Aggregation pheromone 4-vinylanisole promotes the synchrony of sexual maturation in female locusts

  1. Dafeng Chen
  2. Li Hou
  3. Jianing Wei
  4. Siyuan Guo
  5. Weichan Cui
  6. Pengcheng Yang
  7. Le Kang  Is a corresponding author
  8. Xianhui Wang  Is a corresponding author
  1. State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, China
  2. CAS Center for Excellence in Biotic Interactions, University of Chinese Academy of Sciences, China
  3. Beijing Institutes of Life Sciences, Chinese Academy of Sciences, China

Abstract

Reproductive synchrony generally occurs in various group-living animals. However, the underlying mechanisms remain largely unexplored. The migratory locust, Locusta migratoria, a worldwide agricultural pest species, displays synchronous maturation and oviposition when forms huge swarm. The reproductive synchrony among group members is critical for the maintenance of locust swarms and population density of next generation. Here, we showed that gregarious female locusts displayed more synchronous sexual maturation and oviposition than solitarious females and olfactory deficiency mutants. Only the presence of gregarious male adults can stimulate sexual maturation synchrony of female adults. Of the volatiles emitted abundantly by gregarious male adults, the aggregation pheromone, 4-vinylanisole, was identified to play key role in inducing female sexual maturation synchrony. This maturation-accelerating effect of 4-vinylanisole disappeared in the females of Or35-/- lines, the mutants of 4-vinylanisole receptor. Interestingly, 4-vinylanisole displayed a time window action by which mainly accelerates oocyte maturation of young females aged at middle developmental stages (3–4 days post adult eclosion). We further revealed that juvenile hormone/vitellogenin pathway mediated female sexual maturation triggered by 4-vinylanisole. Our results highlight a ‘catch-up’ strategy by which gregarious females synchronize their oocyte maturation and oviposition by time-dependent endocrinal response to 4-vinylanisole, and provide insight into reproductive synchrony induced by olfactory signal released by heterosexual conspecifics in a given group.

Editor's evaluation

This study describes an important aspect of the devastating swarming behaviour of locusts – how gregarious female locusts might synchronise oviposition. It uncovers a role for olfactory signaling. The authors find that the aggregation pheromone 4-vinylanisole released by gregarious males is instrumental in 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.

https://doi.org/10.7554/eLife.74581.sa0

eLife digest

Since 2019, a plague of flying insects known as migratory locusts has been causing extensive damage to crops in East Africa. Migratory locusts sometimes live a solitary lifestyle but, if environmental conditions allow, they form large groups containing millions of individuals known as swarms that are responsible for causing locust plagues.Locusts are able to maintain such large swarms because they can aggregate and synchronize.

When they live in swarms, individual locusts produce odors that are sensed by other individuals in the group. For example, an aggregation pheromone, called 4-vinylanisole, is known to help keep large groups of locusts together. However, it is less clear how odors synchronize the reproductive cycles of the females in a swarm so that they are ready to mate with males and lay their eggs at the same time.

To address this question, Chen et al. examined when female locusts reached sexual maturity after they were exposed to odors produced by other locusts living alone or in groups. The experiments found that only 4-vinylanisole, which was abundantly released by adult male locusts living in groups, stimulated female locusts to reach sexual maturity at the same time. This odor increased the levels of a hormone known as juvenile hormone in less-developed females to help them reach sexual maturity sooner.

These findings demonstrate that when migratory locusts are living in swarms, male locusts promote the female locusts to reach sexual maturity at the same time by promoting less-developed females to ‘catch up’ with other females in the group. A next step will be to investigate the neural and molecular mechanisms underlying the ‘catch up’ effect induced by 4-vinylanisole.

Introduction

Reproductive synchrony, characterized by a pronounced temporal clustering of births, estrus, or mating, widely occurs in the animal kingdom, especially in group-living species (Ims, 1990). Several prominent cases are best known for their extreme manifestations, for example, sea turtle oviposition, firefly flashing, and fish spawning, involving a mass of individuals with the same reproductive state at certain time windows (Buck and Buck, 1968; Harrison et al., 1984; Kelly and Sork, 2002). Reproductive synchrony may offer adaptive advantages for group-living species, such as predation swamping and inbreeding avoidance (Janzen, 1971). Therefore, understanding how reproductive cycle is synchronized among individuals would provide insight to the biological flexibility in group-living animals.

Reproductive synchrony is a complex process that requires the integration of extra- and endo-signals to coordinate the timing of reproductive cycles between individuals in a group (Kobayashi et al., 2002; Dey et al., 2015). In fact, intra-group variation in developmental status can be induced by many factors, including different nutrition, temperature, and order of eclosion (Ward and Webster, 2016), which essentially makes synchronous reproduction between all members an apparent improbability. Social interaction is considerably critical for triggering reproductive synchrony of individuals in group-living species (French and Stribley, 1985; Ims and Steen, 1990; Jovani and Grimm, 2008). A well-known example is the Whitten effect which is induced by the presence of males in rodents, ewes, and monkey (Vandenbergh, 1967; Cahill et al., 1974; Gattermann et al., 2002). Various kinds of signals, odors, touch, or voice can act as social clues to underpin synchronization with reproduction (Rekwot et al., 2001; Kobayashi et al., 2002; Noguera and Velando, 2019). Endo-signals, such as hormone release, gene expression, and epigenetic modification, have also been suggested to be involved in these interaction processes (Engel et al., 2016; Noguera and Velando, 2019). However, the mechanisms by which social cue/hormone interaction synchronizes the reproductive cycles of individuals within local breeding groups remain largely unknown.

Locusts often form large swarms with hundreds to thousands of individuals, regarded as one of the most extraordinary examples of coordinated behavior (Ariel and Ayali, 2015; Buhl and Rogers, 2016). 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 as 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., 2015; 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, 1997; Guo and Xia, 1964; Norris and Richards, 1964). The accelerating effects of several prominent volatiles released by gregarious mature males in male maturation have been examined in the desert locust. Four volatile pheromones (benzaldehyde, veratrole, phenylacetonitrile [PAN], and 4-vinylveratrole) have significantly stimulatory effects on sexual maturation of male adults, with PAN having the most pronounced effect (Mahamat et al., 1993; Assad et al., 1997). However, how conspecific interactions affect female sexual maturation remain unclear and the pheromones those contribute to maturation synchrony of females have not been determined so far.

In this study, we investigated mechanisms underlying sexual maturation synchrony of female adults in the migratory locust by comparing phase-related maturation patterns of females using multi-disciplinary studies, including physiology, chemical ecology, genomics, and gene manipulation. Unexpectedly, we found that aggregation pheromone, 4-vinylanisole, induced sexual maturation synchrony of female adult locusts. Our results highlight a parsimonious role of olfactory cues in the formation of locust swarms by triggering aggregation behavior and sexual maturation synchrony.

Results

Olfactory signals from gregarious male adults trigger the synchrony of female sexual maturation in locusts

We first investigated whether there was a difference of reproduction synchrony between gregarious and solitarious female locusts by determining the distribution of first oviposition date. The curve of the first oviposition time of gregarious females was much narrower than that of solitarious females (60% decrease in the standard deviation [SD], Figure 1A), implying that the first reproductive cycle was more consistent among gregarious female individuals. As an essential premise of egg laying, sexual maturation states, indicated by the length of terminal oocyte relative to the final mature size, were then measured. The development of terminal oocyte increased with ages of female adults of both phases. Gregarious female adults displayed more uniform and rapid patterns than that of solitarious females after 4 days post adult eclosion (PAE 4 days) (Figure 1B and Figure 1—figure supplement 1). These results indicate that gregarious female adults display significant sexual maturation synchrony and higher maturation rate.

Figure 1 with 2 supplements see all
Olfactory signals from gregarious male adults trigger the synchrony of female sexual maturation in locusts.

(A) Distribution of the first oviposition time of gregarious and solitarious phases. The first oviposition date was recorded after 6 days post adult eclosion (PAE 6 days) when individuals began to mate. To ensure the consistency of mating time in gregarious and solitarious locusts, females that did not successfully mate with 24 hr after pairing were excluded. For gregarious locusts, females were individually marked, and their first oviposition times were recorded by collecting egg pods every 4 hr per day after mating. Females those laid new eggs could be easily distinguished by much thinner abdomen with white foam around ovipositor. Ages of first oviposition were indicated by days post eclosion. (B) The maturity of gregarious and solitarious females from PAE 2 to 8 days. The sexual maturity was presented as the length of terminal oocyte relative to the final mature size. (C) The maturity of gregarious females reared with gregarious males or females, separately. (D) The maturity of solitarious females reared with solitarious males or females, separately. (E) The maturity of gregarious females stimulated by volatiles released from gregarious males or females. (F) The maturity of solitarious females stimulated by volatiles released from gregarious or solitarious males. (G) Distribution of the first oviposition time in wild-type (WT) females and Orco female mutants (Orco-/-). (H) The maturity of WT females and Orco-/- females reared with gregarious males. (I) The maturity of WT females and Orco-/- females stimulated by volatiles released from gregarious males. Only virgin females were used in all experiments refer to sexual maturation examination. Dark lines in violin plots indicate median value. White dotted lines indicate upper and lower quartiles, respectively. Consistency analysis was analyzed using Levene’s test. The number of biological replicates and p values were shown in the figures.

Figure 1—source data 1

Raw data for first oviposition time and sexual maturity of gregarious, solitarious, and Orco-/- female adults.

https://cdn.elifesciences.org/articles/74581/elife-74581-fig1-data1-v1.xlsx

We next investigated whether conspecific interactions can induce sexual maturation synchrony of female adults (Figure 1—figure supplement 2A and B). We found that the maturation synchrony of terminal oocytes of females was significantly retarded by the removal of male adults in gregarious phase (Figure 1C). However, raised with either solitarious female or male did not affect sexual maturation of solitarious females (Figure 1D). The exposure of odor blends from gregarious male adults significantly advanced the maturation synchrony of gregarious females and solitarious females, whereas no effects were observed when exposed to the background air, female odors, or odors from solitarious males (Figure 1E and F and Figure 1—figure supplement 2C, D).

To further explore the roles of olfactory cues in females’ sexual maturation process, we examined the performance of loss-of-function mutants of olfactory receptor co-receptor gene (Orco-/-) established by CRISPR/Cas9, which display significant olfactory deficiency (Li et al., 2016). The best-fit normal curve of the first oviposition date was much wider in Orco-/- females than in wild-type (WT) females, when they were reared together with gregarious males (with 63% increase in the SD, Figure 1G). When reared together with gregarious male adults or exposure to their odor blends, the sexual maturation of Orco-/- females was less synchronous than that of WT females (Figure 1H, I and Figure 1—figure supplement 2E, F). Thus, olfactory signals from gregarious male adults are essential for triggering the synchrony of female sexual maturation in migratory locusts.

4-Vinylanisole abundantly released by gregarious male adults mediates sexual maturation synchrony of female locusts

To identify key active compounds that can promote sexual maturation synchrony of female locusts, we compared the volatile emission dynamics of gregarious male adults, gregarious female adults, and solitarious male adults from PAE 1 to 8 days. In total, 14 chemicals were identified in the volatiles released by male adults (Figure 2A). After PAE 4 days, only five compounds displayed considerably higher abundance in gregarious male adults, compared to that released by gregarious female adults and solitarious male adults, which showed no accelerating effects on female sexual maturation synchrony. Thus, these five kinds of gregarious male adult-abundant volatiles, including PAN, guaicol, 4-vinylanisole (4-VA), vertrole, and anisole (Figure 2A and Figure 2—figure supplement 1), might serve as candidate pheromones for female maturation acceleration.

Figure 2 with 3 supplements see all
4-Vinylanisole abundantly released by gregarious male adults promotes sexual maturation synchrony of female locusts.

(A) Dynamic changes in volatiles released from gregarious male adults, gregarious female adults, and solitarious male adults from post adult eclosion (PAE) 1 to 8 days. (B) The maturity of gregarious females stimulated with different volatile mixtures containing gregarious male-abundant compounds. Ten gregarious virgin females were stimulated by the mixed odor blend (phenylacetonitrile [PAN], guaiacol, 4-vinylanisole [4-VA], vertrole, and anisole) or paraffin oil from PAE 1 to 6 days. (C) The maturity of gregarious females treated with five kinds of gregarious male-abundant volatiles or 4-VA alone. (D) Dosage effects on the maturity of gregarious females after 4-VA stimulation. (E) Distribution of the first oviposition time in wild-type (WT) and Or35-/- females. (F) The maturity of WT and Or35-/- females reared with gregarious male adults. (G) The maturity of WT and Or35-/- females with or without 4-VA stimulation. Dark lines in violin plot indicate median value. White dotted lines indicate upper and lower quartile, respectively. Consistency analysis was analyzed using Levene’s test. The number of biological replicates and p values were shown in the figures.

Figure 2—source data 1

Raw data for volatile contents in adults and sexual maturity of females.

https://cdn.elifesciences.org/articles/74581/elife-74581-fig2-data1-v1.xlsx

We exposed gregarious young female locusts for 6 days after fledging to different synthetic blends of those five compounds (PAN, guaicol, 4-VA, vertrole, and anisole). The full blend of five components was effective in promoting the synchrony of oocyte development. Only the omission of 4-VA, but not other four compounds, from the full blend lost the accelerating effects on sexual maturation synchrony of gregarious females (Figure 2B). Moreover, the exposure to 4-VA can induce similar effects on female sexual maturation synchrony to the full blend (Figure 2C). In addition, the accelerating effects of 4-VA on maturation synchrony displayed a dose-threshold pattern, with an effective concentration more than 100 ng (Figure 2D and Figure 2—figure supplement 2). We further examined the performance of Or35-/- females that cannot sense 4-VA (Guo et al., 2020). Compared to WT females, the best-fit normal curve of the first oviposition date of Or35-/- females was much wider (52% increase in the SD, Figure 2E). The sexual maturation in Or35-/- females was more uneven than that of WT females when they were reared together with gregarious males (Figure 2F) or exposed to the odors of gregarious males (Figure 2—figure supplement 3). Moreover, the synchronous effects of 4-VA completely disappeared in Or35-/- females (Figure 2G).

4-VA action needs a critical time window on sexual maturation synchrony in young females

In fact, intra-group variation generally exists in the maturation period of female locusts due to differences in nymph experience, nutrition, and fledging time (Uvarov, 1977). We hypothesized that there is a differential effect of 4-VA on the maturation rate for female individuals with distinct developmental statuses to achieve maturation synchrony. To test this, we determined the accelerating effects of 4-VA on young females at three different ages after fledging: PAE 1–2 days, 3–4 days, and 5–6 days, respectively. We found that female maturation synchrony was significantly enhanced only when young females were treated by 4-VA at PAE 3–4 days, while did not change at PAE 1–2 days or PAE 5–6 days, indicating the time window of 4-VA action on sexual maturation of females (Figure 3A). Moreover, we compared the effects of gregarious males with different ages on female maturation. The maturation synchrony of females was significantly enhanced by gregarious males aged at PAE 3–4 days and PAE 5–6 days (Figure 3—figure supplement 1), which could release more 4-VA (Figure 2—figure supplement 1). By contrast, rearing together with the fifth instar of gregarious males and male adults aged at PAE 1–2 days did not significantly affect the maturation synchrony of gregarious female adults (Figure 3—figure supplement 1).

Figure 3 with 6 supplements see all
4-Vinylanisole (4-VA) promotes sexual maturation synchrony in young females by enhancing juvenile hormone/vitellogenin (JH/Vg) signaling pathway at post adult eclosion (PAE) 3–4 days.

(A) The 4-VA effects on sexual maturity of gregarious virgin females at different developmental stages. (B) Dosage effects on electroantennography (EAG) responses of females to 4-VA at different developmental stages. EAG responses to 4-VA with different concentrations were recorded in the antennae of female adults aged at PAE 2 days, PAE 4 days, and PAE 6 days, respectively (n = 7–11). (C) Volcano plot of RNA-seq in the corpus cardiacum-corpora allatum (CC-CA) complex of gregarious females after 4-VA stimulation at PAE 3–4 days. Red dots indicate genes related to JH metabolism. (D) Expression changes of JH metabolism-related genes in the CC-CA by 4-VA stimulation. Red and blue indicate upregulated and downregulated, respectively. (E) JH titers in the hemolymph, (F) the mRNA levels, (G) the protein levels of Vg in the fat body, and (H) the protein levels of Vg in the ovary of gregarious females after 4-VA stimulation at different developmental stages. Dark lines in violin plot indicate median value. White dotted lines indicate upper and lower quartiles, respectively; columns show means ± SEM. Consistency analysis of maturity was analyzed using Levene’s test. The mRNA and protein levels were analyzed using Student’s t-test. The number of biological replicates and p values were shown in the figures. n.s., not significant.

Figure 3—source data 1

Raw data for sexual maturity, juvenile hormone (JH) titer, gene expression, and protein level in 4-vinylanisole (4-VA)-treated females.

https://cdn.elifesciences.org/articles/74581/elife-74581-fig3-data1-v1.xlsx

Gene expression profiles in fat body tissue have been demonstrated to correlate tightly with the sexual maturation of female locusts (Guo et al., 2014). Therefore, we further evaluated the time window effects of 4-VA on female sexual maturation at the transcriptomic level. Through RNA-seq, we verified that the gene expression profiles of fat body displayed more remarkable changes in female adults exposed to 4-VA at PAE 3–4 days (1700 differentially expressed genes [DEGs]) than those at PAE 1–2 days (505 DEGs) and at PAE 5–6 days (582 DEGs) (Figure 3—figure supplement 2). Meanwhile, Kyoto encyclopedia of genes and genomes enrichment analysis showed that there were more signal pathways affected by 4-VA treatment at PAE 3–4 days than PAE 1–2 days and PAE 5–6 days. Notably, genes related to energy metabolism, such as retinol metabolism, glycerolipid metabolism, pyruvate metabolism, as well as fatty acid biosynthesis, which play essential roles in ovary development, were significantly activated by 4-VA treatment at PAE 3–4 days (Figure 3—figure supplement 2). Thus, PAE 3–4 days should be a critical time window for 4-VA-induced acceleration of female sexual maturation.

JH/Vg signaling pathway mediates the accelerating effect of 4-VA on sexual maturation synchrony in young females

To explore the regulatory mechanism underlying the time window effects of 4-VA on the sexual maturation synchrony of female locusts, we examined the performance of major signaling pathways involved in the sexual maturation of female locusts. First, we determined whether females display time-dependent electrophysiological response to 4-VA by performing electroantennography (EAG) and single sensilla response (SSR) experiments. We found that 4-VA-induced EAG and SSRs of female adults displayed obvious dose-dependent effects (Figure 3B and Figure 3—figure supplement 3). However, there was no difference of EAG and SSRs of females among the ages of PAE 2 days, PAE 4 days, and PAE 6 days, although LmigOr35 expression levels were dynamic during ovary development (Figure 3B and Figure 3—figure supplements 3 and 4). These results suggested that peripheral olfactory perception may not be involved in the time window effects of 4-VA.

We then compared gene expression profiles of two main neuroendocrinal tissues, the brain and corpus cardiacum-corpora allatum (CC-CA) complex, between the controls and 4-VA-exposed females at PAE 3–4 days. Notably, gene expression profiles in CC-CA significantly changed upon 4-VA treatment, with 290 DEGs, much more than that in the brain (89 DEGs) (Figure 3C and Figure 3—figure supplement 5), implying the molecular and physiological activities in CC-CA might be remarkably affected by 4-VA stimuli. Moreover, a series of DEGs of CC-CA involved in juvenile hormone (JH) metabolism were enriched. There was significantly higher expression of genes related to JH synthesis but lower expression of genes associated with JH degradation (Figure 3C and D and Figure 3—figure supplement 6 and Supplementary file 1). These results indicated a potential role of JH signaling pathway in mediating the effects of 4-VA at PAE 3–4 days.

We therefore tested whether 4-VA exposure can affect the hemolymph JH titer in immature females. As expected, the JH titer was significantly elicited by 4-VA exposure in females aged at PAE 3–4 days, rather than PAE 1–2 days or PAE 5–6 days (Figure 3E). Similarly, the expression levels of vitellogenin (Vg), a key downstream component of JH signaling triggering ovary development in locusts (Song et al., 2014), were prominently increased in fat body and ovary of females aged at PAE 3–4 days upon 4-VA stimuli (Figure 3F–H). By comparison, JH titer did not significantly change in Or35-/- females exposed to 4-VA, contrast to over twofold increase in WT females (Figure 4A). Similar patterns were observed for the expression levels of Vg in fat body (Figure 4B and Figure 4—figure supplement 1A) and ovary (Figure 4C). To verify the critical roles of the JH/Vg pathway in mediating the effect of 4-VA, we further carried out rescue experiments by the injection of JH analog (methoprene) in Or35-/- females. Methoprene-injected Or35-/- females displayed more uniform sexual maturation (Figure 4D). Meanwhile, the expression levels of Vg in fat body and ovary significantly increased in methoprene-injected Or35-/- females (Figure 4E and F, and Figure 4—figure supplement 1B). Results of rescue experiments on WT females indicated that inhibition of JH synthesis by destroying CA using precocene I blocked the 4-VA-accelarated female sexual maturation and Vg expression, which could be recovered by JH III application after precocene treatment (Figure 4G–I). These results provide clear evidence that the JH/Vg signaling pathway can mediate the time-dependent accelerating effects of 4-VA on sexual maturation synchrony in female locusts.

Figure 4 with 1 supplement see all
Juvenile hormone/vitellogenin (JH/Vg) pathway indeed mediates the stimulatory effects of 4-vinylanisole (4-VA) on female sexual maturation.

(A) JH titers in the hemolymph of wild-type (WT) and Or35-/- females after stimulation by 4-VA at post adult eclosion (PAE) 3–4 days. (B) The mRNA levels of Vg in the fat body and (C) protein levels of Vg in the ovaries of WT and Or35-/- females after stimulation by 4-VA at PAE 3–4 days. (D) The effects of JH analog treatments on the maturity of Or35-/- females exposed to 4-VA. (E) The mRNA level of Vg in the fat body and (F) protein level of Vg in the ovary of Or35-/- females with 4-VA stimulation and JH analog treatments at PAE 3–4 days. (G) Sexual maturity in WT females treated by 4-VA, precocene I, and JH III. (H) The mRNA level of Vg in the fat body and (I) protein level of Vg in the ovary of WT females treated by 4-VA, precocene I, and JH III. All insects used were virgin females. Boxplots depict median and upper and lower quartiles. Lines in droplet diagram indicate median value; columns show means ± SEM. One-way ANOVA, p < 0.05. Columns labeled with different letters indicate a significant difference between these groups. The number of biological replicates is shown in the figure.

Figure 4—source data 1

Raw data for juvenile hormone (JH) titer, gene expression, protein level, and sexual maturity in wild-type (WT) and Or35-/- females.

https://cdn.elifesciences.org/articles/74581/elife-74581-fig4-data1-v1.xlsx

Discussion

Our current study demonstrates conclusively that aggregation pheromone, 4-VA, acts to promote female maturation synchrony in locusts. The pheromone is abundantly released from gregarious male adults and speeds up oocyte development of females aged at PAE 3–4 days through activating JH synthesis and vitellogenesis (Figure 5). Our findings highlight a ‘catch-up’ strategy of reproductive synchrony by a time window effect combined with extra- and endo-signals in group-living animals.

Schematic mechanisms underlying 4-vinylanisole (4-VA)-induced synchrony of female sexual maturation.

4-VA released from gregarious male locusts can significantly accelerate the ovary development of females with less-developed ovaries (approximately before post adult eclosion [PAE] 4 days) but not well-developed ovaries (after PAE 4 days). Mechanistically, after recognition by Or35 expressed in antennae, 4-VA promoted juvenile hormone (JH) synthesis in the CC and vitellogenesis in the fat body, thus accelerating female sexual maturation. The time-dependent stimulatory effects of 4-VA on ovary development finally led to the synchrony of female sexual maturation. CC-CA, corpora cardiaca and corpora allata; FB, fat body.

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., 2011), young adults of both sexes lived in gregarious conditions prefer 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, PAN, and 4-vinylveratrole) showed stimulatory effects on male maturation (Mahamat et al., 2011). 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.

We prefer that 4-VA acts as a critical multi-functional pheromone for the formation of large locust swarms. Earlier, we have demonstrated that 4-VA is mainly released by gregarious nymphs and male adults (Wei et al., 2017) and can induce strong attraction behavior of both gregarious and solitarious phases (Guo et al., 2020), indicating its releaser pheromone role in in keeping locust individuals living together. Meanwhile, the present study shows that 4-VA, acting as a primer pheromone, promotes the maturation synchrony of young female adults, which might facilitate simultaneous oviposition and egg hatching to reduce the predation risk of an individual via the dilution effect (Ward and Webster, 2016). Thus, a dual role of 4-VA, including both primer and releaser pheromones, could be proposed in triggering the formation of locust swarms. The maintenance and coordination of locust swarming require elaborate communication mechanisms behind the interaction among individuals (Pener and Simpson, 2009; Wang and Kang, 2014). It is likely an effective and optimized strategy of group-living animals to use a single chemical pheromone to elicit both behavioral and endocrine responses in conspecifics (Rekwot et al., 2001). The action of 4-VA displays a remarkable context-dependent manner, such as phase-, sex-, dose-, and time-dependent, reflecting physiological adaption of locusts to the highly dynamic nature of population density.

A dose-dependent manner was found for the maturation synchrony effect of 4-VA. We find that only gregarious males aged after PAE 3 days have the accelerating effects on female maturation synchrony, which may be attributed to their significantly increased 4-VA content during adult development. Although gregarious nymphs (the fifth instar) and female adults can release relatively small amount of 4-VA (Wei et al., 2017), they did not promote female maturation based on our current results. Thus, the accelerating effects on female maturation synchrony induced by gregarious male adults may depend largely on 4-VA content they released. The ineffectiveness of the fifth nymphs and females in maturation acceleration of female adults may due to their low 4-VA content under efficient threshold. In fact, the fifth nymphs have been shown to display inhibiting effects on male maturation in S. gregaria (Assad et al., 1997). Therefore, the mechanisms underlying pheromone-mediated sexual maturation may differ between different locust species. Recently, 4-VA has been identified in the volatiles released by male adults of S. gregaria (Torto et al., 2021), whether this volatile has maturation-accelerating effect in this locust species needs further validation.

Our results reveal that JH signaling pathway presents as the critical endocrinal factor mediating the accelerating effect of 4-VA on female maturation. This finding is consistent with the role of JH as the major gonadotropin modulating Vg biosynthesis in the fat body and its uptake by the growing oocytes in the migratory locust (Jindra et al., 2013; Guo et al., 2014; Song et al., 2014). It is also supported by the significance of CA (a major JH biosynthesis tissue) in pheromone-induced maturation process in the desert locust (Odhiambo, 2009). Interesting, it has been suggested that the release of the maturation-accelerating pheromone by adult males is under the control of CA (Loher, 1997). Thus, there should be a complex feedback interaction between 4-VA and JH signaling pathway. Extensive studies have established the central roles of JH signaling in mediating the effects of social interactions on reproduction in different kinds of insect species, including eusocial insects (Robinson and Vargo, 1997; Korb, 2015), the burying beetle, Nicrophorus vespilloides (Engel et al., 2016), the German cockroach, Blattella germanica (Uzsák and Schal, 2012), and so on. Such an interaction between social clues and internal hormonal signals that coordinates ovary development is also common among group-living vertebrates (Drickamer, 1977; McClintock, 1978; Berger, 1992).

We demonstrate that 4-VA stimulates sexual maturation of young females within a distinct developmental time window. Compared to the females aged at PAE 1–2 days and 5–6 days, the females aged at PAE 3–4 days were more sensitive to 4-VA stimuli. This point was strongly supported by several lines of evidence from temporal-dependent comparisons of oocyte development, gene expression profiles, JH titer, as well as Vg biosynthesis. It has been shown that JH titers, Vg expression, the size of terminal oocytes, dramatically increased at PAE 3–4 days, implies the PAE 3–4 days is an essential time window for JH-regulated ovary development in female locusts (Luo et al., 2017; Wu et al., 2018). The finding that 4-VA accelerates maturation of less-developed females rather than more-developed females supports a ‘catch-up’ model in achievement of female maturation synchrony in locusts. 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 successively emerge to adults in a couple of days. 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 maturation synchrony of female locusts, which could reduce male competitions for mate selection.

Peripheral and central neural sensitivity to olfactory clues have been demonstrated to vary with developmental stages or physiological statuses (Guo et al., 2011; Gadenne et al., 2016). Given this, sensory processing sensitivity or JH biosynthesis activity might be involved in the stage-dependent sensitivity of females to 4-VA stimuli. However, peripheral olfactory neuron might not be involved in the stage-specific sensitivity to 4-VA stimuli, because we did not detect significant changes of peripheral electrophysiological response during female ovary development. A possible explanation is that signaling factors responsible for JH synthesis might be turned on specifically at Mid-PAE of female locusts upon 4-VA stimuli, such as GPCRs and transcription factors (Bendena et al., 2020). Although there are only a few DEGs in the brain of females exposed to 4-VA, we cannot exclude the involvement of the central nerve system pathway by other regulatory mechanisms, for example, neurotransmitter release, or post-transcription regulation (Nouzova et al., 2018). Further studies should elucidate detailed mechanisms of the linking between 4-VA and JH biosynthesis in female locusts.

In summary, we revealed a catch-up strategy of female reproductive synchrony in locust swarms, whereby 4-VA acts as a maturation-accelerating pheromone hastening less-developed females through triggering JH biosynthesis. Our findings provide novel insight into the mechanisms underlying individual interaction during aggregation in group-living animals.

Materials and methods

Experimental insects

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All insects used in experiments were reared in the same locust colonies at the Institute of Zoology, Chinese Academy of Sciences, Beijing, China. Briefly, gregarious locusts were reared in cages (30 cm × 30 cm × 30 cm) with 800–1000 first-instar insects per cage in a well-ventilated room. Solitarious locusts were individually raised in a ventilated cage (10 cm × 10 cm × 25 cm). All locusts were cultured under the following conditions: a L14:D10 photoperiod, temperature of 30°C ± 2°C, relative humidity of 60% ± 5%, and a diet of fresh greenhouse-grown seedlings and bran.

Oocyte length measurement

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All insects used for sexual maturity determination were virgin females. The ovary of tested females was dissected and placed in locust saline, and the terminal oocytes were isolated. The lengths of terminal oocytes were photographed and measured under the Leica DFC490 stereomicroscope (Leica, Germany). Given that the maximum length of terminal oocytes in gregarious females is much longer than that in solitarious females (Chen et al., 2015), the sexual maturity was presented as the length of terminal oocyte relative to the maximum length.

Recording of the distribution of first oviposition time in female locusts

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Individuals between 0 and 24 hr after adult molting are referred as PAE 1 day adults, with each subsequent day representing an additional 24 hr period. Given that both gregarious and solitarious locusts begin to mate at PAE 6 days, the first oviposition time was recorded after PAE 6 days. For gregarious locusts, 10 females and 10 males at PAE 6 days were placed in a cage (30 cm × 30 cm × 30 cm). The females were individually marked, and their first oviposition times were recorded by collecting egg pods every 4 hr per day after mating. Females those laid new eggs could be easily distinguished by much thinner abdomen with white foam around ovipositor. For solitarious locusts, each female was reared together with a single male at PAE 6 days, and the first oviposition time was recorded by collecting egg pods every day after mating. To ensure the consistency of mating age in gregarious and solitarious locusts, females that did not successfully mate within 24 hr after paired rearing were excluded in both phases. The distribution curve of the first oviposition time was calculated based on data collected from all females.

The effects of conspecifics interaction on female sexual maturation

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Given that the difference of female sexual maturation synchrony between gregarious and solitarious phases appeared at PAE 6 days, the lengths of terminal oocytes of virgin females were detected at PAE 6 days after each treatment in subsequent experiments. For the stimulation of gregarious females, 10 gregarious females were reared with 10 gregarious males or 10 gregarious females in a same cage (15 cm × 15 cm × 10 cm) from PAE 1 to 6 days (Figure 1—figure supplement 2A). For the stimulation of solitarious females, one solitarious female was reared with one solitarious male or one solitarious female in a same cage (15 cm × 15 cm × 10 cm) from PAE 1 to 6 days (Figure 1—figure supplement 2B). The ovaries of treated females were dissected in locust saline and the lengths of terminal oocytes were measured as described above.

The effect of locust volatiles on female sexual maturation

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To determine the effect of locust volatiles on female sexual maturation, virgin female adults were separately reared with females or male adults by a breathable partition. For gregarious phase, 10 gregarious females were reared with 10 gregarious males or 10 gregarious females in a breathable partition cage (15 cm × 15 cm × 10 cm) from PAE 1 to 6 days (Figure 1—figure supplement 2C). For solitarious phase, one solitarious female was reared with 10 gregarious males or 10 solitarious males in a breathable partition cage (15 cm × 15 cm × 10 cm) from PAE 1 to 6 days (Figure 1—figure supplement 2D). The ovaries of treated females were dissected in locust saline and the lengths of terminal oocytes were measured as described above.

SPME and GC-MS-MS

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The volatiles of gregarious male adults, gregarious female adults, and solitarious male adults at PAE 1, 2, 4, 6, 8 days were collected by solid phase microextraction (SPME) for 30 min following our previously study (Wei et al., 2017). In detail, a fiber (PDMS/DVB 65 μm) was introduced into a glass jar (10.5 cm high ×8.5 cm internal diameter) to absorb odors. The SPME volatiles collected from an empty glass jar for 30 min served as the control. Eight biological replicates were performed for each treatment. The fibers with absorbed odors were subjected to chemical analyses with GC-MS/MS. A Bruker GC system (456-GC) coupled with a triple quadrupole (TQ) mass spectrometer (Scion TQ MS/MS, Inc, German) equipped with an DB-1MS column (30 m × 0.25 mm ID ×0.25 μm film thickness, Agilent Technologies) was used to quantify the volatile compounds in the SPME samples. Bruker chemical analysis MS workstation (MS Data Review, Data Process, version 8.0) was used to analyze and process the data. Mixed samples consisting of standard compounds in different dosages (0.1, 1, 5, 10, and 20 ng/μl) were used as external standards to develop the standard curves to quantify the volatiles. The same thermal program and Multiple Reaction Monitoring (MRM) method were used for standard compound detection.

Odor treatment assay

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For mixture treatment, 10 gregarious virgin females were stimulated by the mixed odor blend (the concentrations of PAN, guaiacol, 4-VA, vertrole, and anisole were 1, 10, 3, 2, 3 μg/μl, respectively) or paraffin oil from PAE 1 to 6 days. In detail, a breathable vial containing the mixture or paraffin oil was placed with 10 virgin females in a cage for 6 days. The vial was replaced by newly diluted compounds every day. The 4-VA treatment assay was performed by the same method, and the dose of 4-VA used is 100 ng/μl, and the concentration of 4-VA released was measured as 3.1–40 ng/0.5 hr within 24 hr exposure.

To determine the time-dependent effect of 4-VA, control females were treated by paraffin oil from PAE 1 to 6 days. In parallel, paraffin oil was placed by 4-VA at PAE 1–2, 3–4, 5–6 days, respectively. The ovaries were dissected and the lengths of terminal oocytes were measured as described above. The brains, CC-CA, and fat body of females were dissected and stored immediately in liquid nitrogen for further experiments.

EAG assays

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An aliquot of odor was dissolved in paraffin oil (w/v) and loaded with 10 μl on a 5 × 40 mm filter paper strip (Whatman), which was placed inside a Pasteur pipette. This odor was used on subsequent EAG assay. Hexane was tested as negative controls. The antennae of the adult locusts were cut at the bases of the flagella and distal antennal. Segments were cut off 2 mm and then fixed between two electrodes with electrode gel Spectra 360 (Parker, Orange, NJ). The EAG signals were amplified, monitored, and analyzed with the EAG-Pro software (IDAC4, Syntech, the Netherlands; EAG software v2.6c). A continuous air flow of 30 ml/s was produced by a stimulus controller (Syntech CS-05). Stimulation duration was 1 s and the intervals were 1 min. The blank was applied at the start and end of the stimulation series. The average EAG amplitude was subtracted from that of the blank.

SSRs assay

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SSRs were recorded and analyzed, and stimuli were prepared as previously described (Li et al., 2016). The locust was placed in a plastic tube 1 cm in diameter, and its head and antennae were fixed with dental wax. A tungsten wire electrode was electrolytically sharpened by 10% NaNO2. The recording electrode was inserted into the bottom of the sensilla through a micromanipulator (Narishige, Japan). The reference electrode was inserted into the eye. Recording electrodes were connected to amplifiers (IDAC4, Syntech, the Netherlands). The frequency variation of each pulse at 0.2 s was calculated by using automatic frequency meter software. Signals were recorded for 10 s, starting 1 s before stimulation. The preparation is held in a humidified continuous air flow delivered by the Syntech Stimulus controller (CS-55 model, Syntech) at 1.4 l/min. Chemical substances as SSR stimulants included mineral oil as the blank, which was used to dilute the 4-VA at 1, 10, 100, 1000, 10,000, 100,000, 1,000,000 ng/μl, respectively. A piece of filter paper (Whatman, UK) was placed in a 15 cm Pasteur glass tube and 10 μl of volatile solution was added to the filter paper. Responses were calculated by counting the number of action potentials 1 s after stimulation.

Total RNA extraction, RNA-seq, and quantitative real-time PCR

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Total RNA from different tissues were extracted using the TRIzol reagent (Invitrogen TRIzol Reagent, Cat. 15596026) and treated with DNase I following the manufacturer’s instructions.

For RNA-seq, three independent replicates were performed for each sample. The RNA-seq data reported here have been deposited in the Genome Sequence Archive (Genomics, Proteomics & Bioinformatics 2017) in National Genomics Data Center (Nucleic Acids Res 2020), Beijing Institute of Genomics (China National Center for Bioinformation), Chinese Academy of Sciences, under accession number CRA003038 that are publicly accessible at https://bigd.big.ac.cn/gsa.

RNA integrity

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cDNA libraries were prepared according to Illumina’s protocols. Raw data were filtered, corrected, and mapped to locust genome sequence using TopHat2 software. The number of total reads was normalized by multiple normalization factors. Transcript levels were calculated using the reads per kb million mapped reads criteria. The differences between the test and control groups were represented by p values. DEGs were detected by using edgeR package with significance levels at p < 0.05. Principal component analysis (PCA) was accomplished using the princomp and pca functions. Enrichment analysis of the Gene Ontology (GO) was carried out based on an algorithm presented by GOstat.

For qPCR, cDNA was reverse transcribed with 2 μg of total RNA using M-MLV Reverse Transcriptase (Promega, Madison, WI). The relative mRNA levels of targeting genes were quantified by Real Master Mix Kit (Tiangen) with LightCycler 480 instrument (Roche). Melting curve analysis was performed to confirm the specificity of amplification. The primers used for qPCR were presented in Supplementary file 2.

Protein preparation and Western blot analysis

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Ovaries and fat body of tested females were collected and homogenized in TRIzol reagent (5 individuals/sample, 6 biological repeats/treatment). Total protein was extracted following manufacturer’s instructions. Total protein (100 μg) were separated by gel electrophoresis and then transferred onto polyvinylidene difluoride membranes (Millipore). Non-specific binding sites on the membranes were blocked with 5% bovine serum albumin. The blots were incubated with the primary antibodies (rabbit anti-Vg serum, 1:500, Beijing Protein Innovation Co., Ltd., BPI) in TBST overnight at 4°C. After incubation, the membranes were washed, incubated with anti-rabbit IgG secondary antibody (1:5000) (EASYBIO, China) for 1 hr at room temperature, and then washed again. Protein bands were detected by chemiluminescence (ECL kit, CoWin). The antibodies were stripped from the blots, re-blocked, and then probed with an anti-GAPDH antibody (1:5000) (Wang et al., 2013). Protein bands were detected by chemiluminescence (ECL kit, Thermo Scientific). The intensities of the Western blot signals were quantified using densitometry.

JH titer measurement

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Twenty microliters of hemolymph were added to a 1.5 ml tube with 100 μl of 70% methanol and thoroughly mixed. Then, 200 μl of hexane was added to the solution and thoroughly mixed again. The mixture was centrifuged at 5000 rcf for 10 min at 4°C. Then, 150 μl of supernatant was placed into a new tube, and the JH precipitate was dried by nitrogen. The JH precipitate was dissolved in 50% methanol, mixed by vortexing, and centrifuged at 13,000 rpm for 10 min at 4°C. JH III in the supernatant was detected using the rapid resolution liquid chromatography system (ACQUITY UPLC I-Class, Waters, Milford, MA).

An ACQUITY UPLC BEH C18 column (50 × 2.1 mm, 1.7 μm) was used for LC separation. The autosampler was set at 10°C, using gradient elution with 0.1% formic acid methanol as solvent A and 0.1% formic acid water as solvent B. The flow rate was set at 0.2 ml/min. Mass spectrometry detection was performed on an AB SCIEX Triple Quad 4500 (Applied Biosystems, Foster City, CA) with an electrospray ionization source (Turbo Ionspray). The detection was performed in positive electrospray ionization mode. The [M + H] of the analyte was selected as the precursor ion. The quantitation mode was MRM mode using the mass transitions (precursor ions/product ions). The MRM (m/z) of JH III was 267.2/235.2. Data acquisition and processing were performed using AB SCIEX Analyst 1.6 Software (Applied Biosystems).

JH rescue experiment

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For rescue experiment in Or35 mutants, the active JH analog, S-(+)-methoprene (Santa Cruz Biotech Dallas, TX) was topically applied to the pronotum of locusts (150 µg per locust) from PAE 3 to 4 days according to previously published work (Song et al., 2013; Wu et al., 2016), and acetone was used as the control. Meanwhile, the treated females were stimulated with 4-VA. The treated females were dissected at PAE 6 day, and the lengths of terminal oocytes were measured as previously described. The ovaries and fat bodies of females at PAE 4 days were dissected and stored immediately in liquid nitrogen for further Western blot experiments.

For rescue experiment in WT females, precocene I (Sigma) dissolved in acetone (100 μg/μl) was added to the dorsal neck membrane of locusts (500 μg/locust) within 12 hr after eclosion to inactive the corpora allata. JH III dissolved in acetone (20 μg/μl) was topically applied at 100 μg per locust aged at PAE 3 day to restore the JH activity. All females were treated by 4-VA at PAE 3–4 days. The treated females were dissected at PAE 6 day, and the lengths of terminal oocytes were measured, the mRNA level and protein level of Vg were detected to validate the effect of JH in 4-VA-accelerated sexual maturation and vitellogenesis.

Statistical analyses

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For the measurement of oviposition time and sexual maturation, individuals were randomly allocated into experimental group and control group, and no restricted randomization was applied.

The data that do not meet normal distribution was excluded for the analysis of sexual maturity, mRNA levels, protein levels, as well as JH titer measurement. The distribution of the first oviposition time and the consistency of sexual maturation (represented by the length of terminal oocytes) were analyzed using Levene’s test according to previous studies (Rohner et al., 2013; He et al., 2016). The mean value of the first oviposition time between two groups was analyzed using Student’s t-test. One-way ANOVA followed by Tukey’s post hoc test was used for multi-group comparisons. All data were statistically analyzed using GraphPad Prism 5 software and SPSS 17 software. All experiments were performed with at least three independent biological replicates.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting file; Source Data files have been provided for Figures 1, 2, 3, and 4.

The following data sets were generated
    1. Yang P
    (2020) bigd
    ID CRA003038. locust RNA-Seq with 4-vinylanisole treatment.

References

    1. Guo F
    2. Xia B
    (1964)
    The male locust secretes substances that promote the maturation of the female locust’s ovaries
    Chinese Science Bulletin 01:3.
    1. Norris MJ
    (1952)
    Reproduction in the desert locust (schistocerca gregaria forsk.) in relation to density and phase
    Anti-Locust Bulletin 1:49.
    1. Norris MJ
    (1954)
    Sexual maturation in the desert locust (schistocerca gregaria forsk.) with special reference to the effects of grouping
    Anti-Locust Bull 28:27.
  1. Book
    1. Uvarov BP
    (1977)
    Grasshoppers and Locusts: A Handbook of General Acridology
    London, UK: Centre for Overseas Pest Research, Cambridge University Press.
    1. Ward A
    2. Webster M
    (2016) The evolution of group living
    Sociality: The Behaviour of Group-Living Animals pp. 191–216.
    https://doi.org/10.1007/978-3-319-28585-6_10

Decision letter

  1. Sonia Sen
    Reviewing Editor; Tata Institute for Genetics and Society, India
  2. K VijayRaghavan
    Senior Editor; National Centre for Biological Sciences, Tata Institute of Fundamental Research, India
  3. Sonia Sen
    Reviewer; Tata Institute for Genetics and Society, India
  4. Amir Ayali
    Reviewer; Tel Aviv University, Israel

Our editorial process produces two outputs: i) public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.

Decision letter after peer review:

Thank you for submitting your article "Aggregation pheromone 4-vinylanisole promotes the synchrony of sexual maturation in female locusts" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, including Sonia Sen as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by K VijayRaghavan as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Amir Ayali (Reviewer #2).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

While appreciating the general rigor and breadth of this study, we had a few concerns, which you can hopefully address in a revision. These are listed below:

Essential revisions:

1. Solitary female data: Since the focus of this study is the effect of 4VA on sexual maturation of gregarious females we recommend that the authors focus on this. We are concerned that the solitary female data presented in figures 1D and F pose a potentially problematic comparison.

2. Mating status: Have the authors considered that mating might play a role in synchrony in oviposition, since, in other insects, sex peptide is known to influence JH release from CC-CA post mating? Could the authors test this by using virgin females?

3. Age mismatch: Could the authors discuss the age-mismatch between the age of maximal 4VA production by G-males and the age of maximum effect in females?

4. Concentration of 4VA: Could the authors address what concentrations of 4VA are used with respect to physiological concentrations of 4VA produced? Which dose represents the levels released by 10 adult males at PAE 3-4 days?

5. Introduction and Discussion: Please revisit these sections to incorporate literature that is relevant to this study but has not been referred to here.

6. Choice of 4VA: We assume that part of the decision to chase 4VA was based on prior work. If this is true, could the authors emphasise this. The manner in which this choice is currently presented raises some issues. For example, would not differential analysis of volatiles between gregarious females and males be a better (or an additional) comparison? In general, could the authors make clear their rational and methodology and for the selection of the volatiles?

7. As the haemolymph JH titres in figure 3E are quite low, could the authors also include GAPDH quantification (for this and the subsequent figures), as internal controls, perhaps as a supplementary figure?

8. Allatectomy: We recommend that the authors perform this in the JH rescue experiment.

Reviewer #1 (Recommendations for the authors):

The data are clean and support the claims made, and the manuscript is well written. We have a few comments for the authors.

– The two experiments that support the idea of the critical window are the acceleration of sexual maturity at 3-4 DPE (not younger or older) in response to 4VA, and the RNAseq experiment from age matched fat bodies to show that most transcriptomic changes occur at 3-4 DPE (not younger or older) in response to 4VA. While these are convincing. We found it surprising that the effect of 4VA at this age appears distinctly attenuated compared to that presented in figure 2C, which is also assayed upon the presentation of 4VA alone. Is this true? Are the 4VA concentrations at a physiological range? Could the authors support this central claim by performing this experiment with appropriately aged gregarious males, instead of the volatile?

– In Figure 2B, are the 4VA concentrations used of physiological relevance? We're not sure if we're interpreting Figure 2-sup 1 correctly, but it suggests that the concentrations used in the experiments are far higher than what's emitted by males. Can the authors show the physiological quantification of 4-VA and clarify whether the 4-VA stimulations used in all the subsequent experiments are within physiological ranges?

– We assume that the CC-CA don't accelerate JH sysnthesis in response to 4VA at an earlier age as JH haemolymph titre is unaffected at that age upon 4VA exposure. This would suggest that the CC-CA of both gregarious (and solitary?) females of this age must be primed to increasing JH synthesis in response to 4VA. How could this be? The authors discuss that it might be GPCRs. Have they considered mating to have a role, since, in other insects, sex peptide is known to influence JH release from CC-CA?

– In general, can they comment on role of mating on sexual maturity? They say that both gregarious and solitary males "begin" to mate at PAE 6 days (line 341), is it possible that the dramatic effect seen post- PAE 4d is due to increased mating? This is further supported by the fact that the sexual maturity in Figure 1C (G-females co-housed with G-males) is much higher than Fig1E (G-females exposed to G-males volatiles). Is this difference also significant in WT females in 1H and 1I?

– We found it interesting that S-females reared G-male volatiles have comparable sexual maturity as compared to G-females with G-males (Figure 1F). Perhaps this supports that mating primes them for an altered 4VA response?

– Since the authors claim that the critical window allows a subset of the females' eggs to catch up with older ones so as to have synchronous oviposition it would be nice to have an oviposition read out of this phenomenon. Have the authors tested this? Perhaps by excluding this age group from an experiment such as that seen in C? One would expect no catch up here and therefore a widely distributed oviposition.

– As the haemolymph JH titres in figure 3E are quite low, could the authors also include GAPDH quantification (for this and the subsequent figures), as internal controls, perhaps as a supplementary figure?

– Females of all ages respond similarly to 4VA. Despite that, only the female of a certain age accelerates oogenesis. This is very interesting! Do the authors want to consider presenting this as one of the main figures?

Reviewer #2 (Recommendations for the authors):

The abstract can be improved to better present the findings and their significance.

LL92-93: Anstey et al. has little to do with coordinated behavior. There are other much more relevant studies (e.g. Buhl, Ayali, others).

The introduction should do a much better job in presenting the advantages of reproductive synchrony for locusts.

LL 117-118 I would rather the authors would have first investigated whether there is any synchronization effect associated with the gregarious phase per se, only than adding the comparative information on the solitary phase (where no synchronization is to be expected).

The experimental procedures behind Figure 1 are not presented in a clear enough manner. For example, what exactly is the Sexual maturity index? Why not show length of the terminal oocyte?

LL 355-357 "To avoid the effects caused by asynchronous mating." Not clear.

LL 130-131 "by the removal of male adults in gregarious phase (Figure 1C), but not in solitarious phase (Figure 1D)." Not clear. What was the manipulation conducted on Solitary females how and why is it comparable to that conducted with the gregarious?

Figure 1C vs E: what explains the major difference in the response of the females?

Including the solitary locusts data in Figure 1 adds very little!

LL 148-150. I was under the impression that this was already done in previous studies

LL 211-212 In locusts there really is no CA-CC complex, like in other insects. The CA are easily distinguished and are those attributed with a role in JH/Vg signaling pathway. Not sure why were the CC included.

Figure 3F-H n=4?

Figure 5 – It is not clear what is the difference between the females in the bottom left vs. right.

LL 268-269 ?

Reviewer #3 (Recommendations for the authors):

I think the discussion lacks depth in relation to the biology of gregarious locusts because of the scope of the results which focused on only one locust sex (females). It would be more interesting to investigate sexual maturation in both sexes and the underlying mechanisms. One more thing, I think the authors may have missed the new literature on the composition of odors of the desert locust which reports 4-vinyl anisole as an adult-male specific volatile (see. https://doi.org/10.1016/j.jinsphys.2021.104296). Hence the statement by the authors, "Given that 4-VA has not been detected in S. gregaria (Torto et al., 1996), whether this volatile has maturation-accelerating effect in this locust species needs further validation," must be rephrased.

https://doi.org/10.7554/eLife.74581.sa1

Author response

Essential revisions:

1. Solitary female data: Since the focus of this study is the effect of 4VA on sexual maturation of gregarious females we recommend that the authors focus on this. We are concerned that the solitary female data presented in figures 1D and F pose a potentially problematic comparison.

We understand the reviewer’s concern. However, we think the solitarious phase data is very important for our findings. The aim of this study is to explore the mechanism underlying sexual maturation synchrony by comparing phase- and sex-dependent conspecific interactions in locusts. Given that solitarious males have no stimulatory effect on sexual maturation, phase-dependent comparison of volatile contents is helpful for us to screen candidate volatiles responsible for the acceleration of sexual maturation synchrony in females. According to the suggestion of Reviewer 2, we have added the comparison of volatile contents between gregarious males and gregarious females for effectively screening of targeting volatiles (see revised Figure 2A).

2. Mating status: Have the authors considered that mating might play a role in synchrony in oviposition, since, in other insects, sex peptide is known to influence JH release from CC-CA post mating? Could the authors test this by using virgin females?

We understand the reviewer’s concerns. We have indeed taken the effects of mating on sexual maturation and oviposition into consideration in current study. To eliminate the effects of different mating age on oviposition, only females successfully mate at 6-7 days post eclosion (PAE 6-7 days) were used in both gregarious and solitarious phases for measurement of synchrony in first oviposition time. In addition, only virgin females were used for in all the experiments related to measurement of sexual maturation synchrony in both phases. We have provided these method details in the Figure legend and Materials and methods (lines 372).

3. Age mismatch: Could the authors discuss the age-mismatch between the age of maximal 4VA production by G-males and the age of maximum effect in females?

We accept the reviewer’s suggestions. According to the suggestion, we have provided additional discussion on the age-mismatch between the age of maximal 4-VA production by G-males and the age of maximum effect in females. 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 successively emerge to adults in a couple of days. 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” (lines 336-345).

4. Concentration of 4VA: Could the authors address what concentrations of 4VA are used with respect to physiological concentrations of 4VA produced? Which dose represents the levels released by 10 adult males at PAE 3-4 days?

Thanks. Our analysis revealed that the physiological concentration of 4-VA is 0.18-1.00 ng/0.5 h/locust in gregarious male adults aged at 4 days (1.80-10.00 ng/0.5 h for 10 individuals). Results from dosage effects of 4-VA on sexual maturity in females indicated that the effective dose is 100 ng/ul (the concentration of 4-VA released was 3.1−40 ng/0.5 h within 24 h exposure). Thus, the 4-VA volatile quantity used is within the physiological range of 4-VA production. Detailed information has been added to the methods part (line 431-432).

5. Introduction and Discussion: Please revisit these sections to incorporate literature that is relevant to this study but has not been referred to here.

We have revised the contents and replaced the literature following the reviewer’s suggestions. The importance of reproductive synchrony and the research progress in regulatory mechanisms underlying sexual maturation in locusts have been added to the introduction, details were shown as: “Locusts often form large swarms with hundreds to thousands of individuals, regarded as one of the most extraordinary examples of coordinated behavior (Ariel and Ayali, 2015, Buhl and Rogers, 2016). 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” (lines 92-114). Moreover, the importance of reproductive synchronization in female locusts as well as age mis-match between maximal 4-VA release from gregarious males and sensitive developmental stage to 4-VA of females have also been added in the discussion (see details in following section).

6. Choice of 4VA: We assume that part of the decision to chase 4VA was based on prior work. If this is true, could the authors emphasise this. The manner in which this choice is currently presented raises some issues. For example, would not differential analysis of volatiles between gregarious females and males be a better (or an additional) comparison? In general, could the authors make clear their rational and methodology and for the selection of the volatiles?

We understand the reviewer’s query. Actually, here the functional study of 4-VA in sexual maturation is not to chase our previous work (Guo et al., 2020, Nature, 4-vinylanisole is an aggregation pheromone in locusts). Instead, we aim to explore the mechanisms underlying sexual maturation synchrony by comparing phase- and sex-dependent conspecific interactions in locusts. Given that the volatiles released by gregarious males, rather than gregarious females and solitarious males, have the accelerate effects on female sexual maturation, a comparative analysis of volatile contents among these three groups (G-males, G-females, and S-males) was performed in the revision process (revised Figure 2A). 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 these 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 abundantly released by gregarious male adults played major roles in promoting female sexual maturation synchrony. The logic of selection of candidate volatiles accelerating sexual maturation synchrony has been described as: “To identify key active compounds that can promote sexual maturation synchrony of female locusts, we compared the volatile emission dynamics of gregarious male adults, gregarious female adults, and solitarious male adults from PAE 1-8 days. In total, 14 chemicals were identified in the volatiles released by male adults (Figure 2A). Only 5 compounds displayed significantly higher abundance in gregarious-male adults, compared to volatile released by gregarious female adults and solitarious male adults, which showed no accelerating effects on female sexual maturation. Thus, these five kinds of gregarious male adult-abundant volatiles, including phenylacetonitrile (PAN), guaicol, 4-vinylanisole (4-VA), vertrole, and anisole (Figure 2A and Figure 2—figure supplement 1), might serve as candidate pheromones for female maturation acceleration” (lines 156-165).

7. As the haemolymph JH titres in figure 3E are quite low, could the authors also include GAPDH quantification (for this and the subsequent figures), as internal controls, perhaps as a supplementary figure?

The JH titers measured in this work is at the same level with that reported in previous work in locusts (0-5 ng/ml, Guo et al., 2020, PLoS Genet; Guo et al., 2019, FASEB Journal). And, GAPDH is commonly used as internal reference in western blot experiments examining the abundance of target proteins, not used for HPLC methods that we used for juvenile hormone quantification.

8. Allatectomy: We recommend that the authors perform this in the JH rescue experiment.

Thanks. Allatectomy is a classical method to abolish the capacity of CA for JH synthesis in many early studies. Besides, precocene treatment is now commonly used for effective inhibition of JH synthesis (Wu et al., 2016, The Journal of Biological Chemistry). During the revision process, 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 application can obviously recover the sexual maturation rate and Vg expression (Figure 4G-I).

Reviewer #1 (Recommendations for the authors):

The data are clean and support the claims made, and the manuscript is well written. We have a few comments for the authors.

– The two experiments that support the idea of the critical window are the acceleration of sexual maturity at 3-4 DPE (not younger or older) in response to 4VA, and the RNAseq experiment from age matched fat bodies to show that most transcriptomic changes occur at 3-4 DPE (not younger or older) in response to 4VA. While these are convincing. We found it surprising that the effect of 4VA at this age appears distinctly attenuated compared to that presented in figure 2C, which is also assayed upon the presentation of 4VA alone. Is this true? Are the 4VA concentrations at a physiological range? Could the authors support this central claim by performing this experiment with appropriately aged gregarious males, instead of the volatile?

Thanks for the reviewer’s comments. We understand the reviewer’s concerns about the seemingly nonconsistency of the effects of 4-VA treatments on sexual maturity between Figures including Figure 2C and Figure 3A. However, the comparison with the absolute value of sexual maturity between different figures is not meaningful because the data of different figures were obtained from independent experiments, in which their oocyte developments might vary due to batch effects. In each experiment, the control was designed for avoiding the batch effect. The significant effect of 4-VA treatment was demonstrated from the comparison with the control in all of experiments. So, it is not true that the effect of 4-VA at this age appears distinctly attenuated in Figure 3A compared to that presented in figure 2C, just due to batch effect. And the 4VA concentrations that we used should be at a physiological range. Based on our data form GC-MS-MS, the physiological concentration of 4-VA from ten gregarious male adults aged at 4 days is 1.8-10 ng/0.5 h. The concentration of 4-VA (100 ng/μl) released was measured as 3.1−40 ng/0.5 h within 24 h exposure. Thus, the 4-VA volatile quantity used is within the physiological range of 4-VA production. Detailed information has been added to the methods part (lines 431-432). To further clarify reviewer’s concerns, we have performed the experiment suggested by the reviewer by comparing the effects of gregarious males with different ages on female maturation. As shown in Figure 3—figure supplement 1, the maturation synchrony of females was significantly enhanced by gregarious males aged at PAE 3-4 days and PAE 5-6 days.

– In Figure 2B, are the 4VA concentrations used of physiological relevance? We're not sure if we're interpreting Figure 2-sup 1 correctly, but it suggests that the concentrations used in the experiments are far higher than what's emitted by males. Can the authors show the physiological quantification of 4-VA and clarify whether the 4-VA stimulations used in all the subsequent experiments are within physiological ranges?

The 4-VA volatile quantity that we used should be within the physiological range. The detailed reason can be seen above.

– We assume that the CC-CA don't accelerate JH synthesis in response to 4VA at an earlier age as JH haemolymph titre is unaffected at that age upon 4VA exposure. This would suggest that the CC-CA of both gregarious (and solitary?) females of this age must be primed to increasing JH synthesis in response to 4VA. How could this be? The authors discuss that it might be GPCRs. Have they considered mating to have a role, since, in other insects, sex peptide is known to influence JH release from CC-CA?

Thanks for the reviewer’s nice suggestions. We don’t think that mating plays a role in the accelerating effect of JH synthesis observed in our studies because only virgin females were used in all our experiments refers to sexual maturation examination, JH titer determination, and gene expression analysis (See the method for oocyte length measurement, lines 372-377). Certainly, it will be a valuable subject to explore how mating or sex peptides affect female sexual maturation and JH synthesis in future work.

– In general, can they comment on role of mating on sexual maturity? They say that both gregarious and solitary males "begin" to mate at PAE 6 days (line 341), is it possible that the dramatic effect seen post- PAE 4d is due to increased mating? This is further supported by the fact that the sexual maturity in Figure 1C (G-females co-housed with G-males) is much higher than Fig1E (G-females exposed to G-males volatiles). Is this difference also significant in WT females in 1H and 1I?

Thanks for the reviewer’s comments. Just seen our response above, in fact, virgin females were used in all experiments refers to sexual maturation examination in Figure 1. Therefore, the role of mating in phase-dependent sexual maturation in the current study could be excluded. And, about the variance of sexual maturity between figures, batch effects can be explained (Seen our above responses). To further validate the role of gregarious male adults rather than gregarious female adults in accelerating sexual maturation of females, we have repeated the experiments in Figure 1C. The results conform the maturation-accelerating effect of gregarious male adults (revised Figure 1C).

– We found it interesting that S-females reared G-male volatiles have comparable sexual maturity as compared to G-females with G-males (Figure 1F). Perhaps this supports that mating primes them for an altered 4VA response?

Thanks for the reviewer’s suggestion. Just seen our above response about the role of mating. In fact, we used virgin females in all experiments refers to sexual maturity examination to eliminate the potential effects of mating. To avoid the misunderstanding, we have added the information about mating status of females used in each experiment in the figure legends and methods.

– Since the authors claim that the critical window allows a subset of the females' eggs to catch up with older ones so as to have synchronous oviposition it would be nice to have an oviposition read out of this phenomenon. Have the authors tested this? Perhaps by excluding this age group from an experiment such as that seen in C? One would expect no catch up here and therefore a widely distributed oviposition.

We thank the reviewer’s suggestions. Female ovarian maturation is an essential prerequisite for oviposition, and is thus commonly used as an indicator for reproductive activity in locusts (Wu et al., 2016, J Bio Chem, Song et al., 2018, Development; Gijbels et al., 2019, Sci Rep). The prime objective of current study is to explore the regulatory mechanism underlying sexual maturation synchrony in locusts. The time window-dependent accelerating effects of 4-VA on sexual maturation of female locusts were conceivably supported by several lines of evidence, including significantly changed sexual maturity, gene expression profiles, JH synthesis, and vitellogenesis resulted from 4-VA treatment at PAE 3-4 days. Based on these findings, the further validation of time-window effects of 4-VA on oviposition might not be necessary in the current study.

– As the haemolymph JH titres in figure 3E are quite low, could the authors also include GAPDH quantification (for this and the subsequent figures), as internal controls, perhaps as a supplementary figure?

Thanks for the reviewer’s suggestion. In fact, the JH titers measured in this work is at the same level with that reported in previous work in locusts (0-5 ng/ml, Guo et al., 2020, PLoS Genetics; Guo et al., 2019,FASEB Journal ). And GAPDH is commonly used as internal reference in western blot experiments examining the abundance of target proteins. Because HPLC method was used for JH hormone quantification in this study, GAPDH is not useful for hormone quantification.

– Females of all ages respond similarly to 4VA. Despite that, only the female of a certain age accelerate oogenesis. This is very interesting! Do the authors want to consider presenting this as one of the main figures?

We appreciate the reviewer’s helpful suggestion. Now we have presented the EAG response to 4-VA at different development stage in the main figure (Figure 3B).

Reviewer #2 (Recommendations for the authors):

The abstract can be improved to better present the findings and their significance.

The abstract has been revised as: “Reproductive synchrony generally occurs in various group-living animals. However, the underlying mechanisms remain largely unexplored. The migratory locust, Locusta migratoria, a worldwide agricultural pest species, displays synchronous maturation and oviposition when forms huge swarm. The reproductive synchrony among group members is critical for the maintenance of locust swarms and population density of next generation. Here, we showed that gregarious female locusts displayed more synchronous sexual maturation and oviposition than solitarious females and olfactory deficiency mutants. Only the presence of gregarious male adults can stimulate sexual maturation synchrony of female adults. Of the volatiles emitted abundantly by gregarious male adults, the aggregation pheromone, 4-vinylanisole, was identified to play key role in inducing female sexual maturation synchrony. This maturation-accelerating effect of 4-vinylanisole disappeared in the females of Or35-/- lines, the mutants of 4-vinylanisole receptor. Interestingly, 4-vinylanisole displayed a time-window action by which mainly accelerates oocyte maturation of young females aged at middle developmental stages (3-4 days post adult eclosion). We further revealed that juvenile hormone/vitellogenin pathway mediated female sexual maturation triggered by 4-vinylanisole. Our results highlight a “catch-up” strategy by which gregarious females synchronize their oocyte maturation and oviposition by time-dependent endocrinal response to 4-vinylanisole, and provide insight into reproductive synchrony induced by olfactory signal released by heterosexual conspecifics in a given group”.

LL92-93: Anstey et al. has little to do with coordinated behavior. There are other much more relevant studies (e.g Buhl, Ayali, others).

We have added the references as the reviewer’s suggestion. Details were shown as “Locusts often form large swarms with hundreds to thousands of individuals, regarded as one of the most extraordinary examples of coordinated behavior (Ariel and Ayali, 2015, Buhl and Rogers, 2016)” (lines 93-94).

The introduction should do a much better job in presenting the advantages of reproductive synchrony for locusts.

We have revised the introduction as the reviewer’s suggestion: “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 ensure 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., 2015, 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 that contribute to maturation synchrony of females have not been determined so far” (lines 98-114).

LL 117-118 I would rather the authors would have first investigated whether there is any synchronization effect associated with the gregarious phase per se, only than adding the comparative information on the solitary phase (where no synchronization is to be expected).

As descripted above, 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 in current work. 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.

The experimental procedures behind Figure 1 are not presented in a clear enough manner. For example, what exactly is the Sexual maturity index? Why not show length of the terminal oocyte?

Thanks for the reviewer’s comments. The sexual maturity index is expressed as the length of terminal oocyte relative to the maximum length. Detailed experimental procedures have been added in the revised method: “Given that the maximum length of terminal oocyte in gregarious females is much longer than that in solitarious females (Chen et al., 2015), the sexual maturity was presented as the length of terminal oocyte relative to the maximum length” (lines 375-377).

LL 355-357 "To avoid the effects caused by asynchronous mating." Not clear.

Thanks, we have revised as “To ensure the consistency of mating time in gregarious and solitarious locusts, females that did not successfully mate within 24 h after paired rearing were excluded in both phases” (lines 387-389).

LL 130-131 "by the removal of male adults in gregarious phase (Figure 1C), but not in solitarious phase (Figure 1D)." Not clear. What was the manipulation conducted on Solitary females how and why is it comparable to that conducted with the gregarious?

Thanks, we have revised as “We found that the maturation synchrony of terminal oocytes of females was significantly retarded by the removal of male adults in gregarious phase (Figure 1C). However, whether raised with either solitarious female or male does not affect sexual maturation of solitarious females (Figure 1D)” (137-140).

Figure 1C vs E : what explains the major difference in the response of the females? Including the solitary locusts data in Figure 1 adds very little!

Figure 1C showed that reared with gregarious males, not females, could significantly promotes sexual maturation synchrony of gregarious females; Figure 1E showed that stimulated by volatile emissions of gregarious males enhanced sexual maturation synchrony of gregarious females. For the solitary locust data in Figure 1 D and E, we provide further evidence indicating that the effective compounds for sexual maturation synchrony should be from gregarious male adults, but not solitarious males, or solitarious females. Based on this evidence, the phase- and sex-dependent comparisons of volatile contents were then used for screening candidate sexual maturation accelerating pheromone (Figure 2A).

LL 148-150. I was under the impression that this was already done in previous studies

In the previous work of our lab, Wei et al. analyzed the emission dynamics of locust volatiles associated with development, sexes, and phase changes (Wei et al., 2016, Insect science). However. the objective of this study is different from our previous work. In the current study, emission dynamics of locust adults during sexual maturation period (PAE 1, PAE 2, PAE4, PAE6, PAE8) were continuously monitored to identify candidate volatiles with maturation-accelerating effects. Besides, the age of experimental insects used in these two studies are different.

LL 211-212 In locusts there really is no CA-CC complex, like in other insects. The CA are easily distinguished and are those attributed with a role in JH/Vg signaling pathway. Not sure why were the CC included.

We accept the reviewer’s query. The aim of RNA-seq experiments is to investigate the effects of 4-VA on neuroendocrinal tissues involved in reproduction control, including brain, CC, and CA, not just CA. So, we did not dissect CC and CA tissues, separately. The data from RNA-seq demonstrated that the expression levels of genes related to JH-metabolism were significantly affected upon 4-VA treatment.

Figure 3F-H n=4?

Thanks, we have revised it. For Figure 3F and G, n=4; For Figure 3H, n=5. The numbers of biological replicate are indicated in the figures.

Figure 5 – It is not clear what is the difference between the females in the bottom left vs. right.

Thanks, we have revised the figure to make it clear to be understood. The females in the bottom left have similar sexual states to that of the females in the right, indicating more synchronous sexual maturation states among individuals.

LL 268-269 ?

We have removed the incomplete description.

Reviewer #3 (Recommendations for the authors):

I think the discussion lacks depth in relation to the biology of gregarious locusts because of the scope of the results which focused on only one locust sex (females). It would be more interesting to investigate sexual maturation in both sexes and the underlying mechanisms. One more thing, I think the authors may have missed the new literature on the composition of odors of the desert locust which reports 4-vinyl anisole as an adult-male specific volatile (see. https://doi.org/10.1016/j.jinsphys.2021.104296). Hence the statement by the authors, "Given that 4-VA has not been detected in S. gregaria (Torto et al., 1996), whether this volatile has maturation-accelerating effect in this locust species needs further validation," must be rephrased.

Thanks for the reviewer’s suggestion. (1) we have provided additional discussion on sexual maturation synchrony in both sexes and the underlying mechanism. Details were shown as: “Reproduction synchrony involves consistence in maturation, mating, and egg laying, among which sexual maturation synchrony serve 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 crowing 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 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 on maturation-accelerating pheromones: multicomponent 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” (lines 259-281). (2) We have revised the sentence as “Recently, 4-VA has been identified in the volatiles released by male adults of S. gregaria (Torto et al., 2021), whether this volatile has maturation-accelerating effect in this locust species needs further validation” (lines 309-311).

https://doi.org/10.7554/eLife.74581.sa2

Article and author information

Author details

  1. Dafeng Chen

    State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
    Contribution
    Conceptualization, Data curation, Investigation, Methodology, Writing – original draft
    Contributed equally with
    Li Hou
    Competing interests
    No competing interests declared
  2. Li Hou

    1. State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
    2. CAS Center for Excellence in Biotic Interactions, University of Chinese Academy of Sciences, Beijing, China
    Contribution
    Data curation, Funding acquisition, Investigation, Methodology, Writing – original draft
    Contributed equally with
    Dafeng Chen
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6727-7053
  3. Jianing Wei

    State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
    Contribution
    Investigation, Methodology
    Competing interests
    No competing interests declared
  4. Siyuan Guo

    State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
    Contribution
    Data curation, Methodology, Software
    Competing interests
    No competing interests declared
  5. Weichan Cui

    State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
    Contribution
    Data curation, Investigation, Methodology
    Competing interests
    No competing interests declared
  6. Pengcheng Yang

    Beijing Institutes of Life Sciences, Chinese Academy of Sciences, Beijing, China
    Contribution
    Methodology, Software
    Competing interests
    No competing interests declared
  7. Le Kang

    1. State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
    2. CAS Center for Excellence in Biotic Interactions, University of Chinese Academy of Sciences, Beijing, China
    3. Beijing Institutes of Life Sciences, Chinese Academy of Sciences, Beijing, China
    Contribution
    Conceptualization, Project administration, Supervision, Writing – review and editing
    For correspondence
    lkang@ioz.ac.cn
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4262-2329
  8. Xianhui Wang

    1. State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
    2. CAS Center for Excellence in Biotic Interactions, University of Chinese Academy of Sciences, Beijing, China
    Contribution
    Conceptualization, Funding acquisition, Project administration, Supervision, Writing – review and editing
    For correspondence
    wangxh@ioz.ac.cn
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8732-829X

Funding

National Natural Science Foundation of China (31930012)

  • Xianhui Wang

National Natural Science Foundation of China (32070497)

  • Li Hou

Chinese Academy of Sciences (152111KYSB20180036)

  • Le Kang

Youth Innovation Promotion Association of the Chinese Academy of Sciences (2021079)

  • Li Hou

National Natural Science Foundation of China (32088102)

  • Le Kang

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

This study was supported by the National Natural Science Foundation of China (Grant no. 31930012, 32088102, and 32070497) and grants from Chinese Academy of Sciences (nos. 152111KYSB20180036) and Youth Innovation Promotion Association CAS (no. 2021079).

Senior Editor

  1. K VijayRaghavan, National Centre for Biological Sciences, Tata Institute of Fundamental Research, India

Reviewing Editor

  1. Sonia Sen, Tata Institute for Genetics and Society, India

Reviewers

  1. Sonia Sen, Tata Institute for Genetics and Society, India
  2. Amir Ayali, Tel Aviv University, Israel

Publication history

  1. Received: October 10, 2021
  2. Preprint posted: October 26, 2021 (view preprint)
  3. Accepted: February 15, 2022
  4. Version of Record published: March 8, 2022 (version 1)

Copyright

© 2022, Chen et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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  1. Dafeng Chen
  2. Li Hou
  3. Jianing Wei
  4. Siyuan Guo
  5. Weichan Cui
  6. Pengcheng Yang
  7. Le Kang
  8. Xianhui Wang
(2022)
Aggregation pheromone 4-vinylanisole promotes the synchrony of sexual maturation in female locusts
eLife 11:e74581.
https://doi.org/10.7554/eLife.74581

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    Tiantian Wei et al.
    Research Article Updated

    The dual-specificity tyrosine phosphorylation-regulated kinase DYRK2 has emerged as a critical regulator of cellular processes. We took a chemical biology approach to gain further insights into its function. We developed C17, a potent small-molecule DYRK2 inhibitor, through multiple rounds of structure-based optimization guided by several co-crystallized structures. C17 displayed an effect on DYRK2 at a single-digit nanomolar IC50 and showed outstanding selectivity for the human kinome containing 467 other human kinases. Using C17 as a chemical probe, we further performed quantitative phosphoproteomic assays and identified several novel DYRK2 targets, including eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) and stromal interaction molecule 1 (STIM1). DYRK2 phosphorylated 4E-BP1 at multiple sites, and the combined treatment of C17 with AKT and MEK inhibitors showed synergistic 4E-BP1 phosphorylation suppression. The phosphorylation of STIM1 by DYRK2 substantially increased the interaction of STIM1 with the ORAI1 channel, and C17 impeded the store-operated calcium entry process. These studies collectively further expand our understanding of DYRK2 and provide a valuable tool to pinpoint its biological function.

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Lukas P Feilen et al.
    Research Article

    Cleavage of membrane proteins in the lipid bilayer by intramembrane proteases is crucial for health and disease. Although different lipid environments can potently modulate their activity, how this is linked to their structural dynamics is unclear. Here we show that the carboxy-peptidase-like activity of the archaeal intramembrane protease PSH, a homolog of the Alzheimer's disease-associated presenilin/γ-secretase is impaired in micelles and promoted in a lipid bilayer. Comparative molecular dynamics simulations revealed that important elements for substrate binding such as transmembrane domain 6a of PSH are more labile in micelles and stabilized in the lipid bilayer. Moreover, consistent with an enhanced interaction of PSH with a transition-state analog inhibitor, the bilayer promoted the formation of the enzyme´s catalytic active site geometry. Our data indicate that the lipid environment of an intramembrane protease plays a critical role in structural stabilization and active site arrangement of the enzyme-substrate complex thereby promoting intramembrane proteolysis.