In this work, we isolated, heterologously expressed and functionally characterized three out of five OR-subunits translated from alternative mRNA transcript variants of the D. suzukii OR69a locus. Starting with an SSR-screening of candidate ligands based on orthologues of D. melanogaster , specific compounds were selected to study ligand-binding characteristics by performing dose-response experiments and to confirm their identity as ligands of these subunits by measuring their effects at the nanogram-level by an optimized GC-SSR. To our knowledge, this is among the first contributions where the use of the GC-SSR method helps the functional characterization of insect ORs expressed in empty neurons of D. melanogaster .
Based on the screening of synthetic ligands (Fig. 1D, Table 1), 3-octanol, (Z)-4-nonenal, R-carvone and S-carvone displayed significant activation of all the DsuzOR69a subunits we have tested. Interestingly, the unsaturated (Z)-4-nonenal was the sole aldehyde tested from the panel that we have identified to be active on all tested DsuzOR69as. To compare pharmacological features among these agonists, we performed a comparative dose-response analysis.
Based on the pharmacological parameters elaborated from normalized dose-response effects, DsuzOR69a subunits demonstrated that they are in general more sensitive to R-carvone than 3-octanol and (Z)-4-nonenal (Table 2). However, R-carvone seems to be only a partial agonist for DsuzOR69aA and DsuzOR69aC, given the reduced amplitude of the curve from the approximated Hill-equation when normalized to the 3-octanol effect and compared with 3-octanol and (Z)-4-nonenal (Fig. 2). Conversely, R-carvone demonstrated stronger agonism for DsuzOR69aB. Interestingly, for this subunit, R-carvone shared very similar pharmacological parameters with (Z)-4-nonenal (Table 2, Fig. 2), while (Z)-4-nonenal was always associated with among the highest Fmax, as a possible indication of a real DsuzOR69aB agonism .
To investigate further on the activation of DsuzOR69a subunits by these agonists, we coupled SSR with the gas-chromatographic equipment (see methods) to perform GC-SSR analysis testing compounds at nanogram-aliquots (Fig. 3) upon calibrating the equipment by adjusting glass tubing and airflow until effects were recordable at the lowest possible nanogram-doses (Additional File 4: Fig. S2). In GC-SSR experiments we included also methyl salicylate given the evidence from our SSR-screening demonstrating activation to this ligand by only the DsuzOR69aB subunit, where together with R-carvone, it is one of the two most active ligands (Fig. 1D, Additional file 2: Raw Data File 2).
Contrary to SSR (Fig. 1D, Fig. 2) no effects were recorded by testing 10.0 ng 3-octanol on DsuzOR69aB by GC-SSR (Fig. 3A). Not surprisingly, dose-response for DsuzOR69aB demonstrated the lowest efficacy for this compound when compared with R-carvone and (Z)-4-nonenal (Fig. 2A), as further evidence of its possible partial agonism [45–47]. While these results may indicate that doses of 10 ng were not sufficient to enhance a minimal activation of DsuzOR69aB for 3-octanol, they support that the GC-SSR method is more reliable than the SSR method in the deorphanization of ORs to their agonists. Indeed, upon puffing identical doses of different ligands for detection by SSR, spiking may be biased by their different volatility , by the loss of calibration during serial puffing, by the type of solvent, or by possible contaminants . We expect these effects to be by-passed or at least reduced at minimum when compounds are released from the gas-chromatographer and directly provided to the antenna.
Instead, analysing DsuzOR69aA and DsuzOR69aC, 10.0 ng of 3-octanol were sufficient to enhance activation. Results from an SSR-comparison on OR69a subunits from several species of the genus Drosophila, which will be part of a different publication (Gonzalez et al. in preparation), demonstrated similar response spectrum to the ligands tested between the OR69aCs of D. simulans and D. suzukii, which included 3-octanol and R-carvone, that were also active on the OR69aAs of these Drosophilas. Possibly, the existence of an additional subunit such as OR69aC may increase the repertoire of ligands sensed by OR69aA and OR69aB. Indeed, although observed for the different class of IRs odorant receptors, different spectrums of ligand activation have been demonstrated among Drosophila neurons housing in ac2 and ac3 sensilla expressing different IR75-chemosensory subunits  that are translated from three splice-forms (IR75c, IR75b, IR75a) transcribed from the same locus (IR75cba, ). Future studies testing DsuzOR69aD and DsuzOR69aE subunits, as additional transcript variants from the DsuzOR69a-locus, will validate if this hypothesis holds true.
In analysing the effect to R-carvone by GC-SSR we observed activation for both DsuzOR69aA and DsuzOR69aB subunits. Taken with dose-response experiments, our results may suggest this ligand as real agonists for DsuzOR69aA and DsuzOR69aB. In analysing the effect to (Z)-4-nonenal by GC-SSR we observed activation for the sole DsuzOR69aB subunits. Pharmacological features and plotting of the approximated Hill equations for (Z)-4-nonenal and R-carvone were proximal to identity (Table 2, Fig. 2A), further suggesting (Z)-4-nonenal as a real agonist for DsuzOR69aB.
GC-SSR analysis demonstrated methyl salicylate to be active solely on the DsuzOR69aB subunit. Interestingly, the response to this ligand provides the highest spiking frequency among the other ligands tested and it is active up to doses lower than 1.0 ng (Fig. 3B, C). Methyl salicylate is the most active ligand we have found for DsuzOR69aB and, possibly, the main agonist for cation channels constituted with this transmembrane protein. Activation of DsuzOR69aB to methyl salicylate to 1.0 ng, but not lower aliquots, is in accordance of the optimization limit of our GC-SSR method, where doses of 1.0 ng represented the lowest detectable quantities by our assembled equipment (Fig. 3B, C and Additional File 4: Fig. S2). The ecological role of methyl salicylate is renowned among several plant mechanisms: from systemic acquired resistance to plant-plant interactions, as well as plant-insect interactions, including attraction of pollinators, repellency for phytophagous insects or attraction of their parasitoids . Since long ago, methyl salicylate has been reported among the bouquet of several floral scents  and it can also be found in the headspace of fruits from strawberries , Rubus species and cherries , that are among the renowned hosts of D. suzukii. Previous investigations demonstrated significant antennal responses to methyl salicylate by GC-EAD when D. suzukii were tested with headspaces emitted by strawberries fruits and leaves, even from fruits when this volatile was emitted in small quantities . A more recent investigation characterized methyl salicylate among the main volatiles emitted by intact berries of mistletoe (Viscum album subsp. laxum), representing a food source for winter and spring seasons for D. suzukii . Interestingly, this study reports higher attraction and ovipositional behavior for mated D. suzukii when berries are artificially wounded and emitting significantly reduced levels of methyl salicylate. Furthermore, other studies reported methyl salicylate among the most toxic compounds for D. suzukii, demonstrating among the lowest LD50s when fumigant toxicity experiments were conducted on males and females . All together, these data suggest methyl salicylate is deserving of investigations to verify its potential semiochemical properties towards the control of D. suzukii, in particular, to test repellency to this compound, taking evidences of the enhanced oviposition from its reduced emission from hosts . To this hypothesis, activation of OR69aB in D. suzukii by a repellent would represent quite an evolutionary shift from D. melanogaster, for which the orthologue was demonstrated in our previous investigation to be tuned by the (Z)-4-undecenal pheromone reported to mediate attraction . To validate this hypothesis, taking advantage of the ongoing development of CRISPR-cas9 technologies for D. suzukii  future behavioral experiments may compare sensing to methyl salicylate between wild type flies and CRISPR-knock out lines generated by inducing frame-shift editing within the start codon of the OR69aB-exon (Fig. 1A, Additional File 1: Raw Data File 1).
Our SSR-screening of DsuzOR69as is in accordance with the previous studies  and with evidence from an SSR-comparison of OR69a subunits from several species of the genus Drosophila (Gonzalez et al. in preparation). This study in preparation will demonstrate OR69aAs being mostly responsive to monoterpene alcohols and ethers, including linalool oxide and R-α-terpineol, representing the most shared ligands among the various orthologues. OR69aBs, instead, are mostly responsive to R- and S-carvones, that in our study we reported to be active on all the D. suzukii subunits we tested (Fig. 1D).
However, among our SSR analysis, we did not identify any significant activation for DsuzOR69as to (Z)-4-undecenal, which is instead active on the orthologues of D. melanogaster. Indeed, in Lebreton et al.  we demonstrated the existence of (Z)-4-undecenal in headspaces collected from D. melanogaster. The existence in D. melanogaster of (Z,Z)-7,11-heptacosadiene [(Z,Z)-7,11-HD] as the most abundant cuticular hydrocarbon , and experimental evidences of its autoxidation to the emission of aldehydes led us to hypothesize that (Z)-4-undecenal is the result of the (Z,Z)-7,11-HD autoxidation. OR69a subunits binding for both (Z)-4-undecenal and other odorants suggested a role of this receptor of D. melanogaster as a sensor for both pheromones and kairomones. In accordance, in the same investigation we reported chemical analysis on D. melanogaster and headspaces indicating small traces of (Z)-4-nonenal, associated with the presence of the (Z,Z)-5,9-HD isomer in limited quantities on the cuticular of these Drosophila [57–59]. Interestingly, (Z,Z)-5,9-HD is more abundant on the cuticle of the D. melanogaster subspecies Zimbabwe [59–61], and more recent investigations supported the autoxidation hypothesis demonstrating the emission of (Z)-4-nonenal from the Zimbabwe D. melanogaster .
Based on the most recent publications, there is no evidence in D. suzukii of the presence of cuticular hydrocarbons, which autoxidation may result in the emission of (Z)-4-nonenal [63–64], however, the specific effect of this ligand on DsuzOR69aB is compelling. In theory, if (Z)-4-nonenal would be emitted by D. suzukii, by the use of the GC-SSR method we optimized we would have recorded activation of DsuzOR69as upon testing respective headspaces collected from the female insects. However, volatile collection from 2- to 5-day-old D. suzukii females did not result in any effect on DsuzOR69as (Fig. 4). In any case, we cannot state whether (Z)-4-nonenal is emitted or not by D. suzukii. Indeed, apart from the possible absence of aldehyde ligands in the headspaces we have collected, final quantities of collected aldehydes may range below the detectable sensitivity of our equipment (< 1.0 ng, Additional File 4: Fig. S2, Fig. 3C), or may have difficulties to be released by our GC-equipment given their polar incompatibility with the HP-5 column we have utilized. These various scenario seems to be more reliable considering additional trials we performed by expressing OR69a orthologues of the D. melanogaster subspecies Zimbabwe (Additional File 7: Fig. S3). Testing headspaces from female insects, expecting to contain (Z)-4-nonenal [59–62], we did not record any effect for OR69aB. To shed more light on this aspect, additional experiments may be conducted by testing samples of Zimbabwe by GC-SSR using a different GC-column from HP-5 and adopting a different pheromone collection protocol . On the other hand, activation of DsuzOR69aB to (Z)-4-nonenal, not expected to be emitted by D. suzukii, but identified in the emissions of D. melanogaster  and of its subspecies Zimbabwe , may suggest some sort of mechanisms of inter-specific communication among different insects of the genus Drosophila. For example, complex mechanisms exist at the base of inter-specific discrimination among insects within this genus  that are based on the different composition of cuticular hydrocarbons . Although speculative, the detection of aldehyde pheromones by OR69a receptors upon their emission through autoxidation of cuticular hydrocarbons may be at the base of these inter-specific communication mechanisms. Not surprisingly, we have already demonstrated the existence of similar chemosensory systems in other insects where conserved pheromone receptors detect the main sex odors from other species  and the same kairomones from their hosts (Cattaneo, Witzgall and Walker in preparation). To verify possible emission of (Z)-4-nonenal from D. suzukii and its involvement in DsuzOR69a-based chemosensory communication, additional goals will be aimed at characterization of the rate of emission between males or females at different ages, virgin or mated, proposing (when possible) a similar approach used by Snellings et al.  Taking another possible scenario, (Z)-4-nonenal may be emitted by hosts of D. suzukii or may derive from different sources involved in the ecological relations with this insect, deserving future studies to validate alternative origins of the ligand. Indeed, a similar context exists for the D. melanogaster OR69a-ligand (Z)-4-undecenal identified in Clementine essential peel oil .
Apart from binding (Z)-4-nonenal, with an expected role as pheromone, evidences of the DsuzOR69as in binding kairomones may come from additional GC-SSR data demonstrating activation of the three DsuzOR69a subunits in proximity of GC-peaks of injected headspace collections from H. uvarum (Fig. 5, Additional File 8: Fig. S4). H. uvarum is one of the main yeast symbionts of berries, it is renowned for its ecological relations with D. suzukii and interferes with the behavior of the insect [16–17, 43, 68–70]. Although the compounds from H. uvarum headspace with activity on the DsuzOr69a-subunits were not identified, by invoking responses, it demonstrates involvements of DsuzOR69as in binding compounds emitted by this yeast, and it further remarks the ecological relations between this microorganism and the D. suzukii insect. In the frame of upcoming projects, GC-MS efforts will characterize the active ligands we have identified in H. uvarum headspace collections.
In analyzing expression of DsuzOR69a subunits in antennal sensory neurons of D. suzukii, we did not notice a significant difference between males and females, neither in the neuronal number nor in their overall distribution (Fig. 6). Our fluorescent data seems to converge on two scenarios, demonstrating that single OR69a transcript variants may rather be identified in different neurons, as well as co-existent within the same neurons. The latter scenario seem to be coherent with expectations up to now reported for D. melanogaster suggesting co-existence of both OR69aA and OR69aB transcript variants within the same ab9a neurons [30–34]. However, the study of Couto et al.  demonstrated that the two OR69a subunits may be expressed through different promoters, which cannot exclude their possibility to be present among different sub-populations of neurons. For this reasons, co-expression of OR69a-subunits remains an open and current research question. To our knowledge, this represents the first in situ hybridization study conducted on D. suzukii neurons. Our results may represent the starting point for future projects performing deeper in situ hybridization by combining different probes all in once, to validate convergence of OR subunits within the same OSNs, to consider if their chimeric cation channel may deserve to be tested by SSR. Indeed, in Lebreton et al.  co-expression of OR69aA and OR69aB in the same empty neurons did not report neither qualitative nor quantitative differences in neuronal activity. However, the existence of five antennal OR69a variants in D. suzukii (Fig. 1A, B; Walker et al. in preparation), may suggest different functional dynamics deserving better investigation in future research.
Apart from the heterologous expression, the identification in the antennae of D. suzukii of neurons expressing DsuzOR69a-subunits may add to the recent electrophysiological studies conducted in vivo on this insect : a more deepened GC-SSR analysis will investigate on these neurons the effects of the same active ligands we have identified in the course of our investigation.