In most moth species, females release a unique mixture of chemicals, called a sex pheromone, in an attempt to attract males, which specifically recognize it from far away (Kaissling, 2014). All the pheromonal signals released into the air are immersed in an olfactory environment: plants also emit their own volatile molecules, which have diverse functions including communication with other organisms. In addition, the trajectory of pheromones can be quite erratic because the air can have an unpredictable and turbulent movement. The pheromonal signal is therefore quite fragmented and drowned in a very rich odor background, which has been shown to modulate pheromone detection (Deisig et al., 2014; Renou et al., 2015).

Moth pheromone compounds, like any odorant molecule, are recognized by transmembrane receptors located in the dendrite membrane of olfactory receptor neurons (ORNs), located in several thousand sensilla on the antenna (Fleischer and Krieger, 2018). In insects, the major family of such receptors are the odorant receptors (ORs). Each ORN usually expresses only one type of OR, detecting a limited number of chemicals, in addition to the obligatory co-receptor named Orco (Larsson et al., 2004), but various lines or evidence indicate that each ORN can express more than just one OR and Orco (Couto et al., 2005; Fishilevich and Vosshall, 2005; Koutroumpa et al., 2014; Schultze et al., 2014; McLaughlin et al., 2021; Herre et al., 2022; Task et al., 2022). In moths, pheromone compounds are detected by receptors belonging to particular OR lineages (Zhang and Löfstedt, 2015; Bastin-Héline et al., 2019; Montagné et al., 2021). Whereas the majority of ORs have a broad activation spectrum, i.e., they can recognize several odorant molecules, pheromone receptors (PRs) are often very specific to their ligand or at least narrowly tuned to similar chemicals (Zhang and Löfstedt, 2015). Once interaction occurs between an odorant and the corresponding OR-Orco complex, the signal is transformed by the ORN into an electrical signal that is transmitted along the antennal nerve to the brain primary olfactory centers, the antennal lobes (ALs). There, peripheral inputs are processed, enabling the insect to extract relevant information and adopt the correct behavior (Masse et al., 2009; Touhara and Vosshall, 2009).

It has been demonstrated that volatile plant compounds (VPCs) can modulate the male pheromone response at ORNs, ALs, and behavioral level. At the behavioral level, both field and laboratory studies have shown a synergistic effect of VPC/pheromone mixtures on pheromone trapping (Light et al., 1993; Landolt and Phillips, 1997; Deng et al., 2004) and on male orientation in wind tunnel experiments (Schmidt-Busser et al., 2009; Varela et al., 2011). Such synergism has also been evidenced within the male macroglomerular complex (a set of enlarged glomeruli dedicated to the process of pheromones in the ALs) in Cydia pomonella (Trona et al., 2013). At the pheromone-responsive ORN level, studies are more contrasted. Addition of a VPC to the pheromone usually results in a suppressive effect on the pheromone response (Den Otter et al., 1978; Pophof and van der Goes van Naters, 2002). For instance, a background of linalool (one of the most common VPCs in nature) appears to halve the response of ORNs tuned to the major component of the sex pheromone in Spodoptera littoralis (Party et al., 2009). Similarly, a background of some VPCs – namely linalool, (Z)3-hexenyl acetate, hexanal and heptanal – reduces the firing rate of pheromone-responsive ORNs in the black cutworm moth Agrotis ipsilon (Renou et al., 2015). However, the effects have been shown to be VPC-dependent in Heliothis virescens. In this species, VPCs like linalool, geraniol or (Z)3-hexenyl acetate diminish the response of (Z)11-16:Ald-specific ORNs and this suppressive effect has been shown to occur at the level of the PR protein (Hillier and Vickers, 2010; Pregitzer et al., 2012). By contrast, a mixture of (Z)11-16:Ald and β-caryophyllene appears to enhance the activity of the (Z)11-16:Ald ORNs (Hillier and Vickers, 2010). In Helicoverpa zea, mixtures of linalool or (Z)3-hexenol and the major sex pheromone component also synergize the response of pheromone-responsive ORNs (Ochieng et al., 2002). Moreover, it has unexpectedly been shown in A. ipsilon that ORNs tuned to (Z)7-12:OAc – one of the three components of the sex pheromone – can respond to stimulation with VPCs alone, notably linalool, (Z)3-hexenyl acetate and heptanal (Rouyar et al., 2015; Conchou et al., 2021). Consequently, backgrounds of these volatiles mask pheromone responses and impact male behavior, notably by altering the perception of the ratio of pheromone constituents (Dupuy et al., 2017; Hoffmann et al., 2020). However, the mechanisms underlying this response of pheromone-sensitive ORNs to VPCs remain unknown.

Several hypotheses can be put forward. Although it is generally accepted that ORNs express only one type of OR, many exceptions have been documented (Couto et al., 2005; Fishilevich and Vosshall, 2005; Koutroumpa et al., 2014; Schultze et al., 2014; McLaughlin et al., 2021; Herre et al., 2022). First, A. ipsilon (Z)7-12:OAc-responsive ORNs could express several ORs, one specific for the pheromone and other(s) recognizing VPCs. Second, some moth PRs could be less specific than expected and able to bind certain VPCs in addition to pheromone compounds. In order to test these hypotheses and determine whether activation by VPCs occurs at the level of the PR or not, the aim of the present work was to identify the A. ipsilon receptor tuned to (Z)7-12:OAc, taking advantage of its recently published genome (Wang et al., 2021), and to study its functional properties in heterologous systems, isolated from its natural neuronal environment.

Although detection and central processing of sex pheromones and VPCs have been thought for long to be separated from each other, it is now clearly established that interactions between both classes of olfactory cues occur early in the peripheral olfactory system of male moths, usually resulting in a suppressive effect on the pheromone response (Den Otter et al., 1978; Pophof and van der Goes van Naters, 2002; Party et al., 2009). This is the case of pheromone-responsive ORNs of A. ipsilon males, for which a background of some VPCs – including heptanal – reduces the firing rate in response to a pulse of pheromone (Renou et al., 2015). The underlying mechanisms are not clearly understood, but another study conducted in A. ipsilon has demonstrated that some VPCs activate the pheromone ORNs by themselves (Rouyar et al., 2015), although it is not known if this phenomenon is direct or indirect. In this study, native A. ipsilon (Z)7-12:OAc-ORNs responded to heptanal starting at a 1% dilution with a spike frequency of around 60 spikes·s⁻¹, reaching 125 spikes·s⁻¹ at 10%. Interestingly, a previous report has already revealed that high doses of VPCs activate pheromone-responsive ORNs in a closely related species, A. segetum (Hansson et al., 1989). As it is generally accepted that ORNs express only one type of OR (but see exceptions to this, Couto et al., 2005; Fishilevich and Vosshall, 2005; Koutroumpa et al., 2014; Schultze et al., 2014; McLaughlin et al., 2021; Herre et al., 2022; Task et al., 2022), and considering that PRs are highly specific to pheromones, activation of pheromone ORNs by VPCs is puzzling. Would pheromone ORNs express more than one OR type? Would A. ipsilon PRs be less specific than previously thought?

To test these hypotheses, we worked at the level of the PR tuned to the pheromone compound (Z)7-12:OAc. First, we identified this receptor as AipsOR3. It belongs to the classical moth PR clade and is the orthologue of AsegOR4, also tuned to (Z)7-12:OAc in A. segetum (Zhang and Löfstedt, 2013). Interestingly, whereas some closely related PRs can respond to different pheromone compounds (Montagné et al., 2021), we have here a clear example of functional conservation. A large screening with 26 pheromone compounds revealed that AipsOR3, when expressed in Drosophila ORNs, was quite specific to (Z)7-12:OAc and did not respond to any of the four VPCs known to activate (Z)7-12:OAc ORNs in A. ipsilon. Moreover, a background of heptanal had no effect on the pheromone response of AipsOR3-expressing Drosophila ORNs, contrary to what has been observed in A. ipsilon (Z)7-12:OAc ORNs. Thus, heterologous expression of the receptor tuned to (Z)7-12:OAc does not recapitulate the functional properties of A. ipsilon (Z)7-12:OAc ORNs to VPCs.

The simpler interpretation of these results is that (Z)7-12:OAc ORNs express another OR responsible for the detection of VPCs. In fact, some cases of co-expression of several ORs within a single ORN have been revealed in different species. For instance, double in situ hybridization showed that at least two ORs are co-expressed in some Anopheles gambiae ORNs (Schultze et al., 2014). In D. melanogaster, some OR pairs are co-expressed in a single ORN and are both functional (Ebrahim et al., 2015; Lebreton et al., 2017). In the moth Ostrinia nubilalis, some pheromone-responsive ORNs co-express up to four different PR genes, and the broad tuning of these ORNs strongly suggest that at least several of these PRs are functional (Koutroumpa et al., 2014). Alternatively, non-OR membrane receptors could be responsible for VPC responses. In insects, olfactory detection is indeed performed not only via ORs but also via ionotropic receptors (IRs; Wicher and Miazzi, 2021). Whereas ORs and IRs are generally expressed in different ORN populations, a recent study in the mosquito Aedes aegypti has unexpectedly shown that IRs could be co-expressed with ORs in the same ORNs, and that both receptor types were functional (Herre et al., 2022). In Drosophila, Orco and the three IR co-receptors extensively overlap in expression (Task et al., 2022). It is plausible that A. ipsilon (Z)7-12:OAc ORNs express IRs in addition to AipsOR3, yet IRs are not the best candidates to explain VPC detection because several VPCs used in our study are known to be detected by ORs in moths (de Fouchier et al., 2017; Guo et al., 2021).

Although our results suggest that at least one OR is co-expressed with AipsOR3 in A. ipsilon ORNs, we cannot rule out other possibilities. First, our study may suffer from a protocol bias, as the stimulation system we used (Pasteur pipettes) differs from that used in Rouyar et al., 2015 (vials). Thus, the amount of heptanal reaching the antennae is difficult to compare. However, in our heptanal background experiment, we used exactly the same stimulation system as in Rouyar et al., 2015 and no response of AipsOR3-expressing Drosophila ORNs to 1% heptanal could be noticed at the onset on the background delivery. In addition, we found no effect of this background on the pheromone response of these ORNs, contrary to what has been observed for A. ipsilon (Z)7-12:OAc ORNs. Another explanation could be that AipsOR3 is in fact less specific in vivo than what we observed when expressed in Drosophila ORNs. Indeed, it is known that the sensillum environment can affect OR response profiles. For instance, odorant-binding proteins (OBPs) and/or chemosensory proteins (CSPs) are proposed to transport odorants within the sensilla lymph to the ORN membrane, with implications for ORN sensitivity and specificity (Große-Wilde et al., 2007; Forstner et al., 2009; Pelosi et al., 2014). Moreover, it has been shown that some OBPs can modulate olfactory physiology and the behavior that it drives (Xiao et al., 2019). Sensory Neuron Membrane Proteins (SNMPs) may also be part of the pheromone reception pathway (Jin et al., 2008). OBPs, CSPs and/or SNMPs, housed in Drosophila at1 sensilla, may strongly differ from those naturally occurring in A. ipsilon pheromone sensitive sensilla, and they could have affected the response of ORNs expressing AipsOR3. To test this hypothesis, we used another functional assay to isolate AipsOR3 from components of the sensillum environment (OBPs, CSPs, SNMPs), which consisted of in vitro expression in Xenopus oocytes coupled to two-electrode voltage clamp. In this system, odorants (pheromones and VPCs) were solubilized in water and carried to the OR via the use of dimethyl sulfoxide. Thus, VPCs should not encounter any solubilization nor transport issues from insect OBPs/CSPs/SNMPs to reach the OR, although the Xenopus oocyte solution used to solubilize odorants might probably not exactly recapitulate OBP function. By doing so, we confirmed that AipsOR3 responded strongly to (Z)7-12:OAc only, and no response was found for any of the four VPCs.

Taken all together, our results demonstrate that activation of (Z)7-12:OAc-sensitive ORNs by VPCs in A. ipsilon does not occur at the level of the AipsOR3 protein. A compelling explanation is that at least another receptor – which remains to be identified – is co-expressed in these neurons and participate in modulating pheromone responses in the presence of plant odors, but we cannot exclude other hypotheses. For instance, the exact function of OBPs/CSPs is still under study (Rihani et al., 2021) and these proteins may change the OR response threshold (Xiao et al., 2019), making the VPC responses not detectable in our expression systems. Apart OBPs/CSPs, other yet unknown elements of the A. ipsilon pheromone sensilla – not present in Drosophila at1 sensilla nor in the Xenopus oocyte aqueous environment – may also confer responses to heptanal in A. ipsilon pheromone ORNs. These could include sensillum shape, cuticle structure or lymph biochemistry, as recently reviewed by Schmidt and Benton, 2020. Alternatively, the response to heptanal in the native system may result from ORN interactions. Such functional interactions between ORNs grouped in the same sensillum have been evidenced earlier in D. melanogaster (Su et al., 2012; Zhang et al., 2019; Pannunzi and Nowotny, 2021). However, very careful examination of spike shape and size in hundreds single sensillum recordings on A. ipsilon pheromone ORNs revealed a complete recovering of spikes in response to either pheromone or heptanal and a homogenous spontaneous firing in the absence of stimulation. This makes highly improbable the contribution of another ORN type within the pheromone long trichoid sensilla, which are located on antennal branches. In addition, responses of non-pheromonal ORNs to heptanal and other plant volatiles were recorded from another category of olfactory sensilla, short hair sensilla, situated on the antennae stem. Conclusive experiments would come from a genome editing approach in A. ipsilon moths, in which AipsOR3 is knocked-out, and testing if the response to heptanal still occurs. Single cell transcriptomics from on A. ipsilon (Z)7-12:OAc-sensitive ORNs, although challenging, would definitively clarify the second OR identity.

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding authors.

PV and RC conducted Drosophila single-sensillum recordings and analyzed data. ZL conducted Xenopus oocyte TEVC recordings and analyzed data. M-CF performed molecular cloning experiments. MR conceived experiments in odorized backgrounds and supervised single-sensillum recordings. NM and RC carried out bioinformatics and Drosophila genetics. EJ-J and NM conceived the study, supervised experiments, analyzed data, and wrote the manuscript. All authors contributed to the article and approved the submitted version.

This work has been funded by the French National Research Agency (ANR-11-BSV7-0026 and ANR-15-CE02-0010), the French Embassy in China (COMIX 2019-2020) and by a Chinese Scholarship Council (CSC) grant to ZL.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.