Use of Microderivatization Techniques in Combination with Thin-Layer Chromatography, Liquid Chromatography and Gas Chromatography-Mass Spectrometry for Investigation of Juvenile Hormones and Related Compounds

  • F. C. Baker
  • C. C. Reuter
  • L. W. Tsai
  • P. A. Brindle
  • D. S. Richard
  • S. S. Tobe
  • D. A. Schooley
Part of the Chromatographic Society Symposium Series book series (CSSS)


The qualitative juvenile hormone (JH) profile in lepidopteran species, as determined by both in vivo and in vitro techniques, is markedly different from that in insects from other orders, the majority of which contain only JH III. A gas chromatographic-mass spectrometric (GC-MS) method for quantification of JHs in vivo was adapted to study the mode of JH biosynthesis by corpora allata incubated in vitro with selected 14C-radiolabeled substrates of high specific activity. We analyzed derivatives of JH using selected ion monitoring to measure ratios of the fragment ion and corresponding ions 2,4, and 6 mass units higher, allowing determination of dilution of the propionate unit which is incorporated as an intermediate. The results showed conclusively that the ethyl branch of JH II from Manduca sexta derives ultimately from metabolism of the branched-chain amino acids isoleucine and valine. A purification procedure for isolation of JHs from biological tissue was modified to allow isolation of JH acids and compounds structurally related to JH, including JH diols, methyl farnesoate (MF), farnesoic acid (FA), and farnesol. To allow analysis, JH acids were methylated and the resulting JHs were detected and quantified, as their d3-methoxyhydrin derivatives, by GC-MS with selected ion monitoring (SIM). A detailed study of fifth stadium Manduca sexta showed that JH I and II acids predominate over the corresponding esters at the prepupal stage. GC-MS methods were devised for quantification of JH diols, MF, FA, and farnesol from arthropods. None of the latter compounds, nor any of the known JHs, could be detected in the hemipteran Oncopeltus fasciatus. Ring glands from a few dipteran species appear to secrete a new JH in vitro, and details are given for the isolation and analysis of this compound. Haemolymph from several crustaceans contain detectable levels of MF, and mandibular organs from crustaceans secrete MF and/or FA in vitro.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    B. J. Bergot, M. Ratcliff and D. A. Schooley, Method for quantitative determination of the four known juvenile hormones in insect tissue using gas chromatography-mass spectroscopy, J. Chromatog., 204:231–244 (1981).CrossRefGoogle Scholar
  2. 2.
    F. C. Baker, Techniques for identification and quantification of juvenile hormones and related compounds in arthropods, in: “Morphogenese Hormones of Arthropods/Recent Advances in Comparative Morphology, Physiology and Development of Arthropods”, A. P. Gupta, ed., Rutgers University Press, New Brunswick, pp. 389–453, in press (1990).Google Scholar
  3. 3.
    D. A. Schooley, F. C. Baker, L. W. Tsai, C. A. Miller and G. C. Jamieson, Juvenile hormones, 0, I and II exist only in Lepidoptera, in: “Biosynthesis, Metabolism and Mode of Action of Invertebrate Hormones”, J. Hoffmann and M. Porchet, eds., Springer-Verlag, Berlin, pp. 373–383 (1984).CrossRefGoogle Scholar
  4. 4.
    G. Bhaskaran, S. P. Sparagana, P. Barrera and K. H. Dahm, Change in corpus allatum function during metamorphosis of the tobacco hornworm Manduca sexta: regulation at the terminal step in juvenile hormone biosynthesis, Arch. Insect Biochem Physiol., 3:321–338 (1986).CrossRefGoogle Scholar
  5. 5.
    F. C. Baker, L. W. Tsai, C. C. Reuter and D. A. Schooley, In vivo fluctuation of JH, JH acid, and ecdysteroid titer, and JH esterase activity, during development of fifth stadium Manduca sexta, Insect Biochem., 17:989–996 (1987).CrossRefGoogle Scholar
  6. 6.
    F. C. Baker, L. W. Tsai, C. C. Reuter and D. A. Schooley, The absence of significant levels of the known juvenile hormones and related compounds in the milkweed bug, Oncopeltusfasciatus, Insect Biochem., 18:453–462 (1988).CrossRefGoogle Scholar
  7. 7.
    A. Dorn, Structure and function of the embryonic corpus allatum of Oncopeltus fasciatus Dallas (Insecta, Heteroptera), Verhandlungen der Deutschen Zoologischen Gesellschaft, pp. 85–89 (1975).Google Scholar
  8. 8.
    M. A. Rankin and L. M. Riddiford, Significance of hemolymph juvenile hormone titer changes in timing of migration and reproduction in adult Oncopeltus fasciatus, J. Insect Physiol., 24:31–38 (1978).CrossRefGoogle Scholar
  9. 9.
    D. S. Richard, S. W. Applebaum, T. J. Sliter, F. C. Baker, D. A. Schooley, C. C. Reuter, V. C. Henrich and L. I. Gilbert, Juvenile hormone bisepoxide biosynthesis in vitro by the ring of Drosophila melanogaster. a putative juvenile hormone in the higher Diptera, Proc. Nat. Acad. Sci. USA, 86:1421–1425 (1989).CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    M. Gadot, A. Goldman, M. Cojocaru and S. W. Applebaum, The intrinsic synthesis of juvenile hormone-III diol by locust corpora allata in vitro, Mol Cell. Endocrinol., 49:99–107 (1987).CrossRefPubMedGoogle Scholar
  11. 11.
    H. Laufer, D. Borst, F. C. Baker, C. Carrasco, M. Sinkus, C. C. Reuter, L. W. Tsai and D. A. Schooley, Identification of a juvenile hormone-like compound in a crustacean, Science, 235:202–205 (1987).CrossRefPubMedGoogle Scholar
  12. 12.
    H. Laufer, M. Landau, E. Homola and D. W. Borst, Methyl farnesoate: its site of synthesis and regulation of secretion in a juvenile crustacean, Insect Biochem., 17:1129–1131(1987).Google Scholar
  13. 13.
    D. W. Borst, H. Laufer, M. Landau, E. S. Chang, W. A. Hertz, F. C. Baker and D. A. Schooley Methyl farnesoate and its role in crustacean reproduction and development, Insect Biochem., 17:1123–1127 (1987).CrossRefGoogle Scholar
  14. 14.
    S. S. Tobe, D. A. Young, H. W. Khoo and F. C. Baker, Farnesoic acid as a major product of release from crustacean mandibular organs in vitro, J. Exp. Zool., 249:165–171 (1989).CrossRefGoogle Scholar
  15. 15.
    P. A. Brindle, F. C. Baker, L. W. Tsai, C. C. Reuter and D. A. Schooley, Sources of propionate for the biogenesis of ethyl-branched insect juvenile hormones: role of isoleucine and valine, Proc. Nat. Acad. Sci. USA, 84:7906–7910 (1987).CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    F. C. Baker and D. A. Schooley, Preparation of (10R.S)-[10–3HJjuvenile hormone III and (10R.S, 11S.R)-[10–3HUuvenile hormone O: conversion of [10–3H]juvenile hormone III to methyl (2E, 6E)-[10–3H]farnesoate and (2E,6E)-[10–3H]farnesol, J. Labeled Comp. Radiopharmaceut, 5:533–543 (1986).Google Scholar
  17. 17.
    W. G. Goodman and B. Adams, Semipreparative synthesis and purification of juvenile hormone acids by high-performance liquid chromatography, J. Chromatog., 294:447–451 (1984).CrossRefGoogle Scholar
  18. 18.
    T. J. Sliter, B. J. Sedlak, F. C. Baker and D. A. Schooley, Juvenile hormone in Drosophila meianogfaster.Identification and titer determination during development, Insect Biochem., 17:161–165 (1987).CrossRefGoogle Scholar
  19. 19.
    P. A. Brindle, D. A. Schooley, L. W. Tsai and F. C. Baker, Comparative metabolism of branched-chain amino acids to precursors of juvenile hormone biogenesis in corpora allata of lepidopterous versus non-lepidopterous insects, J. Biol Chem., 263:10653–10657(1988).Google Scholar
  20. 20.
    D. A. Schooley, K. J. Judy, B. J. Bergot, M. S. Hall and J. Siddall, Biosythesis of the juvenile hormones of Manduca sexta: labeling pattern from mevalonate, propionate and acetate, Proc. Nat. Acad. Sci. USA, 70:2921–2925 (1973).CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    S. S. Tobe, D. A. Young and H. W. Khoo, Production of methyl farnesoate by the mandibular organs of the mud crab, Scylla serrata: validation of a radiochemical assay, Gen Comp. Endocrinol., 73:342–353 (1989).CrossRefPubMedGoogle Scholar
  22. 22.
    H. Rembold and B. Lackner, Convenient method for the determination of picomole amounts of juvenile hormone, J. Chromatog., 323:355–361 (1985).CrossRefGoogle Scholar
  23. 23.
    B. Mauchamp, R. Lafont, M. Hardy and D. Jourdain, Analysis of insect juvenile hormones by gas chromatography mass spectrometry: problems of sample preparation and choice of detection procedure, Biomed. Mass Spec, 6:276–281 (1979).CrossRefGoogle Scholar
  24. 24.
    K. H. Trautmann, P. Masner, A. Schüler, M. Suchy and H.-K. Wipf, Evidence of the juvenile hormone methyl(2E,6E)-10,1 l-epoxy-3,7,1 l-trimethyl-2,6-dodecadieno-ate JH-3) in insects of four orders, Zeitschrift fur Naturforschung., 29c:757–759 (1974).Google Scholar
  25. 25.
    J. P. Edward, B. J. Bergot and G. B. Staal, Effects of three compounds with antijuvenile hormone levels in the tobacco hornworm, Manduca sexta, Insect Physiol., 29:83–89 (1983).CrossRefGoogle Scholar
  26. 26.
    F. C. Baker, C. A. Miller, L. W. Tsai, G. C. Jamieson, D. C. Cerf and D. A. Schooley, The effect of juvenoids, anti juvenile hormone agents, and several intermediates of juvenile hormone biosynthesis on the in vivo juvenile hormone levels in Manduca sexta larvae, Insect Biochem., 16:741–747 (1986).CrossRefGoogle Scholar
  27. 27.
    P. O. Lawrence, F. C. Baker, L. W. Tsai, C. A. Miller, D. A. Schooley and L. Geddes, JH III levels in superparasitized pharate pupae of Anastrepha suspensa (Diptera: tephritidae) and in larvae of the parasitic wasp Biosteres longicaudatus (Hymenoptera: braconidae), Arch, Insect Biochem. Physiol., in press (1990).Google Scholar
  28. 28.
    G. Jones, T. Hanzlik, B. D. Hammock, D. A. Schooley, C. A. Miller, L. W. Tsai and F. C. Baker, The juvenile hormone titer during the penultimate and ultimate larval stadia of Trichoplusia ni, J. Insect Physiol., in press (1990).Google Scholar
  29. 29.
    W. Loher, D. A. Schooley and F. C. Baker, Influence of the ovaries on JH titer in Teleogryllus commodus, Insect Biochem., 17:1099–1102 (1987).CrossRefGoogle Scholar
  30. 30.
    N. A. Granger, S. M. Niemiec, L. I. Gilbert and W. E. Bollenbacher, Juvenile hormone synthesis in vitro by larval and pupal corpora allata of Manduca sexta, Mol. Cell Endocrinol., 28:587–604 (1982).CrossRefPubMedGoogle Scholar
  31. 31.
    M. F. Feldlaufer, W. S. Bowers, D. M. Soderlund and P. H. Evans, Biosynthesis of the sesquiterpenoid skeleton of juvenile hormone 3 by Dysdercus fasciatus corpora allata in vitro, J. Exp. Zool., 223:295–298 (1982).CrossRefGoogle Scholar
  32. 32.
    W. S. Bowers, P. A. Marsella and P. H. Evans, Identification of an hemipteran juvenile hormone: in vitro biosynthesis of JH III by Dysdercus fasciatus, J. Exp. Zool., 228:555–559(1983).Google Scholar
  33. 33.
    K. J. Judy D. A. Schooley, L. L. Dunham, M. S. Hall, B. J. Bergot and J. Siddall, Isolation, structure and absolute configuration of a new natural insect juvenile hormone from Manduca sexta, Proc. Nat. Acad. Sci. USA, 70:1509–1513 (1973).CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    M.-A. Liu, G. L. Jones, J. G. Stoffolano, Jr. and C.-M. Yin, Conditions for estimation of corpus allatum activity in the blowfly, Phormia regina, in vitro, Physiol Entomol, 13:69–79 (1988).CrossRefGoogle Scholar
  35. 35.
    B. Lanzrein, H. Imboden, C. Bürgin, E. Brüning and H. Gfeller, On titers, origin and functions of juvenile hormone III, methyl farnesoate, and ecdysteroids in embryonic development of the ovoviviparous cockroach Nauphoeta cinerea, in: “Biosynthesis, Metaboism and Mode of Action of Invertebrate Hormones”, J. Hoffmann and M. Porchet, eds., Springer-Verlag, Berlin, pp. 454–465 (1984).CrossRefGoogle Scholar

Copyright information

© Plenum Press, New York 1990

Authors and Affiliations

  • F. C. Baker
    • 1
  • C. C. Reuter
    • 1
  • L. W. Tsai
    • 1
  • P. A. Brindle
    • 2
  • D. S. Richard
    • 3
  • S. S. Tobe
    • 4
  • D. A. Schooley
    • 5
  1. 1.Research DivisionSandoz Crop ProtectionPalo AltoUSA
  2. 2.American CyanamidPrincetonUSA
  3. 3.Department of BiologyUniversity of North CarolinaChapel HillUSA
  4. 4.Department of ZoologyUniversity of TorontoTorontoCanada
  5. 5.Department of BiochemistryUniversity of NevadaRenoUSA

Personalised recommendations