Journal of Chemical Ecology

, Volume 38, Issue 7, pp 882–892 | Cite as

Insect Egg Deposition Induces Indirect Defense and Epicuticular Wax Changes in Arabidopsis thaliana

  • Beatrice Blenn
  • Michele Bandoly
  • Astrid Küffner
  • Tobias Otte
  • Sven Geiselhardt
  • Nina E. Fatouros
  • Monika HilkerEmail author


Egg deposition by the Large Cabbage White butterfly Pieris brassicae on Brussels sprouts plants induces indirect defense by changing the leaf surface, which arrests the egg parasitoid Trichogramma brassicae. Previous studies revealed that this indirect defense response is elicited by benzyl cyanide (BC), which is present in the female accessory reproductive gland (ARG) secretion and is released to the leaf during egg deposition. Here, we aimed (1) to elucidate whether P. brassicae eggs induce parasitoid-arresting leaf surface changes in another Brassicacean plant, i.e., Arabidopsis thaliana, and, if so, (2) to chemically characterize the egg-induced leaf surface changes. Egg deposition by P. brassicae on A. thaliana leaves had similar effects to egg deposition on Brussels sprouts with respect to the following: (a) Egg deposition induced leaf surface changes that arrested T. brassicae egg parasitoids. (b) Application of ARG secretion of mated female butterflies or of BC to leaves had the same inductive effects as egg deposition. Based on these results, we conducted GC-MS analysis of leaf surface compounds from egg- or ARG-induced A. thaliana leaves. We found significant quantitative differences in epicuticular waxes compared to control leaves. A discriminant analysis separated surface extracts of egg-laden, ARG-treated, untreated control and Ringer solution-treated control leaves according to their quantitative chemical composition. Quantities of the fatty acid tetratriacontanoic acid (C34) were significantly higher in extracts of leaf surfaces arresting the parasitoids (egg-laden or ARG-treated) than in respective controls. In contrast, the level of tetracosanoic acid (C24) was lower in extracts of egg-laden leaves compared to controls. Our study shows that insect egg deposition on a plant can significantly affect the quantitative leaf epicuticular wax composition. The ecological relevance of this finding is discussed with respect to its impact on the behavior of egg parasitoids.


Tritrophic interactions Plant defense Insect eggs Pieris brassicae Trichogramma brassicae Brassicaceae Epicuticular wax Leaf surface 



We thank Ute Braun for rearing plants and insects. This work was funded by the German Research Foundation (DFG-GRK 837/2-06) and the Netherlands Organization for Scientific Research NWO/ALW Veni grant 863.09.002 (to N.E.F.).

Supplementary material

10886_2012_132_MOESM1_ESM.pdf (39 kb)
Supplementary Table 1 Comparison of cuticular wax composition of leaves of Arabidopsis thaliana in dependence of plant ecotype and extraction method (literature data and own data) (PDF 38 kb)


  1. Abdel-Latief, M. and Hilker, M. 2008. Innate immunity: eggs of Manduca sexta are able to respond to parasitism by Trichogramma evanescens. Insect Biochem. Mol. Biol. 38:136–145.PubMedCrossRefGoogle Scholar
  2. Andersson, J., Borg-Karlson, A.-K., and Wiklund, C. 2003. Antiaphrodisiacs in Pierid butterflies: a theme with variation! J. Chem. Ecol. 29:1489–1499.PubMedCrossRefGoogle Scholar
  3. Avato, P., Bianchi, G., and Pogna, N. 1990. Chemosystematics of surface lipids from maize and some related species. Phytochemistry 29:1571–1576.CrossRefGoogle Scholar
  4. Balbyshev, N. F. and Lorenzen, J. H. 1997. Hypersensitivity and egg drop: a novel mechanism of host plant resistance to Colorado potato beetle (Coleoptera: Chrysomelidae). J. Econ. Entomol. 90:652–657.Google Scholar
  5. Bernays, E. A. and Chapman, R. F. 1997. Host-Plant Selection by Phytophagous Insects. Chapman & Hall, New York.Google Scholar
  6. Beyaert, I., Wäschke, N., Scholz, A., Varama, M., Reinecke, A., and Hilker, M. 2010. Relevance of resource-indicating key volatiles and habitat odour for insect orientation. Anim. Behav. 79:1077–1086.CrossRefGoogle Scholar
  7. Bruce, T. J. A., Midega, C. A. O., Birkett, M. A., Pickett, J. A., and Khan, Z. R. 2010. Is quality more important than quantity? Insect behavioural responses to changes in a volatile blend after stemborer oviposition on an African grass. Biol. Lett. 6:314–317.PubMedCrossRefGoogle Scholar
  8. Chang, G. C., Neufeld, J., Durr, D., Duetting, P. S., and Eigenbrode, S. D. 2004. Waxy bloom in peas influences the performance and behavior of Aphidius ervi, a parasitoid of the pea aphid. Entomol. Exp. Appl. 110:257–265.CrossRefGoogle Scholar
  9. Colazza, S., Aquila, G., De Pasquale, C., Peri, E., and Millar, J. G. 2007. The egg parasitoid Trissolcus basalis uses n-nonadecane, a cuticular hydrocarbon from its stink bug host Nezara viridula, to discriminate between female and male hosts. J. Chem. Ecol. 33:1405–1420.PubMedCrossRefGoogle Scholar
  10. Colazza, S., Lo Bue, M., Lo Giudice, D., and Peri, E. 2009. The response of Trissolcus basalis to footprint contact kairomones from Nezara viridula females is mediated by leaf epicuticular waxes. Naturwissenschaften 96:975–981.PubMedCrossRefGoogle Scholar
  11. Conti, E., Salerno, G., Leombruni, B., Frati, F., and Bin, F. 2010. Short-range allelochemicals from a plant-herbivore association: a singular case of oviposition-induced synomone for an egg parasitoid. J. Exp. Biol. 213:3911–3919.PubMedCrossRefGoogle Scholar
  12. Cooper, L. D., Doss, R. P., Price, R., Peterson, K., and Oliver, J. E. 2005. Application of bruchin B to pea pods results in the up-regulation of CYP93C18, a putative isoflavone synthase gene, and an increase in the level of pisatin, an isoflavone phytoalexin. J. Exp. Bot. 56:1229–1237.PubMedCrossRefGoogle Scholar
  13. Desurmont, G. A. and Weston, P. A. 2011. Aggregative oviposition of a phytophagous beetle overcomes egg-crushing plant defences. Ecol. Entomol. 36:335–343.CrossRefGoogle Scholar
  14. Doss, R. P. 2005. Treatment of pea pods with bruchin B results in up-regulation of a gene similar to MtN19. Plant Physiol. Biochem. 43:225–231.PubMedCrossRefGoogle Scholar
  15. Doss, R. P., Proebsting, W. M., Potter, S. W., and Clement, S. L. 1995. Response of Np mutant of pea (Pisum sativum L.) to pea weevil (Bruchus pisorum L.) oviposition and extracts. J. Chem. Ecol. 21:97–106.CrossRefGoogle Scholar
  16. Doss, R. P., Oliver, J. E., Proebsting, W. M., Potter, S. W., Kuy, S. R., Clement, S. L., Williamson, R. T., Carney, J. R., and Devilbiss, E. D. 2000. Bruchins: insect-derived plant regulators that stimulate neoplasm formation. Proc. Natl. Acad. Sci. USA 97:6218–6223.PubMedCrossRefGoogle Scholar
  17. Dutton, A., Mattiacci, L., Amadò, R., and Dorn, S. 2002. A novel function of the triterpene squalene in a tritrophic system. J. Chem. Ecol. 28:103–116.PubMedCrossRefGoogle Scholar
  18. Edwards, P. B. 1982. Do waxes on juvenile Eucalyptus leaves provide protection from grazing insects? Aust. J. Ecol. 7:347–352.CrossRefGoogle Scholar
  19. Eigenbrode, S. D. 2004. The effects of plant epicuticular waxy blooms on attachment and effectiveness of predatory insects. Arthropod. Struct. Dev 33:91–102.PubMedCrossRefGoogle Scholar
  20. Eigenbrode, S. D. and Espelie, K. E. 1995. Effects of plant epicuticular lipids on insect herbivores. Annu. Rev. Entomol. 40:171–194.CrossRefGoogle Scholar
  21. Espelie, K. E., Bernays, E. A., and Brown, J. J. 1991. Plant and insect cuticular lipids serve as behavioral cues for insects. Arch. Insect Biochem. 17:223–233.CrossRefGoogle Scholar
  22. Fatouros, N. E., Bukovinszkine’Kiss, G., Kalkers, L. A., Soler Gamborena, R., Dicke, M., and Hilker, M. 2005. Oviposition-induced plant cues: do they arrest Trichogramma wasps during host location? Entomol. Exp. Appl. 115:207–215.CrossRefGoogle Scholar
  23. Fatouros, N. E., Broekgaarden, C., Fatouros, N. E., Broekgaarden, C., Bukovinszkine’Kiss, G., Van Loon, J. J. A., Mumm, R., Huigens, M. E., Dicke, M., and Hilker, M. 2008. Male-derived butterfly anti-aphrodisiac mediates indirect plant defense. Proc. Natl. Acad. Sci. USA 105:10033–10038.PubMedCrossRefGoogle Scholar
  24. Fatouros, N. E., Pashalidou, F. G., Aponte Cordero, W. V., Van Loon, J. J. A., Mumm, R., Dicke, M., Hilker, M., and Huigens, M. E. 2009. Anti-aphrodisiac compounds of male butterflies increase the risk of egg parasitoid attack by inducing plant synomone production. J. Chem. Ecol. 35:1373–1381.PubMedCrossRefGoogle Scholar
  25. Feltwell, J. 1982. Large White Butterfly: The Biology, Biochemistry and Physiology of Pieris brassicae (Linnaeus). Dr. W. Junk, The Hague.Google Scholar
  26. Greany, P. D., Tumlinson, J. H., Chambers, D. L., and Boush, G. M. 1977. Chemically mediated host finding by Biosteres (Opius) longicaudatus, a parasitoid of tephritid fruit fly larvae. J. Chem. Ecol. 10:1251–1264.Google Scholar
  27. Hilker, M. and Meiners, T. 2006. Early herbivore alert: insect eggs induce plant defense. J. Chem. Ecol. 32:1379–1397.PubMedCrossRefGoogle Scholar
  28. Hilker, M. and Meiners, T. 2010. How do plants “notice” attack by herbivorous arthropods? Biol. Rev. 85:267–280.PubMedCrossRefGoogle Scholar
  29. Hilker, M. and Meiners, T. 2011. Plants and insect eggs: how do they affect each other? Phytochemistry 72:1612–1623.PubMedCrossRefGoogle Scholar
  30. Jenks, M. A., Tuttle, H. A., Eigenbrode, S. D., and Feldmann, K. A. 1995. Leaf epicuticular waxes of eceriferum mutants in Arabidopsis. Plant Physiol. 108:369–377.PubMedGoogle Scholar
  31. Jenks, M. A., Rashotte, A. M., Tuttle, H. A., and Feldmann, K. A. 1996a. Mutants in Arabidopsis thaliana altered in epicuticular wax and leaf morphology. Plant Physiol. 110:377–385.Google Scholar
  32. Jenks, M. A., Tuttle, H. A., and Feldman, K. A. 1996b. Changes in epicuticular waxes on wild type and eceriferum mutants in Arabidopsis during development. Phytochemistry 42:29–34.CrossRefGoogle Scholar
  33. Jenks, M. A., Eigenbrode, S. D., and Lemieux, B. 2002. Cuticular waxes of Arabidopsis, pp. 1–24, in C. Somerville and E. Meyerowitz (eds.), The Arabidopsis Book 1: e0016. American Society of Plant Biologists, Rockville.Google Scholar
  34. Jetter, E. and Schäffer, S. 2001. Chemical composition of the Prunus laurocerasus leaf surface. Dynamic changes of the epicuticular wax film during leaf development. Plant Physiol. 126:1725–1737.PubMedCrossRefGoogle Scholar
  35. Jetter, R., Schäffer, S., and Riederer, M. 2000. Leaf cuticular waxes are arranged in chemically and mechanically distinct layers: evidence from Prunus laurocerasus L. Plant Cell Environ. 23:619–628.CrossRefGoogle Scholar
  36. Jetter, R., Kunst, L., and Samuels, A. L. 2006. Composition of plant cuticular waxes, pp. 145–181, in M. Riederer and C. Müller (eds.), Biology of the Plant Cuticle. Annual Plant Reviews, Vol. 23. Blackwell Publishing, Oxford.CrossRefGoogle Scholar
  37. Kerstiens, G. 1996. Plant Cuticles: An Integrated Functional Approach. BIOS Scientific Publishers, Oxford.Google Scholar
  38. Knutson, A. 1998. The Trichogramma Manual. b-6071. Texas Agriculture Extension Service Texas A&M University System, College Station.Google Scholar
  39. Koepke, D., Schroeder, R., Fischer, H. M., Gershenzon, J., Hilker, M., and Schmidt, A. 2008. Does egg deposition by herbivorous pine sawflies affect transcription of sesquiterpene synthases in pine? Planta 228:427–438.CrossRefGoogle Scholar
  40. Koepke, D., Beyaert, I., Gershenzon, J., Hilker, M., and Schmidt, A. 2010. Species-specific responses of pine sesquiterpene synthases to sawfly oviposition. Phytochemistry 71:909–917.CrossRefGoogle Scholar
  41. Kosma, D. K., Bourdenx, B., Bernard, A., Parsons, E. P., Lü, S., Joubès, J., and Jenks, M. A. 2009. The impact of water deficiency on leaf cuticle lipids of Arabidopsis. Plant Physiol. 151:1918–1929.PubMedCrossRefGoogle Scholar
  42. Kováts, E. 1965. Gas chromatographic characterization of organic substances in the retention index system. Adv. Chromatogr. 1:229–247.Google Scholar
  43. Kunst, L. and Samuels, A. L. 2003. Biosynthesis and secretion of plant cuticular wax. Prog. Lipid Res. 42:51–80.PubMedCrossRefGoogle Scholar
  44. Little, D., Gouhier-Darimont, C., Bruessow, F., and Reymond, P. 2007. Oviposition by pierid butterflies triggers defense responses in Arabidopsis. Plant Physiol. 143:784–800.PubMedCrossRefGoogle Scholar
  45. Lo Giudice, D., Peri, E., Lo Bue, M., and Colazza, S. 2010. Plant surfaces of vegetable crops mediate interactions between chemical footprints of true bugs and their egg parasitoids. Commun. Integr. Biol. 3:70–74.CrossRefGoogle Scholar
  46. Müller, C. 2006. Plant-insect interactions on cuticular surfaces, pp. 398–422, in M. Riederer and C. Müller (eds.), Biology of the plant cuticle. Annual plant reviews, vol. 23. Blackwell Publishing, Oxford.CrossRefGoogle Scholar
  47. Müller, C. 2008. Resistance at the plant cuticle, pp. 107–129, in A. Schaller (ed.), Induced Plant Resistance to Herbivory. Springer, Berlin.CrossRefGoogle Scholar
  48. Noldus, L. P. J. J., van Lenteren, J. C., and Lewis, W. J. 1991. How Trichogramma parasitoids use moth sex pheromones as kairomones: orientation behaviour in a wind tunnel. Physiol. Entomol. 16:313–327.CrossRefGoogle Scholar
  49. Petzold-Maxwell, J., Wong, S., Arellano, C., and Gould, F. 2011. Host plant direct defence against eggs of its specialist herbivore, Heliothis subflexa. Ecol. Entomol. 36:700–708.CrossRefGoogle Scholar
  50. Pinto, J. D. and Stouthammer, R. 1994. Systematics of the Trichogrammatidae with emphasis on Trichogramma, pp. 1–36, in E. Wajnberg and S. A. Hassan (eds.), Biological Control with Egg Parasitoids. CAB International, Wallingford.Google Scholar
  51. Reifenrath, K., Riederer, M., and Müller, C. 2005. Leaf surface wax layers of Brassicaceae lack feeding stimulants for Phaedon cochleariae. Entomol. Exp. Appl. 115:41–50.CrossRefGoogle Scholar
  52. Riederer, M. and Müller, C. 2006. Biology of the Plant Cuticle. Blackwell Publishing, Oxford.CrossRefGoogle Scholar
  53. Riederer, M. and Schneider, G. 1990. The effect of the environment on the permeability and composition of Citrus leaf cuticles. Planta 180:154–165.CrossRefGoogle Scholar
  54. Rostás, M. and Woelfling, M. 2009. Caterpillar footprints as host location kairomones for Cotesia marginiventris. J. Chem. Ecol. 35:20–27.PubMedCrossRefGoogle Scholar
  55. Rostás, M., Ruf, D., Zabka, V., and Hildebrandt, U. 2008. Plant surface wax affects parasitoid’s response to host footprints. Naturwissenschaften 95:997–1002.PubMedCrossRefGoogle Scholar
  56. Rutledge, C. E. 1996. A survey of identified kairomones and synomones used by insect parasitoids to locate and accept their hosts. Chemoecology 7:121–131.CrossRefGoogle Scholar
  57. Samuels, L., Kunst, L., and Jetter, R. 2008. Sealing plant surfaces: cuticular wax formation by epidermal cells. Annu. Rev. Plant Biol. 59:683–707.PubMedCrossRefGoogle Scholar
  58. Schroeder, R., Wurm, L., Varama, M., Meiners, T., and Hilker, M. 2008. Unusual mechanims involved in learning of oviposition-induced host plant odours in an egg parasitoid? Anim. Behav. 75:1423–1430.CrossRefGoogle Scholar
  59. Seino, Y., Suzuki, Y., and Sogawa, K. 1996. An ovicidal substance produced by rice plants in response to oviposition by the whitebacked planthopper, Sogatella furcifera (Horváth) (Homoptera: Delphacidae). Appl. Entomol. Zool. 31:467–473.Google Scholar
  60. Shapiro, A. M. and de Vay, J. E. 1987. Hypersensitivity reaction of Brassica nigra L. (Cruciferae) kills eggs of Pieris butterflies (Lepidoptera: Pieridae). Oecologia 71:631–632.CrossRefGoogle Scholar
  61. Shepherd, T. and Griffiths, D. W. 2006. The effect of stress on plant cuticular waxes. New Phytol. 171:469–499.PubMedCrossRefGoogle Scholar
  62. Shu, S., Swedenborg, P. D., and Jones, R. L. 1990. A kairomone for Trichogramma nubilale (Hymenoptera: Trichogrammatidae). Isolation, identification and synthesis. J. Chem. Ecol. 16:521–529.CrossRefGoogle Scholar
  63. Sokal, R. R. and Rohlf, J. F. 1969. Biometry. W. H. Freeman and Co., San Francisco.Google Scholar
  64. Suzuki, Y., Sogawa, K., and Seino, Y. 1996. Ovicidal reaction of rice plants against the Whitebacked planthopper Sogatella furcifera Horváth (Homoptera: Delphacidae. Appl. Entomol. Zool. 31:111–118.Google Scholar
  65. Tamiru, A., Bruce, T. J. A., Woodcock, C. M., Caulfield, J. C., Midega, C. A. O., Ogol, C. K. P. O., Mayon, P., Birkett, M. A., Pickett, J. A., and Khan, Z. R. 2011. Maize landraces recruit egg and larval parasitoids in response to egg deposition by a herbivore. Ecol. Lett. 14:1075–1083.PubMedCrossRefGoogle Scholar
  66. Wen, M. and Jetter, R. 2009. Composition of secondary alcohols, ketones, alkanediols, and ketols in Arabidopsis thaliana cuticular waxes. J. Exp. Bot. 60:1811–1821.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Beatrice Blenn
    • 1
  • Michele Bandoly
    • 1
  • Astrid Küffner
    • 1
  • Tobias Otte
    • 1
  • Sven Geiselhardt
    • 1
  • Nina E. Fatouros
    • 2
  • Monika Hilker
    • 1
    Email author
  1. 1.Institute of BiologyFreie Universität BerlinBerlinGermany
  2. 2.Laboratory of Entomology, Department of Plant SciencesWageningen UniversityWageningenThe Netherlands

Personalised recommendations