had identified an aggregating pheromone produced by gregarious male locusts - 4VA. In this MS, they show that 4VA is sensed by females of all ages, but at a certain age, it induces the females to accelerate oogenesis and sexual maturity, leading to a synchronisation of egg laying. This synchrony constitutes part of the devastating swarming behavioural repertoire that make these insects a major agricultural pest.

    Specifically, the manuscript shows that when a female of age 3-4 days (and not younger or older) senses 4VA, her CC-CA organ increases JH synthesis, her haemolymph JH titre increases, and her fat body ovary show increased levels of vitellogenins. The result of all this is the acceleration of sexual maturity and consequent synchrony in oviposition.

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  4. Reviewer #2 (Public Review):

    The reported study includes an overall well-conducted and well-presented set of experiments. Ample data are reported and a clear and conclusive picture of the findings is portrayed.

    1. The Introduction falls short of providing the background needed for fully appreciating the current findings and their importance. The authors don't present the current understanding regarding the role of 4-vinylanisole in locusts (mostly their own work). Nor do they present the accepted knowledge of the control of sexual maturation in locusts (mostly several decades-old work). Moreover, the importance of reproductive synchrony in the life history of gregarious locusts, including its tentative roles in maintenance of the homogeneity and integrity of the swarm, in ensuring high density conditions for the next generation, and more, is also not adequately presented.

    2. Research on pheromonal signaling in locusts have traditionally focused on compounds with a putative role in density-dependent phase-specific behaviors. Hence, it is common to compare the response of crowd-reared vs. solitary locusts to applied chemicals. The challenge, however, is maintaining the density context, while attempting to conduct controlled similar experiments with locusts of the two phases (i.e. keeping the solitary phase locusts isolated, while the gregarious locusts must always be crowded). This is even more challenging when studying reproductive physiology. By the basic nature of the two phases, there can be a multitude of interacting factors (behavioral and/or physiological) affecting the much-desired reproductive synchronization in gregarious locusts, while such synchronization is not expected at all in solitary ones (it may even be claimed to have no fitness-related advantage).

    3. In general, the authors of the current report have dealt well with these challenges, taking extra care to conduct multiple controls and making an effort to specifically test all the possible factors. However, there are several points that raise some uncertainties. For example:

    o If I am not mistaken, females of both phases were included in the study only if already mated by day A+7 (LL355-357). While this is reasonable for gregarious locusts, it may not be suitable for the solitary locusts, imposing an undesired and unequal selection criterion.

    o In the test of the effects of conspecifics interactions, 10 gregarious locusts provided stimulation to the tested gregarious female, while only one insect was the stimulating factor for the solitary female.

    o It is not clear how were egg pods attributed to specific gregarious females (maintained in groups of 10)
    Overall, since the focus of this study is actually not on the comparison between the phases, it might have been beneficial to the readers if the focus was on the gregarious locusts only, with maybe a couple of experiments conducted on solitary insects and presented separately.

    4. Assuming that within a locust group there is overall agreement in the age of males and females, there seem to be a not-fully-explained mismatch between the age of max 4-VA release by males (linearly increasing with age) and the age of max effect in females (critical period at A+3-4)

    5. Similar to the introduction, the discussion section also does not present comprehensive arguments regarding the importance of reproductive synchronization in female locusts. Points that could have been discussed include: females' oviposition disrupting migration, synchronization affecting sexual selection, accelerating intra-sex competition over mates as well as oviposition sites, and more.

    Was this evaluation helpful?
  5. Reviewer #3 (Public Review):

    Strengths: Grouping behavior for marching, sexual maturation, swarming, oviposition and egg hatching in gregarious locusts is complex and it's mediated by a combination of cues-olfactory, tactile, and visual cues to ensure synchronous behavior. The authors show that only olfactory cues released by gregarious adult males mediates maturation synchrony of females. This finding is a confirmatory result of a well-established phenomenon for maturation synchrony in both sexes of adult locusts, although in this study, the authors focused on only females. Further, the authors validated their findings using gene editing techniques to show that maturation synchrony was diffused in Or35-/- mutant adult females but not in wild type females exposed to adult male volatiles and the individual component identified as 4-vinylanisole among five male-abundant volatiles as promoting synchronous sexual maturation in only post adult eclosion females (PAE) 3-4 days old. Use of molecular and single sensillum recordings, followed by physiological experiments focused on the interaction between this specific adult pheromone and juvenile hormone to validate the behavioral results found for females add scientific value to the study.

    Weaknesses: Firstly, synchronous and accelerated sexual maturation of young adults by older pheromone-producing ones, is a primer effect driven by males and this facilitates 'integration and cohesion' of both sexes of adults. In my view, the fact that this study focused on only females but not on both sexes, weakens the contribution of the study towards increased understanding of the biology/ecology of locusts. There are also weaknesses in the methods, such as focusing on only the five-abundant male volatiles based on heat maps. Basically, the decision as to which components in adult male volatiles may be contributing to sexual maturation should be made by antennae of different ages of PAE females and males to avoid selecting only abundant compounds based on artificial intelligence (AI). Since most studies in this subject area have demonstrated that there is no direct correlation between volatile abundance and detection at the periphery or central nervous systems of an insect, I believe that the authors will agree with me that often some of the minor volatile components tend to contribute more to the chemical ecology of an insect than the more abundant components. Without testing minor components identified in male volatiles as a blend or individually, as additional controls to increase the robustness of the study, I am not convinced that the authors have fully achieved their aim in identifying a male-produced volatile that promotes sexual maturation in females.

    JH experiments - My main concern is the lack of proper controls to fully investigate the interactive effect of the male-produced pheromone promoting sexual maturation and juvenile hormone production. JH titers were not measured in females exposed to the other male-abundant compounds including PAN, guaiacol, veratrole and anisole or blend/individual minor components.
    Another notable weakness is the 'JH Rescue Experiment'. The authors did not inhibit JH synthesis in the corpora allata (allalectomized locusts) in treated locusts before injecting the JH-analog methoprene to accelerate maturation and reproduction in females.

    Was this evaluation helpful?