Journal of Chemical Ecology

, Volume 29, Issue 9, pp 2085–2100 | Cite as

Chemical and Olfactory Characterization of Odorous Compounds and Their Precursors in the Parotoid Gland Secretion of the Green Tree Frog, Litoria caerulea

  • Benjamin P. C. Smith
  • Michael J. Tyler
  • Brian D. Williams
  • Yoji Hayasaka


When stressed or challenged by a predator, the Australian green tree frog, Litoria caerulea, emits a characteristic nutty odor from its parotoid glands. This study identifies the source of the odor as the cyclic amide 2-pyrrolidone (2-PyrO). In addition, we demonstrate the presence of 2-PyrO's straight chain form, γ-aminobutyric acid or GABA, in the frog's glandular secretion and propose an odorant–precursor relationship. What role both compounds play in the frog's defensive strategy remains unknown. Prolonged exposure to the odor is shown to result in adverse effects that may be attributed to a GABAergic mechanism. It is our hypothesis, however, that the odor acts as an aposematic signal, indicating the toxicity of the frog's nonvolatile secretion.

Litoria caerulea odorous/volatile secretion nutty odor 2-pyrrolidone GABA 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Albert, A. 1968. Heterocyclic Chemistry, 2nd edn. Athlone Press, University of London, London.Google Scholar
  2. Aleshin, V. V. 1974. Toxicological-hygienic characteristics of pyrrolidone, Med. Probl. Okhr. Vneshn. Sredy 4; Chem. Abstr. 86 26605b. Cited in Ullmann's Encyclopedia of Industrial Chemistry, 6th Edition. (2002), Wiley InterScience, New York [electronic resource].Google Scholar
  3. Anastasi, A., Erspamer, V., and Endean, R. 1968. Isolation and amino acid sequence of Caerulein, the active decapeptide of the skin of Hyla caerulea. Arch. Biochem. Biophys. 125:57–68.PubMedGoogle Scholar
  4. Barthalmus, G. T. 1994. Biological roles of amphibian skin secretions, pp. 382–410, in H. Heatwole, G. T. Barthalmus, and A. Y. Heatwole, (Eds.). Amphibian Biology, Vol. 1. Surrey Beatty, Sydney, NSW, Australia.Google Scholar
  5. Bergström, K. and Gürtler, J. 1971. Trimethylsilylation of amino acids. II: Gas chromatographic and structural studies on trimethylsilyl derivatives of straight chain amino acids. Acta Chem. Scand. 25:175–181.Google Scholar
  6. Bessman, S. P. and Fishbein, W. M. 1963. Gamma-hydroxybutyrate, a normal brain metabolite. Nature 200:1207–1208.PubMedGoogle Scholar
  7. Boulenger, E. G. 1911. On a new tree frog from Trinidad, living in the society's gardens. Proc. Zool. Soc. (London) 2:1082–1083.Google Scholar
  8. Brodie, ED, Jr. 1983. Antipredator adaptations of Salamanders: Evolution and convergence among terrestrial species, pp. 109–133, in N. S. Margaris, M. Arianoutsou-Faraggitaki, and R. J. Reiter (Eds.). Plant, Animal and Microbial Adaptations to Terrestrial Environment, Plenum, New York.Google Scholar
  9. Budzikiewicz, H., Djerassi, C. and Williams, D. H. 1967. Mass Spectrometry of Organic Compounds. Holden-Day, San Francisco, California.Google Scholar
  10. Clark, D. R. 1971. Branding as a marking technique for amphibians and reptiles. Copeia 1971:145–151.Google Scholar
  11. CRCS Inc. 1985. Working draft, EPA contract no. 68–01–650, TSCA Interagency Testing Committee, July 15, 1985. Cited in Ullmann's Encyclopedia of Industrial Chemistry, 6th Edition. (2001), Wiley InterScience, New York [electronic resource].Google Scholar
  12. Daly, J. W. 1995. The chemistry of poisons in amphibian skin. Proc. Nate. Acad. Sci. USA. 92:9–13.Google Scholar
  13. Daly, J. W., Myers, C. W., and Whittaker, N. 1987. Further classification of skin alkaloids from neotropical poison frogs (Dendrobatidae), with a general survey of toxic/noxious substances in the amphibia. Toxicon 25:1023–1095.PubMedGoogle Scholar
  14. Delfinio, G., Brizzi, R., and Calloni, C. 1985. Dermoepithelial interactions during the development of cutaneous gland anlagen in amphibia: A light and electron microscope study on several species with some cytochemical findings. Z. Mikrosk. Anat. Forsch. Leipzig.99 (2s):225–253.Google Scholar
  15. Deml, R. 2001. Influence of food and larval age on the defensive chemistry of Saturnia pyri. Z. Naturforsch.. C56:89–94.Google Scholar
  16. Deml, R. and Dettner, K. 1995. “Balloon hairs” of gipsy moth larvae (Lep., Lymantriidae): Morphology and comparative chemistry. Comp. Biochem. Physiol.. B112:673–681.Google Scholar
  17. Duellman, W. E. and Trueb, L. 1986. Biology of Amphibians. McGraw-Hill, New York.Google Scholar
  18. Erspamer, V. 1994. Bioactive secretions of the amphibian integument, pp. 178–350, in H. Heatwole, G. T. Barthalmus, and A. Y. Heatwole, (Eds.), Amphibian Biology, Vol. 1. Surrey Beatty, Sydney, NSW, Australia.Google Scholar
  19. Fisher, R., Norris, J. W., and Gilka, L. 1974. GABA in Huntington's chorea. Lancet 23:506.Google Scholar
  20. Gans, C. and Northcutt, R. G. 1983. Neural crest and the origin of vertebrates: A new head. Science. 220:268–274.Google Scholar
  21. Gilon, P., Mallefet, J., De Vriendt, C., Pauwels, S., Geffard, M., Campistron, G., and Remacle, C. 1990. Immunocytochemical and autoradiographic studies of the endocrine cells interacting with GABA in the rat stomach. Histochemistry 93:645–654.PubMedGoogle Scholar
  22. Guyton, A. G. and Hall, J. E. 1996. Textbook of Medical Physiology, 9th edn. Saunders, Philadelphia, Pennsylvania.Google Scholar
  23. Habermehl, G. 1981. Venomous Animals and Their Toxins. Springer-Verlag, Berlin.Google Scholar
  24. Juhasz, G., Emri, Z., Kekesi, K., and Pungor, K. 1989. Local perfusion of the thalmus with GABA increases sleep and induces long-lasting inhibition of somatosensory event related potentials in cats. Neurosci. Lett. 103:229–233.PubMedGoogle Scholar
  25. Lebeau, M. A., Montgomery, M. A., Miller, M. L., and Burmeister, S. G. 2000. Analysis of biofluids for gamma-hydroxybutyrate (GHB) and gamma-butyrolactone (GBL) by headspace GC-FID and GC-MS. J. Anal. Toxicol.. 24:421–428.PubMedGoogle Scholar
  26. Le Douarin, N. M. 1986. Cell line segregation during peripheral nervous system ontogeny. Science 231:1515–1522.PubMedGoogle Scholar
  27. Luning, P. A., Roozen, J. P., Moest, R. A. F. J., and Posthumus, M. A. 1991. Volatile composition of white bread using enzyme active soya flour as improver. Food Chem..41:81–91.Google Scholar
  28. Martin, A. A. and Littlejohn, M. J. 1966. The breeding biology and larval development of Hyla jervisiensis (Anura: Hylidae). Proc. Linn. Soc. NSW.91:47–57.Google Scholar
  29. Mende, P., Ziebarth, D., Preussmann, R., and Spieglehalder, B. 1994. Occurrence of the nitrosamide precursor pyrrolidine-(2)-one in food and tabacco. Carcinogenesis 15:733–737.PubMedGoogle Scholar
  30. Mesmer, M. Z. and Satzger, R. D. 1997. Determination of gamma-hydroxybutyrate (GHB) and gamma-butyrolactone (GBL) by HPLC/UV-VIS spectrophotometry and HPLC/thermospray mass spectrometry. J. Forensic Sci. 43:489–492.Google Scholar
  31. Michl, H. and Bachmayer, H. 1964. Free amino acids in the poison of the orange speckled toad, Bombina variegata L. Montash. 95:480–484.Google Scholar
  32. Mueller, G. P., Alpert, L., Reichlin, S., and Jackson, I. M. D. 1980. Thyrotropin-releasing hormone and serotonin secretion from frog skin are stimulated by norepinepherine. Endocrinology 106:1–4.PubMedGoogle Scholar
  33. Noble, G. K. 1931. The Biology of the Amphibia. McGraw-Hill, New York.Google Scholar
  34. Nussbaum, R. A., Brodie, E. D., Jr, and Strom, R. M. 1983. Amphibians and Reptiles of the Pacific Northwest. University of Idaho, Moscow, Idaho.Google Scholar
  35. Pearse, A. G. E. 1976. Peptides in brain and intestine. Nature. 262:92–94.Google Scholar
  36. Pearse, A. G. E. 1977. The diffuse neuroendocrine system and the APUD concept: Related endocrine peptides in brain, intestine, pituitary, placenta, and anuran cutaneous glands. Med. Biol.. 55:115–125.PubMedGoogle Scholar
  37. Pearse, A. G. E. and Takor, T. T. 1976. Neuroendocrine embryology and the APUD concept. Clin. Endocrinol..(Suppl.) 5:229s-224s.Google Scholar
  38. Perry, T. L., Hansen, S., and Urquhart, N. 1974. GABA in Huntington's chorea. Lancet 18: 995–996.Google Scholar
  39. Pianka, E. R. 1978. Evolutionary Ecology, 2nd edn. Harper & Row, New York.Google Scholar
  40. Reynolds, J. E. F. and Prasad, A. B. (Eds.) 1982. Martindale The Extra Pharmacopeia, 28th edn. Pharmaceutical Press, London, England, p. 1678.Google Scholar
  41. Roseghini, M., Erspamer, V., and Endean, R. 1976. Indole-, imidazole-, and phenyl-alkylamines in the skin of one hundred amphibian species from Australia and Papua New Guinea. Comp. Biochem. Physiol.. C54:31–43.Google Scholar
  42. Roth, R. H. and Giarman, N. J. 1969. Conversion in vivo of γ-amino-butyric to γ-hydroxybutyric acid in mammalian brain. Biochem. Pharmacol. 18:247–250.PubMedGoogle Scholar
  43. Salmon, A. L., Johnsen, A. H., Bienert, M., Mcmurray, G., Nandha, K. A., Bloom, S. R., and Shaw, C. 2000. Isolation, structural characterization, and bioactivity of a novel Neuromedin U analog from the defensive skin secretion of the Australasian tree frog, Litoria caerulea. J. Biol. Chem.. 275:4549–4554.PubMedGoogle Scholar
  44. Schildknecht, H., Kunzelmann, P., Krauss, D., and Kuhn, C. 1972. über die Chemie der Spinnwebe. I: Arthropodenabwehrstoffe, LLLVII. Naturwissenschaften. 59:98–99.Google Scholar
  45. Seki, T., Kikuyama, S., and Yanaihara, N. 1989. Development of Xenopus laevis skin glands producing 5-hydroxytryptamine and cerulein. Cell Tissue Res. 258:483–489.PubMedGoogle Scholar
  46. Stebbins, R. C. (1985). A Field Guide to Western Reptiles and Amphibians. Houghton Mifflin, Boston, Massachusetts.Google Scholar
  47. Stebbins, R. C. and Cohen, N. W. 1995. A Natural History of Amphibians. Princeton University Press, Princeton, New Jersey.Google Scholar
  48. Stone, D. J. M., Waugh, R. J., Bowie, J. H., and Wallace, J. C. 1992. Peptides from Australian frogs: Structure of the caerins and caeridin I from Litoria splendida. J. Chem. Soc., Perkin. Trans. 1 1992:3173–3178.Google Scholar
  49. Takeuchi, A. and Takeuchi, N. 1965. Localized action of gamma-aminobutyric acid on the crayfish muscle. J. Physiol. (London). 177:225–238.Google Scholar
  50. Tews, J. K., Repa, J. J., and Harper, A. E. 1987. Acceptability by rats of aqueous solutions amino acid analogs. Pharmacol. Biochem. Behav.. 28:525–528.PubMedGoogle Scholar
  51. Tews, J. K., Repa, J. J., Thornquist, M. D., and Harper, A. E. 1986. Choices by rats between water and solutions of GABA or other amino acids. Physiol. Behav.. 37:919–923.PubMedGoogle Scholar
  52. Toyota, B., Kitamura, T., and Takagi, S. F. 1978. Olfactory Disorders, Olfactometry and Therapy. Igaku-Shoin, Tokyo.Google Scholar
  53. Tyler, M. J. 1968. Papuan hylid frogs of the genus Hyla. Zool. Verh. Rijksmus. Nat. Hist. (Leiden) 96:1–203.Google Scholar
  54. Tyler, M. J. 1976. Frogs. Collins, Sydney, NSW, Australia.Google Scholar
  55. Tyler, M. J., Stone, D. J. M., and Bowie, J. H. 1992. A novel method for the release and collection of dermal, glandular secretions from the skin of frogs. J. Pharmacol. Toxicol. Methods. 28:199–200.PubMedGoogle Scholar
  56. Wassersug, R. J. 1971. On the comparative palatability of some dry-season tadpoles from Costa Rica. Am. Midl. Nat. 86: 101–109.Google Scholar
  57. Waugh, R. J., Stone, D. J. M., Bowie, J. H., Wallace, J. C., and Tyler, M. J., 1993. Peptides from Australian frogs: Structure of the caeridins from Litoria caerulea. J. Chem. Soc., Perkin Trans. 1 1993:573–576.Google Scholar
  58. Waye, H. L. and Shewchuk, C. H. 1995. Scaphiopus intermontanus (Great Basin spadefoot). Production of Odor. Herpetol. Rev. 26:98–99.Google Scholar
  59. Weldon, P. J. and Rappole, J. H. 1997. A survey of birds odorous or unpalatable to humans: Possible indications of chemical defense. J. Chem. Ecol. 23:2609–2633.Google Scholar
  60. White, J. 1790. Journal of a Voyage to New South Wales. Bebrett, London.Google Scholar
  61. Williams, C. R., Brodie, E. D., Jr., Tyler, M. J., and Walker, S. J. 2000. Antipredator mechanisms of Australian frogs. J. Herpetol. 34:431–443.Google Scholar
  62. Yasuhara, T., Nakajima, T., Erspamer, G. F., and Erspamer, V. 1981. New tachykinins, glutamic acid-2-proline-5-kassinin (Hylambates maculatus kassinin) and hylambatin, in the skin of the African rhacophorid frog. Biomed. Res. (Tokyo). 2:613–617.Google Scholar
  63. Ziegleder, D. 1991. Composition of flavour extracts of raw and roasted cocoas. Z. Lebensm.-Unters. Forsch.. 192:521–525.Google Scholar

Copyright information

© Plenum Publishing Corporation 2003

Authors and Affiliations

  • Benjamin P. C. Smith
    • 1
    • 2
  • Michael J. Tyler
    • 1
  • Brian D. Williams
    • 2
  • Yoji Hayasaka
    • 3
  1. 1.Department of Environmental BiologyUniversity of AdelaideAdelaideAustralia
  2. 2.Department of Soil & WaterUniversity of Adelaide, PMB1Glen OsmondAustralia
  3. 3.Australian Wine Research InstituteGlen OsmondAustralia

Personalised recommendations