Research ArticleECOLOGY

Phenylacetonitrile in locusts facilitates an antipredator defense by acting as an olfactory aposematic signal and cyanide precursor

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Science Advances  23 Jan 2019:
Vol. 5, no. 1, eaav5495
DOI: 10.1126/sciadv.aav5495
  • Fig. 1 PAN production patterns and behavioral responses of gregarious locusts under treatment with different dosages of synthetic PAN.

    (A) PAN production by first-instar (G1) to fifth-instar (G5) nymphs (means ± SEM, six biological replicates per sex per developmental stage, n = 6). Data for the two sexes were compared using two-tailed Student’s t test. (B) PAN emission per nymph reared under different population densities (1 to 100 nymphs). Data were analyzed using Kruskal-Wallis ANOVA on ranks following Tukey honestly significant difference (HSD) post hoc test. Different letters above bars represent significant differences among densities [P < 0.05 (means ± SEM, n = 6 biologically independent samples)]. (C) Whole-body PAN production by a gregarious locust. Detailed statistical analyses are shown in fig. S1. (D) Total distance moved (in centimeters) of a fourth-instar gregarious locust in the PAN zone versus in the paraffin oil control zone over 10 min in the dual-choice bioassay. (E) Total time spent (in seconds) over 10 min in the PAN zone versus that in the control zone. (D and E) PAN dosages used in behavioral assays were expressed as LM (1 LM = PAN emitted by one locust over 10 min in the PAN zone of the observation arena; dosages from 1 LM to 1000 LM; for a detailed description, see Materials and Methods). Under each treatment dosage, 25 locusts successfully finished the dual-choice tests (n = 25); C, control treatment. Distance or time data in the PAN zone versus the control zone for every treatment dosage were compared using Wilcoxon signed rank test (***P < 0.001; NS, not significant). All data are presented as the means ± SEM.

  • Fig. 2 Biosynthesis of PAN and characterization of the key enzyme CYP305M2, which catalyzes (Z)-phenylacetaldoxime ([Z]-PAOx) synthesis in the migratory locust.

    (A) GC-MS MRM chromatograms of (E/Z)-PAOx in the head integuments of gregarious (G, red trace) and solitary (S, green trace) locusts and synthetic standards (stds, black trace). (E/Z)-PAOx was identified on the basis of the chemical parent (135) and daughter ions (117 and 90). (B) (Z)-PAOx distribution in the body of a gregarious locust. Detailed statistical analyses are presented in table S1. (C to E) CYP305M2 expression (relative to rp49 gene), protein level (relative to GAPDH), and PAN and (Z)-PAOx production in head integument tissues of gregarious locusts at 48 hours after dsRNA injections and in those of untreated solitary locusts. Data were analyzed using Kruskal-Wallis ANOVA on ranks following Tukey HSD post hoc test (**P < 0.01, ***P < 0.001; means ± SEM, n = 5 biologically independent samples for each bar). (F) Histological localization of CYP305M2 in head integument tissue from dsGFP-injected (left) and dsCYP305M2-injected (right) gregarious locusts. CYP305M2 protein was detected in oenocytes (arrowheads). oe, oenocytes; epi, epidermal cell; fb, fat body; mu, muscle. Green, CYP305M2; blue, nuclear. Scale bars, 50 μm. (G) D8-Phe incorporation into (Z)-PAOx and PAN in dsGFP- or dsCYP305M2-injected gregarious and untreated solitary locusts. Data were analyzed using Kruskal-Wallis ANOVA on ranks following the Tukey HSD post hoc test (***P < 0.001; means ± SEM, n = 4 biologically independent samples). (H) Incorporation of deuterium-labeled (Z)-PAOx incorporation into PAN in dsGFP- or dsCYP305M2-injected gregarious locusts and untreated solitary locusts. ANOVA test (ND, not detected; means ± SEM, n = 6 biologically independent samples).

  • Fig. 3 PAN emission by the migratory locust influences predation by the great tit.

    (A) Typical gregarious and solitary fourth-instar nymphs on a background of green plants. Photo credit: All authors. (B) Total ion GC-MS chromatograms of SPME-trapped headspace volatiles of fourth-instar gregarious (G) (red trace) and solitary (S) (green trace) locusts. Major locust volatiles are listed above the compound peaks. PAN is present as the most abundant component in gregarious locusts but is absent from solitary locusts. (C) In a dual-choice bioassay conducted with paired locusts, the percentage (%) of the great tits selected and fed on hexane-treated solitary locusts (control) or PAN-treated solitary locusts. The first choice, injury rate, and consumption rate of the locusts by the great tits are recorded (means ± SEM, n = 9 birds). (D) First choice, injury rate, and consumption rate for the paired dsGFP-injected gregarious locusts and dsCYP305M2 (dsRNAi)–injected gregarious locusts (means ± SEM, n = 12 birds). (E) Selections of great tits between a hexane-treated dsCYP305M2-injected locust (control) and a PAN-treated dsCYP305M2-injected locust (right) (means ± SEM, n = 10 birds). (C to E) Paired data were compared using Wilcoxon signed rank test. **P < 0.01, ***P < 0.001.

  • Fig. 4 HCN biosynthesis from PAN in gregarious locusts.

    (A) HCN production in the headspaces of vials containing intact undisturbed, bird-attacked, or shake-disturbed (SD) gregarious and solitary locusts in the fourth-instar stage at 48 hours after ecdysis. Data were compared using Mann-Whitney U test (means ± SEM, n = 6 biologically independent samples for intact and bird-attacked locusts and n = 7 for SD locusts; **P < 0.01). (B) HCN contents of the headspaces of vials containing SD gregarious locusts injected with dsGFP or dsCYP305M2 and solitary locusts injected with PAN (1 μg per locust), (Z)-PAOx (1 μg per locust), and solvent control (10 μl of 5% EtOH per locust). Data are expressed as the means ± SEM (n = 6 biologically independent samples). (C) BA production in the head integument of intact undisturbed or SD fourth-instar dsGFP- or dsCYP305M2-injected gregarious (G) and solitary (S) locusts at 48 hours after ecdysis. Data are expressed as the means ± SEM (n = 6 biologically independent samples). (D) D8-Phe incorporation into BA in the headspace of SD dsGFP- or dsCYP305M2-injected gregarious and solitary locusts. Data are expressed as the means ± SEM (n = 5 biologically independent samples). (B to D) Data were statistically analyzed through Kruskal-Wallis ANOVA by ranks following Tukey HSD post hoc test. Different letters show significant differences among treatments (P < 0.05). (C) BA production levels by intact and SD dsGFP-injected gregarious locusts were compared using two-tailed Student’s t test (***P < 0.001).

  • Fig. 5 Model of the biosynthesis of PAN and HCN from phenylalanine, the steps of cyanogenesis, and the responses of bird predators to gregarious and solitary locusts.

    A cytochrome (CYP) gene (CYP305M2) that catalyzes the formation of (Z)-PAOx, a precursor in PAN and HCN biosynthesis from phenylalanine in gregarious locusts, was identified. CYP305M2 is present at barely detectable levels in solitary locusts. Aposematism with PAN can warn and deter consumers from poisoning by HCN generated from PAN. The yellow marker in gregarious locusts denotes the expression of CYP305M2. Against a green background, solitary locusts use camouflage to appear cryptic to predators and are otherwise readily discovered and predated.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/1/eaav5495/DC1

    Supplementary Text

    Fig. S1. PAN accumulation in tissues, organs, and body fluids of gregarious locusts.

    Fig. S2. PAN dominates the six major volatile compounds released in headspace and dissolved in hemolymph of fourth-instar gregarious locusts

    Fig. S3. Cytochrome (CYP) P450 gene transcriptomic analyses in whole body of gregarious and solitary locusts from first- to fifth-instar nymphs.

    Fig. S4. Knockdown of three putative CYP genes by RNAi.

    Fig. S5. Alignment of amino acid sequences of LmCYP305M2 (LM16181 shown in fig. S3B) with other five members in CYP305A subfamily of other insect species.

    Fig. S6. CYP305M2 gene expression levels in different tissues and organs of fifth-instar gregarious locusts.

    Fig. S7. The histological localization of CYP305M2 in tissue slides of head integument of dsGFP.

    Fig. S8. Gregarious locusts are distasteful to the great tit (P. major).

    Fig. S9. Major volatile components in the headspaces of hexane- and PAN-treated solitary locusts.

    Fig. S10. Comparison of major volatile components in headspaces between dsGFP- and dsCYP305M2-injected locusts or between hexane-treated and PAN-treated dsCYP305M2-injected locusts.

    Fig. S11. Perfuming beetle mealworm (T. molitor) with PAN increased the larval survivorship.

    Fig. S12. Perfuming fourth-instar solitary locusts (S4) with BA or phenol did not affect the bird selection and predation.

    Fig. S13. Extracted-ion GC-MS chromatograms of ion 26 of synthetic HCN and HCN in headspaces of locusts.

    Fig. S14. HPLC chromatograms of BA in the head integuments of fourth-instar gregarious (G) and solitary (S) locusts.

    Fig. S15. Confirmation of synthetic (E/Z)-PAOx with NMR and GC-MS.

    Fig. S16. Confirmation of synthetic D8-(E/Z)-PAOx with GC-MS.

    Fig. S17. HPLC chromatogram of D8-(E/Z)-PAOx.

    Fig. S18. Measurement of the average volumes of headspace gas and locusts in a hermetically sealed glass vial (20 ml).

    Fig. S19. Bird hungry and neophobic test before experiments.

    Fig. S20. PAN load of locusts influences predation by the great tit.

    Table S1. (E/Z)-PAOx in hexane extracts of tissues, organs, and body fluids of gregarious fourth-instar locusts.

    Table S2. Primer sequences used for PCR amplification and the dsRNA synthesis of the putative genes in PAN biosynthetic pathway.

    Table S3. MRM precursor and product ions and their collision energies (CE) of compounds.

    Movie S1. Video clip showing a bird preferring to attack a solitary fourth-instar locust as its first choice under light condition.

    Movie S2. Video clip showing a bird preferring to attack a solitary fourth-instar locust (S4) as its first choice under dark condition in a topless birdcage.

    Movie S3. Video clip demonstrating the effect of PAN supplementation on bird choice and predation on solitary locusts.

    Movie S4. Video clip shows that great tits preferentially attacked and consumed a dsCYP305M2-injected gregarious locust (RNAi) over a dsGFP-injected gregarious locust (GFP).

    Movie S5. Video clip showing the effect of PAN supplementation on bird choice and predation on dsCYP305M2-injected gregarious locust.

    Movie S6. Video clip showing bird-attacking behaviors in an observation chamber.

  • Supplementary Materials

    The PDF file includes:

    • Supplementary Text
    • Fig. S1. PAN accumulation in tissues, organs, and body fluids of gregarious locusts.
    • Fig. S2. PAN dominates the six major volatile compounds released in headspace and dissolved in hemolymph of fourth-instar gregarious locusts
    • Fig. S3. Cytochrome (CYP) P450 gene transcriptomic analyses in whole body of gregarious and solitary locusts from first- to fifth-instar nymphs.
    • Fig. S4. Knockdown of three putative CYP genes by RNAi.
    • Fig. S5. Alignment of amino acid sequences of LmCYP305M2 (LM16181 shown in fig. S3B) with other five members in CYP305A subfamily of other insect species.
    • Fig. S6. CYP305M2 gene expression levels in different tissues and organs of fifth-instar gregarious locusts.
    • Fig. S7. The histological localization of CYP305M2 in tissue slides of head integument of dsGFP.
    • Fig. S8. Gregarious locusts are distasteful to the great tit (P. major).
    • Fig. S9. Major volatile components in the headspaces of hexane- and PAN-treated solitary locusts.
    • Fig. S10. Comparison of major volatile components in headspaces between dsGFP- and dsCYP305M2-injected locusts or between hexane-treated and PAN-treated dsCYP305M2-injected locusts.
    • Fig. S11. Perfuming beetle mealworm (T. molitor) with PAN increased the larval survivorship.
    • Fig. S12. Perfuming fourth-instar solitary locusts (S4) with BA or phenol did not affect the bird selection and predation.
    • Fig. S13. Extracted-ion GC-MS chromatograms of ion 26 of synthetic HCN and HCN in headspaces of locusts.
    • Fig. S14. HPLC chromatograms of BA in the head integuments of fourth-instar gregarious (G) and solitary (S) locusts.
    • Fig. S15. Confirmation of synthetic (E/Z)-PAOx with NMR and GC-MS.
    • Fig. S16. Confirmation of synthetic D8-(E/Z)-PAOx with GC-MS.
    • Fig. S17. HPLC chromatogram of D8-(E/Z)-PAOx.
    • Fig. S18. Measurement of the average volumes of headspace gas and locusts in a hermetically sealed glass vial (20 ml).
    • Fig. S19. Bird hungry and neophobic test before experiments.
    • Fig. S20. PAN load of locusts influences predation by the great tit.
    • Table S1. (E/Z)-PAOx in hexane extracts of tissues, organs, and body fluids of gregarious fourth-instar locusts.
    • Table S2. Primer sequences used for PCR amplification and the dsRNA synthesis of the putative genes in PAN biosynthetic pathway.
    • Table S3. MRM precursor and product ions and their collision energies (CE) of compounds.
    • Legends for movies S1 to S6

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    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mov format). Video clip showing a bird preferring to attack a solitary fourth-instar locust as its first choice under light condition.
    • Movie S2 (.mp4 format). Video clip showing a bird preferring to attack a solitary fourth-instar locust (S4) as its first choice under dark condition in a topless birdcage.
    • Movie S3 (.mov format). Video clip demonstrating the effect of PAN supplementation on bird choice and predation on solitary locusts.
    • Movie S4 (.mov format). Video clip shows that great tits preferentially attacked and consumed a dsCYP305M2-injected gregarious locust (RNAi) over a dsGFP-injected gregarious locust (GFP).
    • Movie S5 (.mov format). Video clip showing the effect of PAN supplementation on bird choice and predation on dsCYP305M2-injected gregarious locust.
    • Movie S6 (.mp4 format). Video clip showing bird-attacking behaviors in an observation chamber.

    Files in this Data Supplement:

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