Aggregation pheromone 4-vinylanisole promotes the synchrony of sexual maturation in female locusts
- State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, China
- CAS Center for Excellence in Biotic Interactions, University of Chinese Academy of Sciences, China
- Beijing Institutes of Life Sciences, Chinese Academy of Sciences, China
Figures
Figure 1 with 2 supplements
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.
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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
Figure 1—figure supplement 1
Comparison of sexual maturation rate of gregarious and solitarious females.
The maturity of terminal oocytes in gregarious and solitarious females were measured from post adult eclosion (PAE) 2 to 8 days. Data were analyzed using Student’s t-test. The number of biological replicates and p values were shown in the figures.
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Figure 1—figure supplement 1—source data 1
Raw data for maturation rate of females between gregarious and solitarious phases.
- https://cdn.elifesciences.org/articles/74581/elife-74581-fig1-figsupp1-data1-v1.xlsx
Figure 1—figure supplement 2
Schematic diagram of the stimulation experiments for females of the migratory locust.
(A) Ten gregarious females were reared with 10 gregarious males or 10 gregarious females after emergence, respectively. (B) One solitarious female was reared with one solitarious male or one solitarious female after emergence, respectively. (C) Ten gregarious females were separately reared with 10 gregarious males or 10 gregarious females by a breathable partition after emergence. (D) One solitarious female was separately reared with 10 gregarious males or 10 solitarious males by a breathable partition after emergence. (E) Five Or mutant females (Orco-/- or Or35-/-) were reared with five gregarious wild-type (WT) females and 10 gregarious WT males from post adult eclosion (PAE) 1 to 6 days. (F) Five Or mutant females (Orco-/- or Or35-/-) and five gregarious WT females were separately reared with 10 gregarious WT males by a breathable partition from PAE 1 to 6 days.
Figure 2 with 3 supplements
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.
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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
Figure 2—figure supplement 1
Releasing dynamics of 14 identified volatiles in gregarious male adults, gregarious female adults, and solitarious male adults from post adult eclosion (PAE) 1 to 8 days.
Gregarious male adults release much more phenylacetonitrile (PAN), guaicol, 4-vinylanisole, vertrole, and anisole than gregarious female adults and solitarious male adults after PAE 4 days. Data are shown as means ± SEM (n = 5–8).
Figure 2—figure supplement 2
Dose-dependent effects of 4-vinylanisole (4-VA) on female maturation rate.
Different letters represent significant differences between the two groups (one-way ANOVA, p < 0.05). The number of biological replicates was shown in the figures.
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Figure 2—figure supplement 2—source data 1
Raw data for maturation rate of females treated by 4-vinylanisole (4-VA).
- https://cdn.elifesciences.org/articles/74581/elife-74581-fig2-figsupp2-data1-v1.xlsx
Figure 2—figure supplement 3
The sexual maturity of wild-type (WT) females and Or35-/- females after stimulation by volatiles released from gregarious males (n = 20, Levene’s test, p = 0.02; Student’s t-test, ***p < 0.001).
Lines in droplet diagram indicate median quartile.
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Figure 2—figure supplement 3—source data 1
Raw data for sexual maturity of wild-type (WT) females and Or35-/- females stimulated by volatiles released from gregarious males.
- https://cdn.elifesciences.org/articles/74581/elife-74581-fig2-figsupp3-data1-v1.xlsx
Figure 3 with 6 supplements
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.
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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
Figure 3—figure supplement 1
Effects of gregarious males with different ages on maturation synchrony of female aged at post adult eclosion (PAE) 3–4 days.
Gregarious females aged at PAE 3 days were reared together with the fifth-instar gregarious males, or male adults aged at PAE 1 day, PAE 3 days, PAE 5 days for 2 days, respectively. Data were analyzed by Levene’s test. The number of biological replicates was shown in the figures. Lines in droplet diagram indicate median quartile.
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Figure 3—figure supplement 1—source data 1
Raw data for sexual maturity of females reared with gregarious males with different ages.
- https://cdn.elifesciences.org/articles/74581/elife-74581-fig3-figsupp1-data1-v1.xlsx
Figure 3—figure supplement 2
Enrichment of differentially expressed genes (DEGs) and Kyoto encyclopedia of genes and genomes (KEGG) in the fat body of gregarious females after 4-vinylanisole (4-VA) stimulation at different developmental stages.
Gene expression pattern showed the most dynamic changes in the fat body of females exposed to 4-VA at post adult eclosion (PAE) 3–4 days.
Figure 3—figure supplement 3
Peripheral electrophysiological responses of female locusts to 4-vinylanisole (4-VA).
(A) and (B) Dosage effects on single sensilla responses (SSRs) of basiconic sensillum in females to 4-VA. (C) The SSR of basiconic sensillum in females to 4-VA aged at different developmental stages. Different letters represent significant differences between the two groups (one-way ANOVA, p < 0.05).
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Figure 3—figure supplement 3—source data 1
Raw data for electrophysiological responses of female locusts to 4-vinylanisole (4-VA).
- https://cdn.elifesciences.org/articles/74581/elife-74581-fig3-figsupp3-data1-v1.xlsx
Figure 3—figure supplement 4
The mRNA levels of LmigOr35 during post adult eclosion (PAE) 1–8 days.
Different letters represent significant differences (one-way ANOVA, p < 0.05). Points labeled with different letters indicate a significant difference between these groups. Data are shown as means ± SEM.
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Figure 3—figure supplement 4—source data 1
Raw data for mRNA levels of LmigOr35 during post adult eclosion (PAE) 1–8 days.
- https://cdn.elifesciences.org/articles/74581/elife-74581-fig3-figsupp4-data1-v1.xlsx
Figure 3—figure supplement 5
Volcano plot of RNA-seq data in the brain of females treated by 4-vinylanisole (4-VA) at post adult eclosion (PAE) 3–4 days.
There were 52 downregulated and 37 upregulated genes in the brain of female locusts upon 4-VA treatment, respectively.
Figure 3—figure supplement 6
The mRNA level of JHAMT and JHE upon 4-vinylanisole (4-VA) treatment.
Student’s t-test, n = 4, *p < 0.05, **p < 0.01. Data are shown as means ± SEM.
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Figure 3—figure supplement 6—source data 1
Raw data for mRNA level of JHAMT and JHE upon 4-vinylanisole (4-VA) treatment.
- https://cdn.elifesciences.org/articles/74581/elife-74581-fig3-figsupp6-data1-v1.xlsx
Figure 4 with 1 supplement
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.
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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
Figure 4—figure supplement 1
Validation the role of Or35 in 4-vinylanisole (4-VA)-enhanced vitellogenin (Vg) expression in the fat body of female locusts.
(A) The protein levels of Vg in the fat body of wild-type (WT) females and Or35-/- females with or without 4-VA treatment. Student’s t-test: n = 4, *p < 0.05, n.s. indicates not significant. Data are shown as means ± SEM. (B) The protein levels of Vg in the fat body of WT and Or35-/- females with 4-VA exposure and JHA treatment. Different letters represent significant differences (n = 4, one-way ANOVA, p < 0.05). Columns labeled with different letters indicate a significant difference between these groups. Data are shown as means ± SEM.
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Figure 4—figure supplement 1—source data 1
Raw data for vitellogenin (Vg) expression in the fat body of female locusts stimulated by 4-vinylanisole (4-VA).
- https://cdn.elifesciences.org/articles/74581/elife-74581-fig4-figsupp1-data1-v1.xlsx
Figure 5
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.
Additional files
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Supplementary file 1
List of genes related to juvenile hormone (JH) synthesis and degradation in the corpus cardiacum-corpora allatum (CC-CA) of female adults exposure to 4-vinylanisole (4-VA) at post adult eclosion (PAE) 3–4 days.
- https://cdn.elifesciences.org/articles/74581/elife-74581-supp1-v1.xlsx
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Supplementary file 2
Primers used in qPCR analysis.
- https://cdn.elifesciences.org/articles/74581/elife-74581-supp2-v1.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/74581/elife-74581-transrepform1-v1.docx
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Aggregation pheromone 4-vinylanisole promotes the synchrony of sexual maturation in female locusts
eLife 11:e74581.
https://doi.org/10.7554/eLife.74581
