Cell and Tissue Research

, Volume 327, Issue 2, pp 385–398 | Cite as

Distribution of neuropeptides in the primary olfactory center of the heliothine moth Heliothis virescens

  • Bente G. Berg
  • Joachim Schachtner
  • Sandra Utz
  • Uwe Homberg
Regular Article


Neuropeptides are a diverse widespread class of signaling substances in the nervous system. As a basis for the analysis of peptidergic neurotransmission in the insect olfactory system, we have studied the distribution of neuropeptides in the antennal lobe of the moth Heliothis virescens. Immunocytochemical experiments with antisera recognizing A-type allatostatins (AST-As), Manduca sexta allatotropin (Mas-AT), FMRFamide-related peptides (FaRPs), and tachykinin-related peptides (TKRPs) have shown that members of all four peptide families are present in local interneurons of the antennal lobe. Whereas antisera against AST-As, Mas-AT, and FaRPs give similar staining patterns characterized by dense meshworks of processes confined to the core of all antennal-lobe glomeruli, TKRPs are present only in neurons with blebby processes distributed throughout each glomerulus. In addition to local neurons, a pair of centrifugal neurons with cell bodies in the lateral subesophageal ganglion, arborizations in the antennal lobe, and projections in the inner antenno-cerebral tracts exhibits tachykinin immunostaining. Double-label immunofluorescence has detected the co-localization of AST-As, Mas-AT, and FaRPs in certain local interneurons, whereas TKRPs occurs in a distinct population. MALDI-TOF mass spectrometry has revealed nearly 50 mass peaks in the antennal lobe. Seven of these masses (four AST-As, two N-terminally extended FLRFamides, and Mas-AT) match known moth neuropeptides. The data thus show that local interneurons of the moth antennal lobe are highly differentiated with respect to their neuropeptide content. The antennal lobe therefore represents an ideal preparation for the future analysis of peptide signaling in insect brain.


Antennal lobe Immunocytochemistry Peptides MALDI-TOF mass spectrometry Heliothine moth Heliothis virescens (Insecta) 



We thank Drs. H. Agricola, E. Marder, and J. Veenstra for providing antisera against neuropeptides, Dr. E. Buchner for the gift of the anti-synapsin antiserum, and Drs. R. Predel, C. Wegener, and J. Kahnt for their help with MALDI-TOF mass spectrometry. We are also grateful to Syngenta, Basel, Switzerland for sending insect pupae and to T. Vuttudal for assistance with the figures.


  1. Abercrombie M (1946) Estimation of nuclear population from microtome sections. Anat Rec 94:239–247CrossRefGoogle Scholar
  2. Almaas TJ, Mustaparta H (1991) Heliothis virescens: response characteristics of receptor neurons in sensilla trichodea type 1 and type 2. J Chem Ecol 5:953–972CrossRefGoogle Scholar
  3. Anton S, Homberg U (1999) Antennal lobe structure. In: Hansson BS (ed) Insect olfaction. Springer, Berlin Heidelberg New York, pp 97–124Google Scholar
  4. Audsley N, Weaver RJ (2003a) Identification of neuropeptides from brains of larval Manduca sexta and Lacanobia oleracea using MALDI-TOF massspectrometry and post-source decay. Peptides 24:1465–1474PubMedCrossRefGoogle Scholar
  5. Audsley N, Weaver RJ (2003b) A comparison of the neuropeptides from the retrocerebral complex of adult male and female Manduca sexta using MALDI-TOF mass spectrometry. Regul Pept 116:127–137PubMedCrossRefGoogle Scholar
  6. Audsley N, Matthews J, Weaver RJ (2005) Neuropeptides associated with the frontal ganglion of larval Lepidoptera. Peptides 26:11–21PubMedCrossRefGoogle Scholar
  7. Baker TC, Ochieng SA, Cossé AA, Lee SG, Todd JL, Quero C, Vickers NJ (2004) A comparison of responses from olfactory receptor neurons of Heliothis subflexa and Heliothis virescens to components of their sex pheromone. J Comp Physiol [A] 190:155–165CrossRefGoogle Scholar
  8. Berg BG, Almaas TJ, Bjaalie JG, Mustaparta H (1998) The macroglomerular complex of the antennal lobe in the tobacco budworm moth Heliothis virescens: specified subdivision in four compartments according to information about biologically significant compounds. J Comp Physiol [A] 183:669–682CrossRefGoogle Scholar
  9. Berg BG, Galizia CG, Brandt R, Mustaparta H (2002) Digital atlases of the antennal lobe in two species of tobacco budworm moths, the oriental Helicoverpa assulta (male) and the American Heliothis virescens (male and female). J Comp Neurol 446:123–134PubMedCrossRefGoogle Scholar
  10. Berg BG, Almaas TJ, Bjaalie JG, Mustaparta H (2005) Projections of male-specific receptor neurons in the antennal lobe of the oriental tobacco budworm moth, Helicoverpa assulta: a unique glomerular organization among related species. J Comp Neurol 486:209–220PubMedCrossRefGoogle Scholar
  11. Billimoria CP, Li L, Marder E (2005) Profiling of neuropeptides released at the stomatogastric ganglion of the crab, Cancer borealis, with mass spectrometry. J Neurochem 95:191–199PubMedCrossRefGoogle Scholar
  12. Birse RT, Johnson EC, Taghert PH, Nässel DR (2005) Widely distributed G-protein-coupled receptor (CG7887) is activated by endogeneous tachykinin-related peptides. J Neurobiol 66:33–46CrossRefGoogle Scholar
  13. Blackburn MB, Kingan TG, Raina AK, Ma MC (1992) Colocalization and differential expression of PBAN- and FMRFamide-like immunoreactivity in the subesophageal ganglion of Helicoverpa zea (Lepidoptera: Noctuidae) during development. Arch Insect Biochem Physiol 21:225–238CrossRefGoogle Scholar
  14. Boeckh J, Tolbert LJ (1993) Synaptic organization and development of the antennal lobe in insects. Microsc Res Tech 24:260–280PubMedCrossRefGoogle Scholar
  15. Boer HH, Schot LPC, Roubos EW, Maat A, Lodder JC, Reichelt D (1979) ACTH-like immunoreactivity in two electronically coupled giant neurons in the pond snail Lymnaea stagnalis. Cell Tissue Res 202:231–240PubMedCrossRefGoogle Scholar
  16. Brezina V, Weiss KR (1997) Analyzing the functional consequences of transmitter complexity. Trends Neurosci 20:538–543PubMedCrossRefGoogle Scholar
  17. Christensen TA, Mustaparta H, Hildebrand JG (1991) Chemical communication in heliothine moths. II. Central processing of intra- and interspecific olfactory messages in the male corn earworm moth Helicoverpa zea. J Comp Physiol [A] 180:523–536Google Scholar
  18. Christensen TA, Mustaparta H, Hildebrand JG (1995) Chemical communication in heliothine moths. VI. Parallel pathways for information processing in the macroglomerular complex of the male tobacco budworm moth Heliothis virescens. J Comp Physiol [A] 177:545–557Google Scholar
  19. Davey M, Duve H, Thorpe A, East PD (1999) Characterisation of the helicostatin peptide precursor gene from Helicoverpa armigera (Lepidoptera: Noctuidae). Insect Biochem Mol Biol 29:1119–1127PubMedCrossRefGoogle Scholar
  20. Duve H, Johnsen AH, Maestro J-L, Scott AG, Winstanley D, Davey M, East PD, Thorpe A (1997) Lepidopteran peptides of the allatostatin superfamily. Peptides 18:1301–1309PubMedCrossRefGoogle Scholar
  21. Glasscock JM, Mizoguchi A, Rachinsky A (2005) Immunocytochemical localization of an allatotropin in developmental stages of Heliothis virescens and Apis mellifera. J Insect Physiol 51:345–355PubMedCrossRefGoogle Scholar
  22. Hansson BS, Almaas TJ, Anton S (1995) Chemical communication in heliothine moths. V. Antennal lobe projection patterns of pheromone-detecting olfactory receptor neurons in the male Heliothis virescens (Lepidoptera: Noctuidae). J Comp Physiol [A] 177:535–543Google Scholar
  23. Homberg U, Müller U (1999) Neuroactive substances in the antennal lobe. In: Hansson BS (ed) Insect olfaction. Springer, Berlin Heidelberg New York, pp 181–206Google Scholar
  24. Homberg U, Christensen TA, Hildebrand JG (1989) Structure and function of the deutocerebrum in insects. Annu Rev Entomol 34:477–501PubMedCrossRefGoogle Scholar
  25. Homberg U, Kingan TG, Hildebrand JG (1990) Distribution of FMRFamide-like immunoreactivity in the brain and suboesophageal ganglion of the sphinx moth Manduca sexta and colocalization with SCPB-, BPP-, and GABA-like immunoreactivity. Cell Tissue Res 259:401–419PubMedCrossRefGoogle Scholar
  26. Homberg U, Hoskins SG, Hildebrand JG (1995) Distribution of acetylcholinesterase activity in the deutocerebrum of the sphinx moth Manduca sexta. Cell Tissue Res 279:249–259PubMedGoogle Scholar
  27. Homberg U, Brandl C, Clynen E, Schoofs L, Veenstra JA (2004) Mas-allatotropin/Lom-AG-myotropin I immunostaining in the brain of the locust, Schistocerca gregaria. Cell Tissue Res 318:439–457PubMedCrossRefGoogle Scholar
  28. Hoskins SG, Homberg U, Kingan TG, Christensen TA, Hildebrand JG (1986) Immunocytochemistry of GABA in the antennal lobes of the sphinx moth Manduca sexta. Cell Tissue Res 244:243–252PubMedCrossRefGoogle Scholar
  29. Iwano M, Kanzaki R (2005) Immunocytochemical identification of neuroactive substances in the antennal lobe of the male silkworm moth Bombyx mori. Zool Sci 22:199–211PubMedCrossRefGoogle Scholar
  30. Jenkins AC, Brown MR, Crim JW (1989) FMRF-amide immunoreactivity in a moth larva (Heliothis zea): the cerebral nervous system. Tissue Cell 21:569–579CrossRefGoogle Scholar
  31. Kim M-Y, Lee BH, Kwon D, Kang H, Nässel DR (1998) Distribution of tachykinin-related neuropeptide in the developing central nervous system of the moth Spodoptera litura. Cell Tissue Res 294:351–365PubMedCrossRefGoogle Scholar
  32. Krieger J, Grosse-Wilde E, Gohl T, Dewer YME, Raming K, Breer H (2004) Genes encoding candidate pheromone receptors in a moth (Heliothis virescens). Proc Natl Acad Sci USA 101:11845–11850PubMedCrossRefGoogle Scholar
  33. Lei H, Christensen TA, Hildebrand JG (2002) Spatial and temporal organization of ensemble representations for different odor classes in the moth antennal lobe. J Neurosci 24:11108–11119CrossRefGoogle Scholar
  34. MacLeod K, Laurent G (1996) Distinct mechanisms for synchronization and temporal patterning of odor-encoding neural assemblies. Science 274:976–979PubMedCrossRefGoogle Scholar
  35. Marder E, Calabrese RL, Nusbaum MP, Trimmer B (1987) Distribution and partial characterization of FMRFamide-like peptides in the stomatogastric nervous system of the rock crab, Cancer borealis, and the spiny lobster Panulirus interruptus. J Comp Neurol 259:150–163PubMedCrossRefGoogle Scholar
  36. Nässel DR (2002) Neuropeptides in the nervous system of Drosophila and other insects: multiple roles as neuromodulators and neurohormones. Prog Neurobiol 68:1–84PubMedCrossRefGoogle Scholar
  37. Nässel DR, Homberg U (2006) Neuropeptides in interneurons of the insect brain. Cell Tissue Res (in press)Google Scholar
  38. Nässel DR, Passier PCCM, Elekes K, Dircsen H, Vullings HGB, Cantera R (1995) Evidence that locustatachykinin I is involved in release of adipokinetic hormone from locust corpora cardiaca. Regul Pept 57:297–310PubMedCrossRefGoogle Scholar
  39. Negoescu A, Labat-Moleur F, Lorimier P, Lamarcq L, Guillermet C, Chambaz E, Brambilla E (1994) F(ab) secondary antibodies: a general method for double immunolabeling with primary antisera from the same species. Efficiency control by chemoluminescence. J Histochem Cytochem 42:433–437PubMedGoogle Scholar
  40. Nusbaum MP, Blitz DM, Swensen AM, Wood D, Marder E (2001) The roles of co-transmission in neural network modulation. Trends Neurosci 24:146–154PubMedCrossRefGoogle Scholar
  41. Oeh U, Antonicek H, Nauen R (2003) Myotropic effect of helicokinins, tachykinin-related peptides and Manduca sexta allatotropin on the gut of Heliothis virescens (Lepidoptera: Noctuidae). J Insect Physiol 49:323–337PubMedCrossRefGoogle Scholar
  42. Predel R (2001) Peptidergic neurohemal system of an insect: mass spectrometric morphology. J Comp Neurol 436:363–375PubMedCrossRefGoogle Scholar
  43. Predel R, Herbert Z, Eckert M (2003) Neuropeptides in perisympathetic organs of Manduca sexta: specific composition and changes during development. Peptides 24:1457–1464PubMedCrossRefGoogle Scholar
  44. Sachse S, Galizia CG (2002) Role of inhibition for temporal and spatial odor representation in olfactory output neurons: a calcium imaging study. J Neurophysiol 87:1106–1117PubMedGoogle Scholar
  45. Schachtner J, Trosowski B, D’Hanis W, Stuber S, Homberg U (2004a) Development and steroid regulation of RFamide immunoreactivity in antennal-lobe neurons of the sphinx moth Manduca sexta. J Exp Biol 207:2389–2400PubMedCrossRefGoogle Scholar
  46. Schachtner J, Utz S, Wegener C, Homberg U, Predel R (2004b) Neuropeptides in developing antennal lobe of the sphinx moth Manduca sexta. Program No 41.18. Abstract Viewer/Itinery planer. Society of Neuroscience, Washington DCGoogle Scholar
  47. Schachtner J, Schmidt M, Homberg U (2005) Organization and evolutionary trends of primary olfactory brain centers in Tetraconata (Crustacea+Hexapoda). Arthropod Struct Dev 34:257–299CrossRefGoogle Scholar
  48. Skiri HT, Rø H, Berg BG, Mustaparta H (2005) Consistent organization of glomeruli in the antennal lobes of related species of heliothine moths. J Comp Neurol 491:367–380PubMedCrossRefGoogle Scholar
  49. Sternberger LA (1979) Immunocytochemistry. Wiley, New YorkGoogle Scholar
  50. Stranden M, Røstelien T, Liblikas I, Almaas TJ, Borg-Karlson A-K, Mustaparta H (2003a) Receptor neurones in three heliothine moths responding to floral and inducible plant volatiles. Chemoecology 13:143–154CrossRefGoogle Scholar
  51. Stranden M, Liblikas I, König WA, Almaas TJ, Borg-Karlson A-K, Mustaparta H (2003b) (-)-Germacrene D receptor neurones in three species of heliothine moths: structure-activity relationships. J Comp Physiol [A] 189:563–577CrossRefGoogle Scholar
  52. Teal PEA (2002) Effects of allatotropin and allatostatin on in vitro production of juvenile hormones by the corpora allata of virgin females of the moths of Heliothis virescens and Manduca sexta. Peptides 23:663–669PubMedCrossRefGoogle Scholar
  53. Utz S, Schachtner J (2005) Development of A type allatostatin immunoreactivity in antennal lobe neurons of the sphinx moth Manduca sexta. Cell Tissue Res 320:149–162PubMedCrossRefGoogle Scholar
  54. Veenstra JA, Hagedorn HH (1993) Sensitive enzyme immunoassay for Manduca allatotropin and the existence of an allatotropin-immunoreactive peptide in Periplaneta americana. Arch Insect Biochem Physiol 23:99–109CrossRefGoogle Scholar
  55. Vickers NJ, Christensen TA (2003) Functional divergence of spatially conserved olfactory glomeruli in two related moth species. Chem Senses 28:325–338PubMedCrossRefGoogle Scholar
  56. Vickers NJ, Christensen TA, Hildebrand JG (1998) Combinatorial odor discrimination in the brain: attractive and antagonist odor blends are represented in distinct combinations of uniquely identifiable glomeruli. J Comp Neurol 400:35–56PubMedCrossRefGoogle Scholar
  57. Vitzthum H, Homberg U, Agricola H (1996) Distribution of Dip-allatostatin I-like immunoreactivity in the brain of the locust Schistocerca gregaria with detailed analysis of immunostaining in the central complex. J Comp Neurol 369:419–437PubMedCrossRefGoogle Scholar
  58. Weevers RD (1966) A lepidopteran saline: the effects of inorganic cation concentrations on sensory reflex and motor responses in a herbivorous insect. J Exp Biol 44:163–176PubMedGoogle Scholar
  59. Wilson RI, Laurent G (2005) Role of GABA inhibition in shaping odor-evoked spatiotemporal patterns in the Drosophila antennal lobe. J Neurosci 25:9069–9079PubMedCrossRefGoogle Scholar
  60. Winther ÅM, Acebes A, Ferrús A (2006) Tachykinin-related peptides modulate odor perception and locomotor activity in Drosophila. Mol Cell Neurosci 31:399–406PubMedCrossRefGoogle Scholar
  61. Wood DE, Stein W, Nusbaum MP (2000) Projection neurons with shared cotransmitters elicit different motor patterns from the same neural circuit. J Neurosci 20:8943–8953PubMedGoogle Scholar
  62. Zamboni L, De Martino C (1976) Buffered picric acid-formaldehyde: a new rapid fixative for electron microscopy. J Cell Biol 35:148AGoogle Scholar

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • Bente G. Berg
    • 1
  • Joachim Schachtner
    • 2
  • Sandra Utz
    • 2
  • Uwe Homberg
    • 2
  1. 1.Neuroscience Unit/Department of PsychologyNorwegian University of Science and TechnologyTrondheimNorway
  2. 2.Department of Biology, Animal PhysiologyPhilipps UniversityMarburgGermany

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