Journal of Molecular Evolution

, Volume 86, Issue 6, pp 379–394 | Cite as

Evolution of the Biosynthetic Pathway for Cyanogenic Glucosides in Lepidoptera

  • Mika Zagrobelny
  • Mikael Kryger Jensen
  • Heiko Vogel
  • René Feyereisen
  • Søren Bak
Original Article


Cyanogenic glucosides are widespread defence compounds in plants, and they are also found in some arthropods, especially within Lepidoptera. The aliphatic linamarin and lotaustralin are the most common cyanogenic glucosides in Lepidoptera, and they are biosynthesised de novo, and/or sequestered from food plants. Their biosynthetic pathway was elucidated in the burnet moth, Zygaena filipendulae, and consists of three enzymes: two cytochrome P450 enzymes, CYP405A2 and CYP332A3, and a glucosyl transferase, UGT33A1. Heliconius butterflies also produce linamarin and lotaustralin and have close homologs to CYP405A2 and CYP332A3. To unravel the evolution of the pathway in Lepidoptera, we performed phylogenetic analyses on all available CYP405 and CYP332 sequences. CYP332 sequences were present in almost all Lepidoptera, while the distribution of CYP405s among butterflies and moths was much more limited. Negative purifying selection was found in both CYP enzyme families, indicating that the biosynthesis of CNglcs is an old trait, and not a newly evolved pathway. We compared CYP405A2 to its close paralog, CYP405A3, which is not involved in the biosynthetic pathway. The only significant difference between these two enzymes is a smaller substrate binding pocket in CYP405A2, which would make the enzyme more substrate specific. We consider it likely that the biosynthetic pathway of CNglcs in butterflies and moths have evolved from a common pathway, perhaps based on a predisposition for detoxifying aldoximes by way of a CYP332. Later the aldoxime metabolising CYP405s evolved, and a UGT was recruited into the pathway to establish de novo biosynthesis of CNglcs.


CYP405 CYP332 CYP324 Heliconius Zygaena Cytochrome P450 



Axel Hofmann, Eric Drouet and Marc Nicolle are thanked for providing species of Zygaenoidea. We are grateful to Lene Dalsten for sequencing CYP405A2 from all Zygaenoidea species included in this study. Dr. David R. Nelson is thanked for assigning official names to all unnamed CYPs extracted from Genbank and private transcriptomes according to the general P450 nomenclature. We are grateful to Adam Takos for providing E. postvittana larvae and adults from Australia, and Camilla Knudsen for Pieris brassicae larvae from Copenhagen. This work was supported by the Danish Council for Independent Research (DFF–1323-00088).

Supplementary material

239_2018_9854_MOESM1_ESM.txt (59 kb)
Supplementary File S1 (TXT 58 KB)
239_2018_9854_MOESM2_ESM.docx (17 kb)
Supplementary Table S1 (DOCX 16 KB)


  1. Ahn SJ, Vogel H, Heckel DG (2012) Comparative analysis of the UDP-glycosyltransferase multigene family in insects. Insect Biochem Mol Biol 42:133–147CrossRefPubMedGoogle Scholar
  2. Ai J, Zhu Y, Duan J, Yu Q, Zhang G, Wan F, Xiang ZH (2011) Genome-wide analysis of cytochrome P450 monooxygenase genes in the silkworm, Bombyx mori. Gene 480:42–50CrossRefPubMedGoogle Scholar
  3. Bak S, Tax FE, Feldmann KA, Galbraith DW, Feyereisen R (2001) CYP83B1, a cytochrome P450 at the metabolic branch point in auxin and indole glucosinolate biosynthesis in Arabidopsis. Plant Cell 13:101–111CrossRefPubMedPubMedCentralGoogle Scholar
  4. Bak S, Paquette SM, Morant M, Rasmussen AV, Saito S, Bjarnholt N, Zagrobelny M, Jørgensen K, Hamann T, Osmani S et al (2006) Cyanogenic glycosides; a case study for evolution and application of cytochromes P450. Phytochem Rev 5:309–329CrossRefGoogle Scholar
  5. Baudry J, Li W, Pan L, Berenbaum MR, Schuler MA (2003) Molecular docking of substrates and inhibitors in the catalytic site of CYP6B1, an insect cytochrome P450 monooxygenase. Protein Eng 16:577–587CrossRefPubMedGoogle Scholar
  6. Bjarnholt N, Nakonieczny M, Kedziorski A, Debinski DM, Matter SF, Olsen CE, Zagrobelny M (2012) Occurrence of sarmentosin and other hydroxynitrile glucosides in Parnassius (papilionidae) butterflies and their food plants. J Chem Ecol 38:525–537CrossRefPubMedGoogle Scholar
  7. Brockerhoff EG, Suckling DM, Ecroyd CE, Wagstaff SJ, Raabe MC, Dowell RV, Wearings CH (2011) Worldwide host plants of the highly polyphagous, invasive Epiphyas postvittana (Lepidoptera: Tortricidae). J Econ Entomol 104:1514–1524CrossRefPubMedGoogle Scholar
  8. Brûckner A, Raspotnig G, Wehner K, Meusinger R, Norton RA, Heethoff M (2017) Storage and release of hydrogen cyanide in a chelicerate (Oribatula tibialis). Proc Natl Acad Sci USA 114:3469–3472CrossRefPubMedGoogle Scholar
  9. Calla B, Noble K, Johnson RM, Walden KKO, Schuler MA, Robertson HM, Berenbaum MR (2017) Cytochrome P450 diversification and hostplant utilization patterns in specialist and generalist moths: Birth, death and adaptation. Mol Ecol 26(21):6021–6035CrossRefPubMedGoogle Scholar
  10. Cardoso MZ, Gilbert LE (2007) A male gift to its partner? Cyanogenic glycosides in a spermatophore of longwing butterflies (Heliconius). Naturwissenschaften 94:39–42CrossRefPubMedGoogle Scholar
  11. Chauhan R, Jones R, Wilkinson P, Pauchet Y, ffrench-Constant RH (2013) Cytochrome P450-encoding genes from the Heliconius genome as candidates for cyanogenesis. Insect Mol Biol 22:532–540CrossRefPubMedGoogle Scholar
  12. Chiu TL, Wen Z, Rupasinghe SG, Schuler MA (2008) Comparative molecular modeling of Anopheles gambiae CYP6Z1, a mosquito P450 capable of metabolizing DDT. Proc Natl Acad Sci USA 105:8855–8860CrossRefPubMedGoogle Scholar
  13. Clausen M, Kannangara RM, Olsen CE, Blomstedt CK, Gleadow RM, Jørgensen K, Bak S, Motawie MS, Møller BL (2015) The bifurcation of the cyanogenic glucoside and glucosinolate biosynthetic pathways. Plant J 84:558–573CrossRefPubMedGoogle Scholar
  14. Cojocaru V, Winn PJ, Wade RC (2007) The ins and outs of cytochrome P450s. Biochim Biophys Acta 1770:390–401CrossRefPubMedGoogle Scholar
  15. Dobler S, Petschenka G, Wagschal V, Flacht L (2015) Convergent adaptive evolution—how insects master the challenge of cardiac glycoside-containing host plants. Entomol Exp et App 157:30–39CrossRefGoogle Scholar
  16. Edgar RC (2004) MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics 5:113CrossRefPubMedPubMedCentralGoogle Scholar
  17. Engler HS, Spencer KC, Gilbert LE (2000) Insect metabolism—preventing cyanide release from leaves. Nature 406:144–145CrossRefPubMedGoogle Scholar
  18. Feyereisen R (2011) Arthropod CYPomes illustrate the tempo and mode in P450 evolution. Biochim Biophys Acta 1814:19–28CrossRefPubMedGoogle Scholar
  19. Feyereisen R (2012) Insect CYP Genes and P450 Enzymes. In: Gilbert LI (ed) Insect molecular biology and biochemistry, pp 236–316. Academic Press, CambridgeCrossRefGoogle Scholar
  20. Fürstenberg-Hägg J, Zagrobelny M, Jørgensen K, Vogel H, Møller BL, Bak S (2014a) Chemical defense balanced by sequestration and de novo biosynthesis in a lepidopteran specialist. PLoS ONE 9:e108745CrossRefPubMedPubMedCentralGoogle Scholar
  21. Fürstenberg-Hägg J, Zagrobelny M, Olsen CE, Jørgensen K, Møller BL, Bak S (2014b) Transcriptional Regulation of de novo biosynthesis of cyanogenic glucosides throughout the life-cycle of the burnet moth Zygaena filipendulae (Lepidoptera). Insect Biochem Mol Biol 49:80–89CrossRefPubMedGoogle Scholar
  22. Gôtz S, Garcia-Gomez JM, Terol J, Williams TD, Nagaraj SH, Nueda MJ, Robles M, Talon M, Dopazo J, Conesa A (2008) High-throughput functional annotation and data mining with the Blast2GO suite. Nucl Acids Res 36:3420–3435CrossRefPubMedGoogle Scholar
  23. Hall TA (1999) BioEdit: a user-friendly biological sequence alignment aditor and analysis program for Windows 95/98/NT. Nucl Acids Symp Ser 41:95–98Google Scholar
  24. Hamberger B, Bak S (2013) Plant P450s as versatile drivers for evolution of species-specific chemical diversity. Philos Trans R Soc Lond B 368:20120426CrossRefGoogle Scholar
  25. Hasemann CA, Kurumbail RG, Boddupalli SS, Peterson JA, Deisenhofer J (1995) Structure and function of cytochromes P450: a comparative analysis of three crystal structures. Structure 3:41–62CrossRefPubMedGoogle Scholar
  26. Irmisch S, McCormick AC, Boeckler GA, Schmidt A, Reichelt M, Schneider B, Block K, Schnitzler JP, Gershenzon J, Unsicker SB et al (2013) Two herbivore-induced cytochrome P450 enzymes CYP79D6 and CYP79D7 catalyze the formation of volatile aldoximes involved in poplar defense. Plant Cell 25:4737–4754CrossRefPubMedPubMedCentralGoogle Scholar
  27. Irmisch S, Clavijo MA, Gunther J, Schmidt A, Boeckler GA, Gershenzon J, Unsicker SB, Kollner TG (2014) Herbivore-induced poplar cytochrome P450 enzymes of the CYP71 family convert aldoximes to nitriles which repel a generalist caterpillar. Plant J 80:1095–1107CrossRefPubMedGoogle Scholar
  28. Jensen NB, Zagrobelny M, Hjernø K, Olsen CE, Houghton-Larsen J, Borch J, Møller BL, Bak S (2011) Convergent evolution in biosynthesis of cyanogenic defence compounds in plants and insects. Nat Commun. PubMedPubMedCentralCrossRefGoogle Scholar
  29. Kumar P, Pandit SS, Steppuhn A, Baldwin IT (2014) Natural history-driven, plant-mediated RNAi-based study reveals CYP6B46’s role in a nicotine-mediated antipredator herbivore defense. Proc Natl Acad Sci USA 111:1245–1252CrossRefPubMedGoogle Scholar
  30. Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870–1874CrossRefPubMedGoogle Scholar
  31. Li W, Schuler MA, Berenbaum MR (2003) Diversification of furanocoumarin-metabolizing cytochrome P450 monooxygenases in two papilionids: specificity and substrate encounter rate. Proc Natl Acad Sci USA 100:14593–14598CrossRefPubMedGoogle Scholar
  32. Li X, Baudry J, Berenbaum MR, Schuler MA (2004) Structural and functional divergence of insect CYP6B proteins: from specialist to generalist cytochrome P450. Proc Natl Acad Sci USA 101:2939–2944CrossRefPubMedGoogle Scholar
  33. Mao W, Schuler MA, Berenbaum MR (2007) Cytochrome P450s in Papilio multicaudatus and the transition from oligophagy to polyphagy in the Papilionidae. Insect Mol Biol 16:481–490CrossRefPubMedGoogle Scholar
  34. Misof B, Liu S, Meusemann K, Peters RS, Donath A, Mayer C, Frandsen PB, Ware J, Flouri T, Beutel RG et al (2014) Phylogenomics resolves the timing and pattern of insect evolution. Science 346:763–767CrossRefPubMedGoogle Scholar
  35. Mitter C, Davis DR, Cummings MP (2016) Phylogeny and evolution of Lepidoptera. Annu Rev Entomol 62:265–283CrossRefPubMedGoogle Scholar
  36. Nahrstedt A, Davis RH (1983) Occurrence, variation and biosynthesis of the cyanogenic glucosides linamarin and lotaustralin in species of the Heliconiini (Insecta, Lepidoptera). Comp Biochem Physiol B 75:65–73CrossRefGoogle Scholar
  37. Natarajan C, Hoffmann FG, Weber RE, Fago A, Witt CC, Storz JF (2016) Predictable convergence in hemoglobin function has unpredictable molecular underpinnings. Science 354:336–339CrossRefPubMedPubMedCentralGoogle Scholar
  38. Nelson DR (2011) Progress in tracing the evolutionary paths of cytochrome P450. Biochim Biophys Acta 1814:14–18CrossRefPubMedGoogle Scholar
  39. Nelson DR, Goldstone JV, Stegeman JJ (2013) The cytochrome P450 genesis locus: the origin and evolution of animal cytochrome P450s. Philos Trans R Soc Lond B 368:20120474CrossRefGoogle Scholar
  40. Nishida R (1994) Sequestration of plant secondary compounds by butterflies and moths. Chemoecology 5/6:127–138CrossRefGoogle Scholar
  41. Nishida R (2002) Sequestration of defensive substances from plants by Lepidoptera. Annu Rev Entomol 47:57–92CrossRefPubMedGoogle Scholar
  42. Nishida R, Rothschild M (1995) A cyanoglucoside stored by a Sedum-feeding Apollo butterfly, Parnassius phoebus. Experientia 51:267–269CrossRefGoogle Scholar
  43. Nishida R, Rothschild M, Mummery R (1994) A cyanoglucoside, sarmentosin, from the magpie moth, Abraxas grossulariata, geometridae: Lepidoptera. Phytochemistry 36:37–38CrossRefGoogle Scholar
  44. Nutzmann HW, Osbourn A (2014) Gene clustering in plant specialized metabolism. Curr Opin Biotechnol 26:91–99CrossRefPubMedGoogle Scholar
  45. Pauchet Y, Wilkinson P, Vogel H, Nelson DR, Reynolds SE, Heckel DG, ffrench-Constant RH (2010) Pyrosequencing the Manduca sexta larval midgut transcriptome: messages for digestion, detoxification and defence. Insect Mol Biol 19:61–75CrossRefPubMedGoogle Scholar
  46. Pavelka A, Sebestova E, Kozlikova B, Brezovsky J, Sochor J, Damborsky J (2016) CAVER: algorithms for analyzing dynamics of tunnels in macromolecules. IEEE/ACM Trans Comput Biol Bioinform 13:505–517CrossRefPubMedGoogle Scholar
  47. Petschenka G, Wagschal V, von Tschirnhaus M, Donath A, Dobler S (2017) Convergently evolved toxic secondary metabolites in plants drive the parallel molecular evolution of insect resistance. Am Nat 190:S29–S43CrossRefPubMedGoogle Scholar
  48. Regier JC, Mitter C, Zwick A, Bazinet AL, Cummings MP, Kawahara AY, Sohn JC, Zwickl DJ, Cho S, Davis DR et al (2013) A large-scale, higher-level, molecular phylogenetic study of the insect order Lepidoptera (moths and butterflies). PLoS ONE 8:e58568CrossRefPubMedPubMedCentralGoogle Scholar
  49. Rupasinghe SG, Wen Z, Chiu TL, Schuler MA (2007) Helicoverpa zea CYP6B8 and CYP321A1: different molecular solutions to the problem of metabolizing plant toxins and insecticides. Protein Eng Des Sel 20:615–624CrossRefPubMedGoogle Scholar
  50. Schuler MA (2011) P450s in plant-insect interactions. Biochim Biophys Acta 1814:36–45CrossRefPubMedGoogle Scholar
  51. Sørensen M, Neilson EHJ, Møller BL (2018) Oximes: unrecognized chameleons in general and specialized plant metabolism. Mol Plant 11:95–117CrossRefPubMedGoogle Scholar
  52. Sugawara S, Hishiyama S, Jikumaru Y, Hanada A, Nishimura T, Koshiba T, Zhao Y, Kamiya Y, Kasahara H (2009) Biochemical analyses of indole-3-acetaldoxime-dependent auxin biosynthesis in Arabidopsis. Proc Natl Acad Sci USA 106:5430–5435CrossRefPubMedGoogle Scholar
  53. Suzuki H, Yokokura J, Ito T, Arai R, Yokoyama C, Toshima H, Nagata S, Asami T, Suzuki Y (2014) Biosynthetic pathway of the phytohormone auxin in insects and screening of its inhibitors. Insect Biochem Mol Biol 53:66–72CrossRefPubMedGoogle Scholar
  54. Takos AM, Rook F (2012) Why biosynthetic genes for chemical defense compounds cluster. Trends Plant Sci 17:383–388CrossRefPubMedGoogle Scholar
  55. Takos AM, Knudsen C, Lai D, Kannangara R, Mikkelsen L, Motawia MS, Olsen CE, Sato S, Tabata S, Jørgensen K et al (2011) Genomic clustering of cyanogenic glucoside biosynthetic genes aids their identification in Lotus japonicus and suggests the repeated evolution of this chemical defence pathway. Plant J 68:273–286CrossRefPubMedGoogle Scholar
  56. Vannette RL, Mohamed A, Johnson BR (2015) Forager bees (Apis mellifera) highly express immune and detoxification genes in tissues associated with nectar processing. Sci Rep 5:16224CrossRefPubMedPubMedCentralGoogle Scholar
  57. Vazquez-Albacete D, Montefiori M, Kol S, Motawia MS, Møller BL, Olsen L, Nørholm MH (2017) The CYP79A1 catalyzed conversion of tyrosine to (E)-p-hydroxyphenylacetaldoxime unravelled using an improved method for homology modeling. Phytochemistry 135:8–17CrossRefPubMedGoogle Scholar
  58. Vogel H, Badapanda C, Knorr E, Vilcinskas A (2014) RNA-sequencing analysis reveals abundant developmental stage-specific and immunity-related genes in the pollen beetle Meligethes aeneus. Insect Mol Biol 23:98–112CrossRefPubMedGoogle Scholar
  59. Webb B, Sali A (2014) Comparative protein structure modeling using Modeller. Curr Protoc Bioinform 47:5–32CrossRefGoogle Scholar
  60. Wybouw N, Pauchet Y, Heckel DG, van Leeuwen T (2016) Horizontal gene transfer contributes to the evolution of arthropod herbivory. Genome Biol Evol 8:1785–1801CrossRefPubMedPubMedCentralGoogle Scholar
  61. Xu B, Yang Z (2013) PAMLX: a graphical user interface for PAML. Mol Biol Evol 30:2723–2724CrossRefPubMedGoogle Scholar
  62. Yamaguchi T, Kuwahara Y, Asano Y (2017) A novel cytochrome P450, CYP3201B1, is involved in (R)-mandelonitrile biosynthesis in a cyanogenic millipede. FEBS Open Bio 7:335–347CrossRefPubMedPubMedCentralGoogle Scholar
  63. Yang Z (1998) Likelihood ratio tests for detecting positive selection and application to primate lysozyme evolution. Mol Biol Evol 15:568–573CrossRefPubMedGoogle Scholar
  64. Yang Z (2007) PAML 4: phylogenetic analysis by maximum likelihood. Mol Biol Evol 24:1586–1591CrossRefPubMedGoogle Scholar
  65. Yang Z, Bielawski JP (2000) Statistical methods for detecting molecular adaptation. Trends Ecol Evol 15:496–503CrossRefPubMedGoogle Scholar
  66. Yokoyama C, Takei M, Kouzuma Y, Nagata S, Suzuki Y (2017) Novel tryptophan metabolic pathways in auxin biosynthesis in silkworm. J Insect Physiol 101:91–96CrossRefPubMedGoogle Scholar
  67. Zagrobelny M, Møller BL (2011) Cyanogenic glucosides in the biological warfare between plants and insects: the Burnet moth-Birdsfoot trefoil model system. Phytochemistry 72:1585–1592CrossRefPubMedGoogle Scholar
  68. Zagrobelny M, Bak S, Møller BL (2008) Cyanogenesis in plants and arthropods. Phytochemistry 69:1457–1468CrossRefPubMedGoogle Scholar
  69. Zagrobelny M, Scheibye-Alsing K, Jensen NB, Møller BL, Gorodkin J, Bak S (2009) 454 pyrosequencing based transcriptome analysis of Zygaena filipendulae with focus on genes involved in biosynthesis of cyanogenic glucosides. BMC Genom 10:574CrossRefGoogle Scholar
  70. Zagrobelny M, Motawie MS, Olsen CE, Bak S, Møller BL (2013) Male-to-female transfer of 5-hydroxytryptophan glucoside during mating in Zygaena filipendulae (Lepidoptera). Insect Biochem Mol Biol 43:1037–1044CrossRefPubMedGoogle Scholar
  71. Zagrobelny M, Simonsen HT, Olsen CE, Bak S, Møller BL (2015) Volatiles from the burnet moth Zygaena filipendulae (Lepidoptera) and associated flowers, and their involvement in mating communication. Physiol Entomol 40:284–295CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Plant and Environmental SciencesUniversity of CopenhagenCopenhagenDenmark
  2. 2.Department of EntomologyMax Planck Institute of Chemical EcologyJenaGermany

Personalised recommendations