Analysis of olfactory transduction mechanisms in the behaving hawkmoth Manduca sexta

• Applicants: Professorin Dr. Monika Stengl; Privatdozent Dr. Dieter Wicher
• Subject Area: Cognitive, Systems and Behavioural Neurobiology
• Term: from 2009 to 2017
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 122350432

• Final Report Year: 2018

Final Report Abstract

Manduca sexta females attract their mates with the release of a species-specific sex-pheromone blend with bombykal (E,Z)-10,12-hexadcadienal and (E,E,Z)-10,12,14-hexadecatrienal being the two major components. We searched for the hawkmoth bombykal receptor by testing candidate receptor proteins in heterologous expression systems. The putative pheromone receptor MsexOr1 co-expressed with MsexOrco in Xenopus oocytes elicited dose-dependent inward currents upon bombykal application. Coexpressed in HEK293 and CHO cells bombykal stimulation of receptors elicited rises in the intracellular free Ca2+ concentration. In contrast, MsexOr4 coexpressed with MsexOrco neither responded to bombykal nor to the (E,E,Z)-10,12,14-hexadecatrienal mimic. Thus, MsexOr1, but not MsexOr4, and also not MsexOrco, is the bombykal-binding pheromone receptor in the hawkmoth. Finally, we obtained evidence that phospholipase C- and protein kinase C-activity is involved in the hawkmoth´s bombykal-receptor-mediated Ca2+ signals in HEK293 and CHO cells. The overall progress of our investigations was hampered by the limited success of functional membrane incorporation in the used expression systems. We thus had to focus our attempts to main aspects of function and regulation of pheromone receptor proteins. Since new pharmacological tools were published we changed our original plan of work to concentrate with in vivo pharmacology on the most important question of the role of Orco in pheromone transduction and the question which pheromone transduction cascades are used by insects. With different physiological, biochemical, and pharmacological approaches we succeeded to show that MsexOrco does not underlie an ionotropic mechanism of pheromone transduction. Instead, bombykal transduction is metabotropic and MsexOrco is a voltage-dependent pacemaker channel that controls the membrane potential and thus the spontaneous activity of ORNS. MsexOrco is activated seconds after the pheromone stimulation, when voltage and second messenger levels are rising via bombykal-dependent activation of a phospholipase C cascade. In addition, controlled via daytime-dependent rhythms in cAMP and IP3 levels different second messenger-dependent ion channels are available for pheromone transduction during the course of the day. In addition, different pheromone transduction cascades are being employed, either metabotropic via PLC activation, or non-metabotropic via ligand-gated activation of guanylyl cyclases, depending on stimulus strength and length.

Publications

• (2010) Sex-specific odorant receptors of the tobacco hornworm Manduca sexta. Front Cell Neurosci, Aug.3;4. p 22
  Grosse-Wilde E, Stieber R, Forstner M, Krieger J, Wicher D, Hansson BS
  
• Pheromone transduction in moths. Frontiers Neurosci Dec 31 4:133
  Stengl M
  
• (2011) Antennal transcriptome of Manduca sexta. PNAS 108(18) 7449-7454
  Grosse-Wilde E, Kuebler LS, Bucks S, Vogel H, Wicher D, Hansson BS
  
• (2012) Time of day changes in cyclic nucleotides are modified via octopamine and pheromone in antennae of the Madeira cockroach. J Biol Rhythms 27(5):388-397
  Schendzielorz T, Peters W, Boekhoff I, Stengl M
  
• (2013) In situ tip-recordings found no evidence for an Orco-based ionotropic mechanism of pheromone-transduction in Manduca sexta. PLOS ONE 8(5)e62648
  Nolte A, Funk N, Mukunda L, Gawalek P, Werckenthin A, Hansson BS, Wicher D, Stengl M
  
• (2013) The role of Orco in insect odor transduction. J Comp Physiol A., 199:897-909
  Stengl M, Funk N
  
• (2015) Octopamine regulates antennal sensory neurons via daytime-dependent changes in cAMP and IP3 levels in the hawkmoth Manduca sexta. PLOS ONE Mar 18;10(3):e0121230
  Schenzielorz T, Schirmer K, Stolte P, Stengl M
  
• (2016) No evidence for ionotropic pheromone transduction in the hawkmoth Manduca sexta. PLOS ONE 11(11):e0166060
  Nolte A, Gawalek P, Koerte S, Stengl M
  
• (2016). The mouse receptor transporting protein RTP1S and the fly SNMP1 support the functional expression of the Drosophila odorant coreceptor Orco in mammalian culture cells. J. Neurosci Meth 271, 149-53
  Halty-deLeon L, Miazzi F, Kaltofen S, Hansson B S and Wicher D
  
• (2017) Identification and characterization of the bombykal receptor in the hawkmoth Manduca sexta. J. Exp. Biol., jeb.154260
  Wicher D, Morinaga S, Halty-deLeon L, Funk N, Hansson B, Touhara K, Stengl M