Insects have positive and negative impacts on humans, in terms of health, economy, and food stores. Insects pollinate plants to increase global food production, with 35% of global production of crops depending on animal pollinators ^{1, 2} . Insects also cause significant destruction of crops and food stores ^{3– 5} . Insects can also transmit fatal diseases such as dengue fever ⁶ , malaria ⁷ , yellow fever and epidemic typhus ⁸ . Insects use olfaction to sense their surroundings and to guide important activities, including feeding, mating and oviposition. This makes the insect olfactory system receptors an attractive target for the chemical control of deleterious insect species.

Insects use odorant receptors (ORs) to recognize and distinguish a diverse range of odorants ^{9, 10} . Each OR is composed of two functionally essential parts: a highly conserved co-receptor subunit (Orco) and one of a large number of variable odorant-binding (or “tuning”) subunits ^{11– 17} . These subunits associate in an unknown stoichiometry to form an odorant-gated ion channel ^{18, 19} . ORs have also been proposed to initiate, or be modified by, second messenger cascades ^{13, 18} . While the odorant-binding subunit is responsible for interacting with odorants ^{9, 20, 21} , both the odorant-binding subunits and Orco are involved in forming the ion channel pore ^{21, 22} . Insect ORs are not related to the receptors and channels of humans and other tetrapods ¹⁵ , suggesting that control of detrimental insect activity may be possible through the development of insect OR selective compounds. A current approach to developing these compounds is to identify the particular odorant binding subunits that recognize behaviorally important odorants ^{10, 23– 26} and then conduct large scale ligand screens ^{27, 28} , but high diversity among the odorant binding subunit repertoires of different species makes this approach exceptionally labor intensive ^{29, 30} .

The recent identification of the synthetic compound VUAA1 as a novel OR agonist that acts directly on Orco ²⁷ , suggests that manipulation of insect behavior might be achieved by targeting Orco. Based on the VUAA1 structure, several additional synthetic Orco agonists and a larger, more diverse series of synthetic Orco antagonists have been identified ^{31– 33} . Importantly, several of these Orco antagonists were shown to inhibit odorant activation of ORs through a non-competitive mechanism ^{31– 33} . These findings suggest that Orco antagonists might be useful in altering insect behavior.

Orco subunits are highly conserved across insect species, suggesting that Orco serves an essential function common to all insect ORs ^{15, 34} . This high conservation underlies observations that Orco subunits from different species are functionally interchangeable; an Orco subunit from one species can form functional ORs with an odorant-binding subunit from a different species ^{21, 22} . As the “pharmacology” of synthetic Orco agonists and antagonists has expanded, it has also become clear that Orco subunits from disparate insect species have very similar sensitivities to known Orco ligands ^{27, 31– 33, 35} . This suggested to us that the binding site for Orco ligands may serve as a modulatory site for compounds endogenous to the insects or may be a target of exogenous compounds, such as those generated by plants. Insects use a variety of amines as neurotransmitters and neuromodulators ^{36– 39} . Plants also generate a variety of amines that may play a role in resistance to insect herbivores ^{40– 42} . For these reasons, we screened a panel of biogenic and trace amines for agonist and antagonist activity at insect Orco subunits. We found tryptamine to be a potent Orco antagonist with broad activity at Orco subunits from different species. Tyramine and phenethylamine also function as Orco antagonists, but were substantially less potent than tryptamine. Importantly, we found that tryptamine, acting through Orco, could inhibit odorant activation of a wide range of ORs from a variety of insect species. Our findings suggest a role for Orco as a modulatory site common to all insect ORs and support the development of Orco-directed compounds that can be used to manipulate insect behavior.

Animals use a variety of biogenic and trace amines as neurotransmitters and neuromodulators. These include compounds derived from tyrosine (dopamine, norepinephrine, epinephrine, tyramine, octopamine and phenethylamine), tryptophan (serotonin, melatonin and tryptamine) and histidine (histamine) ⁴⁷ . Dopamine and serotonin play a variety of roles in the insect nervous system ^{48– 50} . In addition, insects use octopamine, histamine and tyramine as neurotransmitters ^{48– 53} . Melatonin also appears to exert neuromodulatory effects in insects ^{54, 55} . Interestingly, many of these amines modulate the olfactory system ^{56– 58} .

Recent reports ^{27, 32} , together with our previous findings ^{31, 33} , have revealed the existence of a ligand-binding site on the Orco subunit and that inhibition of odorant activation through a non-competitive mechanism may be a general property of Orco-directed antagonists. Our current results suggest that endogenous and exogenous natural compounds serve as Orco ligands and modulate insect olfaction. While tyramine is a major neurotransmitter in insects ⁵³ , its low potency in our assay ( Figure 3) suggests that it might not serve as an endogenous OR modulator. However, the function of an endogenous Orco antagonist is unlikely to be the complete block of OR function. Rather, an endogenous Orco antagonist might be used to diminish olfactory sensitivity by inhibiting a fraction of the available receptors. For tyramine, such inhibition could occur at concentrations ranging from 10µM to 30µM. Alternatively, there may be additional, more potent, but as yet uncharacterized, endogenous Orco antagonists that can decrease olfactory sensitivity at lower concentrations.

In contrast to the low potency of tyramine, we found tryptamine to be a high potency Orco antagonist. Tryptamine inhibited odorant activation of an OR with an IC ₅₀ in the low micromolar range ( Figure 5). While it is currently unclear whether tryptamine is endogenous to insects, tryptamine and similar compounds, such as gramine, are produced by a variety of plants and are thought to serve as a defense against insect herbivores ^{42, 59} . Various tryptamine analogs have been proposed as larvicides ⁶⁰ and when tryptamine is caused to accumulate in poplar and tobacco, through ectopic expression of tryptophan decarboxylase, the feeding behavior of insects that target these plants is altered ⁴¹ . Tryptamine-based structures also act on various receptors and transporters, particularly those involved in serotonergic neurotransmission, exerting psychedelic effects in humans. Indeed, many plant derived and synthetic hallucinogens are based on the tryptamine and phenethylamine scaffolds ^{61– 63} . Interestingly, the potency that we observed for tryptamine inhibition of odorant activation of an insect OR ( Figure 5) is similar to the potency for tryptamine inhibition of the D. melanogaster serotonin transporter ⁶⁴ .

Might there also be natural endogenous or exogenous Orco agonists? An endogenous Orco agonist could serve to increase olfactory sensitivity, perhaps in a circadian fashion, to alter behavior during critical foraging or mating periods. An exogenous, plant-derived Orco agonist would, by activating all ORs through Orco, serve as an olfactory “confusant” and might alter the feeding behavior of insect herbivores. The limited screen of 11 compounds that we conducted here did not identify any Orco agonists, but more extensive screening is clearly warranted.

Several synthetic Orco antagonists have been shown to inhibit odorant activation of ORs through an allosteric mechanism ^{31– 33} . The ability of these compounds to inhibit multiple ORs from a variety of species is likely due to the high conservation of Orco across the insects ¹² . Similarly, we found that tryptamine and tyramine, acting as Orco antagonists, could inhibit odorant activation of ORs from insect species chosen from three different orders: Diptera ( An. gambiae), Lepidoptera ( O. nubilalis) and Coleoptera ( M. caryae). Furthermore, when we examined multiple ORs from a single species ( An. gambiae), we found that tryptamine and tyramine blocked odorant activation of each receptor. The action of these compounds through Orco allowed blockade to occur despite the highly diverse odorant-binding subunits used to form the receptors and the different odorant structures used to activate the receptors. Interestingly, while all six receptors were inhibited, the extent of inhibition varied depending on the odorant-binding subunit present and the pattern of variation was similar for tryptamine and tyramine. This suggests differences in allosteric coupling between Orco and the various odorant-binding subunits. Also, while we showed that tryptamine is a potent inhibitor of odorant activation of Agam\Or65+Agam\Orco, the results we present in Figure 6 suggest that tryptamine is even more potent at other ORs, such as those formed by Agam\Or27, Agam\Or31 and Agam\Or39. Our current results with naturally occurring amines, together with previous reports with synthetic compounds ^{27, 31– 33, 35} strongly suggest that: 1) allosteric antagonism of odorant activation of ORs is a general property of Orco antagonists; 2) Orco antagonists are broadly active at ORs of many insect species; and 3) Orco is an important target for the development of novel insect repellants. The broad activity of Orco directed compounds across many insect species that has been observed to date suggests that these compounds may have limited agricultural utility, since both pests and pollinators could be affected. Determining whether species-specific Orco ligands can be developed will require further effort. What is clear, however, is that the pursuit of new, synthetic Orco directed ligands (both agonists and antagonists) is a promising direction for the development of new, more effective insect repellants that can aid in controlling the spread of insect-borne diseases.

The data referenced by this article are under copyright with the following copyright statement: Copyright: � 2014 Chen S and Luetje CW

Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).

figshare: Inhibition of odorant and Orco agonist initiated current responses of oocytes expressing insect odorant receptors by various amines, doi: 10.6084/m9.figshare.977791 ⁶⁵