Research Article: No Evidence for Ionotropic Pheromone Transduction in the Hawkmoth Manduca sexta

Date Published: November 9, 2016

Publisher: Public Library of Science

Author(s): Andreas Nolte, Petra Gawalek, Sarah Koerte, HongYing Wei, Robin Schumann, Achim Werckenthin, Jürgen Krieger, Monika Stengl, Richard David Newcomb.


Insect odorant receptors (ORs) are 7-transmembrane receptors with inverse membrane topology. They associate with the conserved ion channel Orco. As chaperon, Orco maintains ORs in cilia and, as pacemaker channel, Orco controls spontaneous activity in olfactory receptor neurons. Odorant binding to ORs opens OR-Orco receptor ion channel complexes in heterologous expression systems. It is unknown, whether this also occurs in vivo. As an alternative to this ionotropic transduction, experimental evidence is accumulating for metabotropic odor transduction, implicating that insect ORs couple to G-proteins. Resulting second messengers gate various ion channels. They generate the sensillum potential that elicits phasic-tonic action potentials (APs) followed by late, long-lasting pheromone responses. Because it is still unclear how and when Orco opens after odor-OR-binding, we used tip recordings to examine in vivo the effects of the Orco antagonist OLC15 and the amilorides MIA and HMA on bombykal transduction in the hawkmoth Manduca sexta. In contrast to OLC15 both amilorides decreased the pheromone-dependent sensillum potential amplitude and the frequency of the phasic AP response. Instead, OLC15 decreased spontaneous activity, increased latencies of phasic-, and decreased frequencies of late, long-lasting pheromone responses Zeitgebertime-dependently. Our results suggest no involvement for Orco in the primary transduction events, in contrast to amiloride-sensitive channels. Instead of an odor-gated ionotropic receptor, Orco rather acts as a voltage- and apparently second messenger-gated pacemaker channel controlling the membrane potential and hence threshold and kinetics of the pheromone response.

Partial Text

The sense of smell is highly developed in insects. Sensitive detection of intermittent odor stimuli such as sex-pheromones is essential for mating success in various insects. Despite its general importance, insect odor transduction is still not fully understood because contradicting evidence either support ionotropic and/or metabotropic mechanisms even in the same species (reviews: [1, 2]). Ionotropic receptors are ion channels that are directly gated by their specific ligand, while metabotropic receptors are 7-transmembrane (7-TM) receptors that couple to trimeric G-proteins. They modulate the activity of enzymes that generate/deplete second messengers such as cAMP or Ca2+. Insect odorant receptors (ORs) are 7-TM receptors with an inverse topology resulting in an intracellular N-terminus [3–6]. One to three ORs are expressed in each insect olfactory receptor neuron (ORN) together with a conserved ubiquitous ion channel termed Orco (olfactory receptor coreceptor) [6–12]. There is general agreement that ORs but not Orco specifically bind odor ligands [11–14]. Nevertheless, Orco is essential for odor detection because it locates and maintains ORs to the membrane of ORNs [3, 15, 16]. Next to this “chaperon” function, it is generally accepted that Orco forms a spontaneously opening Ca2+-permeable unspecific cation channel in heterologous expression systems [17–19]. Orco is located in membranes of dendritic cilia and soma (Fig 1) and acts as a pacemaker channel controlling spontaneous activity of insect ORNs [6, 15, 19, 20]. An ion channel is termed “pacemaker channel” if it opens at the cell´s negative resting potential, driving a neuron from rest to spike threshold, thus generating spontaneous activity. In contrast to its roles as chaperon and pacemaker channel, Orco´s precise role in insect odor transduction has not been elucidated. Odorant-dependent gating of OR-Orco receptor ion channel complexes in fruitfly, mosquito, and moths studied in heterologous expression systems was taken as evidence for an exclusively ionotropic transduction mechanism in all insect species [1, 17]. Other groups suggested mixed ionotropic and metabotropic odor transduction for fruitflies [18, 21, 22]. They found that in addition to OR-Orco-dependent odor-gated currents, odors elevated cAMP levels that opened Orco after protein kinase C (PKC)-dependent phosphorylation. Finally, evidence for exclusively metabotropic signal transduction with ORs coupling to Gq-proteins which activate phospholipase Cβ were documented for the hawkmoth Manduca sexta [2, 6, 19].

So far hypotheses of Orco function in insect odor transduction were based upon studies in heterologous expression systems (review: [6]).To examine the role of M. sexta Orco (MsexOrco) in pheromone transduction in vivo, we first confirmed with antisera against moth Orco that Orco is present in pheromone-sensitive trichoid sensilla of male hawkmoth antennae. As predicted for a chaperon function and a pacemaker function of Orco, Orco-antibodies stained the sensory cilia, dendrites, and somata, but not the axons of pheromone-specific ORNs in cryostat sections of adult M. sexta male antennae (Fig 1A). Previously, we demonstrated in Ca2+ imaging experiments that Orco activator VUAA1 can activate MsexOrco in heterologous expression systems [19]. Now we examined with Ca2+ imaging, whether Orco agonist VUAA1 activates Orco also in its natural in vivo environment, in pheromone-sensitive ORNs of the hawkmoth. Thus, we tested, whether VUAA1 can also increase intracellular Ca2+ levels in primary cell cultures of M. sexta ORNs at a stage, when they respond to pheromones in vitro [32, 35]. Western blots demonstrated that the Orco-antiserum recognized a protein with the expected mass of Orco in homogenates of the adult antenna of male hawkmoth (Fig 1B). In addition, Orco is already present at early stages of the developing pupal antenna even before ORNs respond to pheromones [36] (Fig 1B). Orco is present throughout larval development, but apparently at varying concentrations. We obtained primary cell cultures of ORNs from male antennae at pupal day 2, when ORNs were just born. The primary cultures were grown for about two weeks in vitro. At this stage they were mature and responded to pheromones, as shown previously [32, 35]. Application of the Orco agonist VUAA1 increased intracellular Ca2+ concentrations dose-dependently in these primary cell cultures of matured hawkmoth ORNs, similar to its effect in heterologous expression (n = 3) (Fig 2). Thus, the Orco agonist VUAA1 recognizes and activates MsexOrco not only in heterologous expression, but also in its natural environment in hawkmoth ORNs.

Two studies in different heterologous expression systems providing contradicting results on odor transduction in the fruitfly Drosophila melanogaster greatly stimulated the field of insect odor transduction research [17, 18]. Sato et al. [17] proposed a purely ionotropic transduction mechanism based upon OR-Orco receptor ion channel heteromers in all insect species, for general odor detection as well as for pheromone detection [17]. In contrast, Wicher et al. [18] hypothesized that in addition to a fast Orco-dependent ionotropic cascade of low sensitivity, ORs couple to Gαs and, thus, support an additional slower, metabotropic transduction cascade of high sensitivity [18].