Chemical Characterization of Territorial Marking Fluid of Male Bengal Tiger, Panthera tigris

  • B. V. Burger
  • M. Z. Viviers
  • J. P. I. Bekker
  • M. le Roux
  • N. Fish
  • W. B. Fourie
  • G. Weibchen
Article

Abstract

The territorial marking fluid of the male Bengal tiger, Panthera tigris, consists of a mixture of urine and a small quantity of lipid material that may act as a controlled-release carrier for the volatile constituents of the fluid. Using gas chromatography and gas chromatography–mass spectrometry, 98 volatile compounds and elemental sulfur were identified in the marking fluid. Another 16 volatiles were tentatively identified. The majority of these compounds were alkanols, alkanals, 2-alkanones, branched and unbranched alkanoic acids, dimethyl esters of dicarboxylic acids, γ- and δ-lactones, and compounds containing nitrogen or sulfur. Several samples of the marking fluid contained pure (R)-3-methyl-2-octanone, (R)-3-methyl-2-nonanone, and (R)-3-methyl-2-decanone, but these ketones were partly or completely racemized in other samples. The γ-lactone (S)-(+)-(Z)-6-dodecen-4-olide and the C8 to C16 saturated (R)-γ-lactones and (S)-δ-lactones were present in high enantiomeric purities. The chiral carboxylic acids, 2-methylnonanoic acid, 2-methyldecanoic acid, 2-methylundecanoic acid, and 2-ethylhexanoic acid were racemates. Cadaverine, putrescine, and 2-acetylpyrroline, previously reported as constituents of tiger urine, were not detected. The dominant contribution of some ketones, fatty acids, and lactones to the composition of the headspace of the marking fluid suggests that these compounds may be important constituents of the pheromone. Although it constitutes only a small proportion, the lipid fraction of the fluid contained larger quantities of the volatile organic compounds than the aqueous fraction (urine). The lipid derives its role as controlled-release carrier of the chemical message left by the tiger, from its affinity for the volatiles of the marking fluid. Six proteins with masses ranging from 16 to 69 kDa, inter alia, the carboxylesterase-like urinary protein known as cauxin, previously identified in the urine of the domestic cat and other felid species, were identified in the urine fraction of the marking fluid.

Keywords

Bengal tiger Panthera tigris Semiochemical Pheromone Territorial marking Mass spectrum Chiral gas chromatography Urinary protein Cauxin Lactone 

Supplementary material

10886_2008_9462_MOESM1_ESM.doc (107 kb)
ESM 1The supplementary information includes descriptions of the syntheses of 3-methyl-2-octanone (10), 3-methyl-2-nonanone (14), (R)-2-methylheptanoic acid, 2-methylnonanoic acid (77), 2-methyldecanoic acid (84), 2-methylundecanoic acid (89), dimethyl succinate (33), dimethyl glutarate (43), dimethyl adipate (55), N-benzylidenepentylamine (51), N-pentylurea (104), N-benzylidene-2-phenylethylamine (117), and (S)-(+)-(Z)-6-dodecen-4-olide (86) and the relevant spectral details (DOC 109 KB)

References

  1. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. 1990. Basic local alignment search tool. J. Mol. Biol. 215:403–410.PubMedGoogle Scholar
  2. Bacchini, A., Gaetani, E., and Cavaggioni, A. 1992. Pheromone binding proteins of the mouse, Mus musculus. Experientia 48:419–421.PubMedCrossRefGoogle Scholar
  3. Balme, G. 2005. Counting cats. Afr. Geogr. 13:37–43.Google Scholar
  4. Bernardo, K., Hurwitz, R., Zenk, T., Desnick, R. J., Ferlinz, K., Schuchman, E. H., and Sandhoff, K. 1995. Purification, characterization, and biosynthesis of human acid ceramidase. J. Biol. Chem. 270:11098–11102.PubMedCrossRefGoogle Scholar
  5. Beynon, R. J., and Hurst, J. L. 2003. Multiple roles of major urinary proteins in the house mouse, Mus domesticus. Biochem. Soc. Trans. 31:142–146.PubMedGoogle Scholar
  6. Biedermann, M., Fiselier, K., and Grob, K. 2005. Injector-internal thermal desorption from edible oils. Part 1: visual experiments on sample desorption on the liner wall. J. Sep. Sci. 28:1550–1557.PubMedCrossRefGoogle Scholar
  7. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248–254.PubMedCrossRefGoogle Scholar
  8. Brahmachary, R. L. 1986. Ecology and chemistry of mammalian pheromones. Endeavour, New Ser. 10:65–68.CrossRefGoogle Scholar
  9. Brahmachary, R. L. 1996. The expanding world of 2-acetyl-1-pyrroline. Curr. Sci. 71:257–258.Google Scholar
  10. Brahmachary, R. L., and Dutta, J. 1979. Phenylethylamine as a biochemical marker of tiger. Z. Naturforsch. 34c:632–633.Google Scholar
  11. Brahmachary, R. L., and Dutta, J. 1987. Chemical communication in the tiger and leopard, pp. 296–302, in R. L. Tilson and U.S. Seal (eds.). Tigers of the World: The Biology, Biopolitics, Management and Conservation. Noyes, Park Ridge, NJ.Google Scholar
  12. Brahmachary, R. L., Poddar-Sarkar, M., and Dutta, J. 1990. The aroma of rice... and tiger. Nature 344:26.PubMedCrossRefGoogle Scholar
  13. Burger, B. V., Marx, B., le Roux, M., and Burger, W. J. G. 2006a. Simplified analysis of organic compounds in headspace and aqueous samples by high-capacity sample enrichment probe. J. Chromatogr. A 1121:259–267.PubMedCrossRefGoogle Scholar
  14. Burger, B. V., Petersen, W. G. B., Ewig, B. T., Neuhaus, J., Tribe, G. D., Spies, H. S. C., and Burger, W. J. G. 2008. Semiochemicals of the Scarabaeinae. VIII. Identification of the active constituents of the abdominal sex-attracting secretion of the male dung beetle, Kheper bonellii, using gas chromatography with flame ionization and electroantennographic detection in parallel. J. Chromatogr. A. 1186:254–253.CrossRefGoogle Scholar
  15. Burger, B. V., Visser, R., Moses, A., and le Roux, M. 2006b. Elemental sulfur identified in urine of cheetah, Acinonyx jubatus. J. Chem. Ecol. 32:1347–1352.PubMedCrossRefGoogle Scholar
  16. Ellerman, J., and Morrison-Scott, T. 1951. Checklist of Palaearctic and Indian Mammals, 1758 to 1946. British Museum, London (reference in G.B. Schaller. 1967. The Deer and the Tiger. University of Chicago Press, Chicago).Google Scholar
  17. Hashimoto, Y., Eguchi, Y., and Arakawa, A. 1963. Historical observation of the anal sac and its glands of a tiger. Jpn. J. Vet. Sci. 25:29–32 (reference in K. F. Andersen and T. Vulpius. 1999. Urinary volatile constituents of the lion, Panthera leo. Chem. Senses 24:179–189).Google Scholar
  18. Hurst, J. L., Thom, M. D., Nevison, C. M., Humphries, R. E., and Beynon, R. J. 2005. MHC odours are not required or sufficient for recognition of individual scent owners. Proc. R. Lond. Soc. B 272:715–724.CrossRefGoogle Scholar
  19. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685.PubMedCrossRefGoogle Scholar
  20. Lazar, J., Rasmussen, L. E., Greenwood, D. R., Bang, I. S., and Prestwich, G. D. 2004. Elephant albumin: a multipurpose pheromone shuttle. Chem. Biol. 11:1093–1100.PubMedCrossRefGoogle Scholar
  21. Leyhausen, P. 1960. Verhaltenstudien an Katzen, in G. B. Schaller. The Deer and the Tiger. University of Chicago Press, Chicago.Google Scholar
  22. Leyhausen, P., and Wolff, R. 1959. Das Revier einer Hauskatze. Z. Tierpsych. 16:666–670.Google Scholar
  23. Maas, B., Dietrich, A., and Mosandl, A. 1994a. Collection of enantiomer separation factors obtained by capillary gas chromatography on chiral stationary phases. J. High Resolut. Chrom. 17:109–115.CrossRefGoogle Scholar
  24. Maas, B., Dietrich, A., and Mosandl, A. 1994b. Collection of enantiomer separation factors obtained by capillary gas chromatography on chiral stationary phases. J. High Resolut. Chrom. 17:169–173.CrossRefGoogle Scholar
  25. Miyazaki, M., Kamiie, K., Soeta, S., Tiara, H., and Yamashita, T. 2003. Molecular cloning and characterization of a novel carboxylesterase-like protein that is physiologically present at high concentrations in the urine of domestic cats (Felis catus). Biochem. J. 370:101–110.PubMedCrossRefGoogle Scholar
  26. Miyazaki, M., Yamashita, T., Hosokawa, M., Tiara, H., and Suzuki, A. 2006a. Species-, sex-, and age-dependent urinary excretion of cauxin, a mammalian carboxylesterase. Comp. Biochem. Physiol. Part B 145:270–277.CrossRefGoogle Scholar
  27. Miyazaki, M., Yamashita, T., Suzuki, Y., Saito, Y., Soeta, S., Tiara, H., and Suzuki, A. 2006b. Major urinary protein of the domestic cat regulates the production of felinine, a putative pheromone precursor. Chem. Biol. 13:1071–1079.PubMedCrossRefGoogle Scholar
  28. Monaco, H. L. 2000. The transthyretin-retinol-binding protein complex. Biochim. Biophys. Acta 1482:65–72.PubMedGoogle Scholar
  29. Mosandl, A., Hener, U., Hagenauer-Hener, U., and Kustermann, A. 1989. Direct enantiomer separation of chiral γ-lactones from food and beverages by multidimensional gas chromatography. J. High Resolut Chromatogr. 12:532–536.CrossRefGoogle Scholar
  30. Müller-Schwarze, D., Ravid, U., Claesson, A., Singer, A. G., Silverstein, R. M., Müller-Schwarze, C., Volkman, N. J., Zemanek, K. F., and Butler, R. G. 1978. The deer lactone: Source, chiral properties, and responses by black-tailed deer. J. Chem. Ecol. 4:247–256.CrossRefGoogle Scholar
  31. Müller-Schwarze, D., Volkman, N. J., and Zemanek, K. F. 1977. Osmetrichia: specialized scent hair in black-tailed deer. J. Ultrastruct. Res. 59:223–230.PubMedCrossRefGoogle Scholar
  32. Novotny, M., Harvey, S., Jemiolo, B., and Alberts, J. 1985. Synthetic pheromones that promote inter-male aggression in mice. Proc. Natl. Acad. Sci. USA 82:2059–2061.PubMedCrossRefGoogle Scholar
  33. Novotny, M. V. 2003. Pheromones, binding proteins and receptor responses in rodents. Biochem. Soc. Trans. 31:117–122.PubMedCrossRefGoogle Scholar
  34. Perkins, D. N., Pappin, D. J., Creasy, D. M., and Cottrell, J. S. 1999. Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20:3551–3567.PubMedCrossRefGoogle Scholar
  35. Poddar-Sarkar, M. 1996. The fixative lipid of tiger pheromone. J. Lipid Mediat. Cell Signal. 15:89–101.PubMedCrossRefGoogle Scholar
  36. Raila, J., Forterre, S., and Schweigert, F. J. 2005. Physiologic and pathophysiologic fundamentals of proteinuria—a review. Berl. Münch. Tierärztl. Wochenschr. 118:229–239.PubMedGoogle Scholar
  37. Reiter, B., Burger, B. V., and Dry, J. 2003. Mammalian exocrine secretions. XVIII: Chemical characterization of interdigital secretion of red hartebeest, Alcelaphus buselaphus caama. J. Chem. Ecol. 29:2235–2252.PubMedCrossRefGoogle Scholar
  38. Sambrook, J., Fritsch, E. F., and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbour Laboratory Press, Cold Spring Harbor, NY.Google Scholar
  39. Schaller, G. B. 1967. The Deer and the Tiger. University of Chicago Press, Chicago.Google Scholar
  40. Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. 1996. Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Anal. Chem. 68:850–858.PubMedCrossRefGoogle Scholar
  41. Smith, J. L. D., McDougal, C., and Miquelle, D. 1989. Scent marking in free-ranging tigers, Panthera tigris. Anim. Behav. 37:1–10.CrossRefGoogle Scholar
  42. Vincenti, M., Guglielmetti, G., Cassani, G., and Tonini, C. 1987. Determination of double-bond position in diunsaturated compounds by mass spectrometry of dimethyl disulfide derivatives. Anal. Chem. 59:694–699.CrossRefGoogle Scholar
  43. Wait, R., Gianazza, E., Eberini, I., Sironi, L., Dunn, M. J., Germeiner, M., and Miller, I. 2001. Proteins of rat serum, urine, and cerebrospinal fluid: VI. Further protein identifications and interstrain comparison. Electrophoresis 22:3043–3052.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • B. V. Burger
    • 1
  • M. Z. Viviers
    • 1
  • J. P. I. Bekker
    • 2
  • M. le Roux
    • 1
  • N. Fish
    • 1
  • W. B. Fourie
    • 3
  • G. Weibchen
    • 4
  1. 1.Laboratory for Ecological ChemistryUniversity of StellenboschMatielandSouth Africa
  2. 2.Institute for PlantbiotechnologyMatielandSouth Africa
  3. 3.Tygerberg ZooKraaifonteinSouth Africa
  4. 4.Institut für Organische ChemieUniversität HamburgHamburgGermany

Personalised recommendations