Functional characterization of a female-biased chemoreceptor of the codling moth (Cydia pomonella) responding to aldehydes and other volatile compounds

doi:10.21203/rs.3.rs-5241030/v1

With the advent of semiochemical-based control strategies used to mitigate damage of agricultural pest moths, many studies have focused on the function of male-specific putative pheromone receptors (PRs). In this investigation, we instead isolated, heterologously expressed, and functionally characterized a female-biased candidate PR, CpomOR22, from the codling moth, Cydia pomonella. Using transgenic Drosophila melanogaster for single sensillum recording (SSR) and gas-chromatographic SSR, we tested both synthetic ligands and various apple headspace extracts, identifying saturated and unsaturated aldehydes (nonanal, decanal, undecanal, dodecanal; (Z)-4-undecenal and (Z)-6-undecenal) among the most active ligands. Parallel experiments expressing CpomOR22 in Xenopus oocytes confirmed the binding of nonanal, decanal and undecanal aldehyde and revealed lactones (gamma-undecalactone and delta-dodecalactone) and several carboxylic acids as additional active compounds. The renowned ecological importance of aldehydes for the codling moth and the potential for newly identified ligands, such as lactones, may inform innovative control strategies based on novel semiochemicals to interfere with the female-specific chemosensory systems of this insect.

Cydia pomonella chemoreceptors

heterologous expression

transgenic Drosophila melanogaster

Single sensillum recording (SSR)

Gas-chromatography-coupled SSR (GC-SSR)

Xenopus oocytes

The Cydia pomonella Odorant receptor OR22 has a female bias in adult antennae and it phylogenetically clusters with pheromone receptors, representing a promising target for control strategies interfering with female codling moth behavior.
Single sensillum recording (SSR) in ab3A neurons of Drosophila melanogaster expressing CpomOR22 unveiled binding to various host-emitted ligands, including aldehydes and pear ester, but not pheromones.
Coupling Gas-Chromatography to SSR (GC-SSR) and combining Gas-Chromatography with Mass Spectromety (GC-MS) demonstrated saturated aldehydes emitted from headspace we isolated from apples among the ligands binding to CpomOR22.
Heterologous expression of CpomOR22 in oocytes from Xenopus laevis unveiled binding to the same aldehyde ligands and identified lactones as additional ligands.
Ligands active on CpomOR22 are promising for semiochemical-based control strategies of C. pomonella.

The codling moth, Cydia pomonella (Lepidoptera: Tortricidae), is a carpophagous global pest of apple, pear, and walnuts. For decades, sustainable methods like male-mating disruption (Witzgall et al. 2008, 2010), and other alternatives based on attract-and-kill (Charmillot et al. 2000, Krupke et al. 2002), have been successful for the management of this pest. Despite this, there is an ongoing and growing concern that these techniques may no longer reduce the damage below economically acceptable thresholds, given their high costs, rendering them less competitive when compared with the most common applications of insecticides (Miller and Gut 2015, Carde and Minks 1995, Gregg et al. 2018).

At the beginning of this millennium, a ripe pear-released volatile, ethyl (E,Z)-2,4-decadienoate, commonly known as pear ester (Light et al. 2001), was identified as invoking olfactory neuronal responses in both male and female C. pomonella (Ansebo et al. 2004, 2005). Incorporating this ligand into codlemone baited traps enabled monitoring of moth flight patterns in orchards but varied across seasons and crops (Landolt et al. 2007, Light et al. 2001, Kutinkova et al. 2005, Thwaite et al. 2004, Knight et al. 2012). Whether kairomones, like pear ester, may modulate food-finding, oviposition site seeking, or both, remain inconclusive. Gravid females of C. pomonella use host kairomones, for example from apple, to locate and select a suitable oviposition site (Landolt et al. 2007, 2008, Preti et al. 2021, Hern and Dorn 2004). In addition, more dated studies showed that mated females of C. pomonella prefer apples infested with conspecific larvae over uninfested apples (Reed et al. 2002, Yan et al. 1999, Hern and Dorn 2002) suggesting that the quality of kairomones changes with arthropod infestation, subsequently impacting pest behaviour. Independently from its possible role as a semiochemical, effects from pear ester are compelling, and contribute to the disruption of male insects, also to lures that are female specific, with the aim to target females, and may be combined with pheromones in baited traps that interfere with both sexes (Knight et al. 2022, Landolt et al. 2007).

Since the discovery of the sex pheromone bombykol in the silk moth Bombyx mori (Butenandt et al. 1959), recent advancements in transcriptomics and bioinformatics have enhanced the understanding of the molecular mechanisms behind chemosensory modalities among the various species belonging to the broad taxonomic group of Lepidoptera (Walker et al. 2023, 2019, Crava et al. 2022, Poivet et al. 2013, Montagné et al. 2021).

While the application of sex pheromones to disrupt males has led to more emphasis on male-specific olfactory receptors (ORs) than on the female-biased ones (Bastin-Heline et al. 2019, Löfstedt et al. 2016), few studies have deciphered female-specific ORs (Wanner et al. 2007, Anderson et al. 2009, Tom et al. 2022). For example, in B. mori, antennal expression of ORs with a female bias is evident for several subunits, including BmorOR19, OR30, and ORs 45 to 48(Wanner et al. 2007). Subsequent functional characterization showed that BmorOR19 and BmorOR45, together with BmorOR47, respond to mulberry leaf volatile components linalool and benzoic acid, respectively (Anderson et al. 2009, Pophof et al. 1997, Heinbockel et al. 1996). In the hawkmoth Manduca sexta, MsexOR6 and MsexOR15 are female-specific ORs, with the former expressed in the antennae and legs and the latter exclusively in the antennae. Additionally, ORs 5, 17, 26, 33, 40, and 87 exhibit biased expression in female antennae (Tom et al. 2022, Koenig et al. 2015). However, the female-biased MsexORs have yet to be functionally characterized.

In the codling moth, antennal transcriptome analysis comparing adults and larvae unveiled four female-specific/biased ORs: CpomOR21, CpomOR22 (also named OR15a: accession number CPOM22347.t1(Wan et al. 2019)), CpomOR30, and CpomOR41 (Walker et al. 2016). Among these, CpomOR21 and CpomOR22 cluster within the traditional lepidopteran pheromone receptor (PR) clade (Krieger et al. 2004, Zhang and Löfstedt 2015). While, to date, ligands for these ORs remain unknown, it is expected that such female-biased or enriched ORs may bind to kairomones. For example, within the PR-clade of the codling moth, an OR which is highly expressed by both males and females, CpomOR3, responds to the pear ester kairomone and to its methyl-analogue methyl(E,Z)-2,4 decadienoate (Bengtsson et al. 2014, Cattaneo et al. 2017). According to functional characterization methods based on Xenopus oocytes, CpomOR3 also responds, to a lesser extent, to the codling moth sex pheromone codlemone, (E,E)-8,10-dodecadien-1-ol (Wan et al. 2019), however, this finding has not been confirmed by expressing the same subunit with other heterologous methods (Cattaneo et al. 2017). Otherwise, only one codling moth PR has been functionally characterized up to now: CpomOR6a, which was reported to respond to various acetates renowned among the female-produced components of sex pheromone communication systems, including (E,E)-8,10-dodecadien-1-yl acetate. (Cattaneo 2018, Cattaneo et al. 2017). For CpomOR22 specifically, putative orthologues exist, based on phylogenetic relatedness, in other tortricids, including Ctenopseustis obliquana, Ctenospeustis herana, and Epiphyas postvittana (Walker et al. 2016, Corcoran et al. 2015, Steinwender et al. 2015). Starting from these lines of eveidence, we focused on this subunit, hypothesizing its possible involvement in perceiving signals that mediate female specific behaviors, such as oviposition, to lead our efforts in attempting its functional characterization.

To test a possible involvement of the female-biased CpomOR22 in host volatile sensing, we heterologously expressed this subunit in the Drosophila empty neuron system and in Xenopus oocytes (Gonzalez et al. 2016, Fleischer et al. 2018), after using qRT-PCR to confirm enriched expression of CpomOR22 in female antennae. Our results illustrate that CpomOR22 provides an olfactory channel for perceiving host-associated cues. Additionally, through assay with structural analogues, we reveal novel compounds previously not reported in the chemosensory ecology of C. pomonella. Altogether, the current study advances our understanding of the mechanisms of olfactory detection in C. pomonella female moth and provides a new perspective for ligand postulation in insect pests.

Quantitative RT-PCR (qRT-PCR)

For each biological sample, antennal pairs from 100 unmated male or unmated female C. pomonella adults were dissected into RNA-Later. Total RNA from all samples was extracted and purified with the RNeasy Plus Mini Kit (Qiagen, Venlo, Netherlands), and cDNA was synthesized with the QuantiTech Reverse Transcription Kit cDNA synthesis kit (Qiagen) according to the manufacturer's protocol. qRT-PCR experiments were carried out with Roche Light Cycler 480 II thermocycler (Roche, Basel, Switzerland). qRT-PCR primers for each gene were designed with the IDT RealTime PCR tool (Integrated DNA Technologies; https://www.idtdna.com/scitools/Applications/RealTimePCR/) (Supplementary Table 1). For each primer assay, amplification was performed on biological triplicates of unmated male and female antennal samples with technical duplicates. For all reactions, the following contents were added: 2.0 μL of cDNA sample, 12.5 μL of enzymatic mix, iTaq SYBR Green supermix with ROX (Bio-Rad Laboratories, Hercules, CA, USA), 5.5 μL water, and 5.0 μL of gene-specific primers (100 nM final concentration of each primer), for a final reaction volume of 25 μL. The qRT-PCR amplification protocol was run as follows: initiation phase: 3 min at 95 °C; amplification phase (40 cycles): 10 s at 95 °C, 40 s at 56 °C; melting curve phase: 40–95 °C gradient, with analysis every 1.0 °C; melting curves were analyzed to verify the specificity of amplification products. Primer efficiencies were calculated for all primer pairs using a Topo4 plasmid (Invitrogen) with an insert of cloned amplicon using the same primers for the qRT-PCR assay, with six serial dilutions ranging from 2.0 ng to 2e-5 ng and a dilution factor of 10. For OR22, relative expression was normalized to the expression of two reference genes, ActR2 and HSP40; these reference genes were selected from a pool of six candidate reference genes(Lü et al. 2018) based upon optimal stability of expression across all biological samples and primer efficiency values as close as possible to 100%. Candidate reference gene sequences were identified in our previously published transcriptome(Walker et al. 2016) or the genome database associated with the published C. pomonella genome (Wan et al. 2019). Relative gene expression differences across male and female samples were assessed by determination of relative quantities (primer efficiency^Delta(CT)) of the OR22 gene relative to the geometric mean of the relative quantities of the two reference genes (Vandesompele et al. 2002). Delta(CT) values for each biological replicate were calculated relative to the average CT value of the three male antennal samples (Supplementary Data File 1). To control for potential differences in olfactory gene expression in male versus female antennae, OR22 expression differences were further compared to the expression of the odorant receptor coreceptor, CpomOrco. Statistical differences were assessed across conditions on binary log-transformed relative quantities using an independent t-test with two-tailed distribution and two samples with equal variance; significance was assessed at (P < 0.05).

Cloning and heterologous expression of CpomOR22 in Drosophila empty neuron system

A synthetic construct of the OR22 ORF of C. pomonella was obtained (Eurofins Genomics, Ebersberg, Germany) as a plasmid insert in pCR2.1-Topo; the ORF was based upon its sequence identified in the antennal transcriptome (Walker et al. 2016)and was codon optimized for expression in Drosophila melanogaster (Supplementary Data File 2). The complete ORF encoding CpomOR22 was amplified by PCR combining specific codon-optimized CDS-primers (Fw: 5'-ATGAAGTTTGAAGAGGCCGAC-3'; Rv: 5´-TTACTCGATAGAGGATTTGAGCATGA-3') with the pCR2.1 plasmid as a template. Purified PCR products were then cloned into the PCR8/GW/TOPO plasmid (Invitrogen, Waltham, MA, USA). Insert integrity and orientation were confirmed by Sanger sequencing, with a 3730xl DNA Analyzer (Eurofins Genomics). Cassettes with inserts were then transferred from their PCR8/GW/TOPO plasmids to the destination vector (pUASg-HA.attB, constructed by E. Furger and J. Bischof, kindly provided by the Basler group, Zürich) using the Gateway LR Clonase II kit (Invitrogen). Insert integrity and orientation were again checked by Sanger sequencing.

Transformant D. melanogaster lines with pUAS-CpomOR22 were generated by Best Gene (Chino Hills, CA, USA), injecting into Best Gene Strain #24749 with genotype M{3xP3-RFP.attP}ZH-86Fb and insertion locus on the third chromosome. Crossings were performed with standard balancer lines and the Δhalo chromosomal background to drive the expression of CpomOR22 in the A neuron of ab3 basiconic sensilla (ab3A OSNs) according to procedures already established in our labs (Gonzalez et al. 2016). The final crossing was performed with w;Δhalo/CyO;pOr22a-Gal4 mutant line (Dobritsa et al. 2003, Hallem et al. 2004), and selection of ∆halo homozygotes was based on the straight wings phenotype. The final strain tested by SSR and GC-SSR had the following genotype: w;Δhalo;pUAS-CpomOR22/pOR22a-Gal4. Insects were reared in our facilities at room temperature (25 ± 2°C) on a sugar-yeast-cornmeal diet (https://bdsc.indiana.edu/information/recipes/bloomfood.html) at a relative humidity of 50 ±5 % and under 12:12 light:dark photoperiod.

Volatile collection from apple headspace

Volatile collections from Hoplocampa testudinea (Hymenoptera:Tenthredinidae) infested branches of apple trees (M. domestica v. 'Discovery', with 10-15 pieces of 1-3 cm diameter apples and 10-20 leaves, sample names abbreviated as Hoplomalus) and branches with non-infested fruits (sample names abbreviated as Malus) were used in the GC-SSR recordings. The H. testudinea insect-infested apples were selected to qualitatively broaden the chemical profile for screening purposes.The volatile collections were done in the laboratory using Porapak Q filled volatile collection traps. The biological samples were placed in 25 x 38 cm polyester oven roasting bags (Look, Teri- nex Ltd., England), and charcoal-filtered air was pumped into the bag at 410 mL/min to keep the bag inflated during sampling. The volatile collection traps were connected to the oven bag and fit with a Teflon tube, while the headspace was drawn through each trap at the rate of 200 mL/min for 2 hours 40 minutes, 6 hours, 14 hours, or 20 hours depending on the sample (2 hours 40 minutes: Malus 559, Hoplomalus 560, Malus 561; 6 hours: Hoplomalus 583, Malus 590;14 hours: Hoplomalus 566, Malus 603; 20 hours Hoplomalus 500). The samples were desorbed from the volatile traps with 200-400 uL hexane, and 1.0 ug heptyl acetate was added as an internal standard to the volatile extracts. The extracts were stored at -40°C in melted glass capillaries.

Single sensillum recordings

CpomOR22 expressed in the A neuron of ab3 basiconic sensilla was tested through single sensillum recordings (SSR), adapting the protocols we recently described (Cattaneo et al. 2023). In brief, three- to eight-day-old female flies were immobilized in 100 μL pipette tips with only the top half of the head protruding. The right antenna of each insect was gently pushed with a glass capillary against a piece of glass. This piece of glass and the pipette tip were fixed with dental wax on a microscope slide. Electrolytically sharpened tungsten electrodes (Harvard Apparatus Ltd, Edenbridge, United Kingdom) were used to penetrate the insect's body: the reference electrode was manually inserted in the right eye of the fly, while the recording electrode was maneuvered with a DC-3K micromanipulator equipped with a PM-10 piezo translator (Märzhäuser Wetzler GmbH, Wetzler, Germany) and inserted in ab3-sensilla. Signals coming from the olfactory sensory neurons were amplified ten times with a probe (INR-02, Syntech, Hilversum, the Netherlands), digitally converted through an IDAC-4-USB (Syntech) interface, and visualized and analyzed with the software Autospike v. 3.4 (Syntech). To carry the odorant stimulus, prevent antennal dryness, and minimize the influence of background odors from the environment, a constant humidified flow of 2.5 L/min charcoal-filtered air was delivered through a glass tube and directed to the preparation. To confirm the expression of CpomOR22-transgenes, basic spiking of ab3-neurons was compared with parental flies Δhalo-homozygous (w;Δhalo;pOr22a-Gal4 and w; Δhalo;+ mutants) (Cattaneo et al. 2023). A panel of 27 odorants (Table 1) was made of synthetic compounds from our collection (Cattaneo et al. 2023, Lebreton et al. 2017), including ligands that have been previously reported among codling moth pheromones, compounds emitted from fruit and yeast (Bengtsson et al. 2014, Bäckman et al. 2000, Bengtsson et al. 2001, Witzgall et al. 2012, Jennings et al. 1964), and a few novel ligands. Among these, we included some primary pheromones and kairomones of C. pomonella based on previous functional studies on its primary pheromone receptors (Bengtsson et al. 2014, Cattaneo et al. 2017). In the screening, we also added (Z, Z)-3,13-Octadecadien-1-yl acetate (CAS: 53120-27-7), a main pheromone compound from the red-belted clearwing moth Synanthedon myophaeformis, for which further investigation will be part of a separate study (Cattaneo et al. in preparation).

Based on the database of odorant responses (http://neuro.uni-konstanz.de/DoOR/content/DoOR.php(Münch and Galizia 2011, Galizia et al. 2010), the panel also included 2-heptanone (CAS 110-43-0) and 3-octanol (CAS: 589-98-0) as positive controls to validate recordings from ab3 sensilla by testing activation of D. melanogaster ab3B. To discriminate ab3 from ab2 sensilla, the ab2A activator ethyl acetate (CAS: 141-78-6) was included as a negative control. To test absence in the ab3A neuron of the wild-type expression of the D. melanogaster OR22a/b-subunits, ethyl hexanoate (CAS 123-66-0) was included as an additional negative control.

To screen the panel, all odorants were diluted in hexane (Sigma Aldrich, St. Louis, MO-USA) at 1.0 μg/μL. Stimuli were prepared by applying 10.0 μL of each dilution on grade 1 - 20 mm circles filter paper (GE Healthcare Life Science, Little Chalfont, United Kingdom), previously inserted into glass Pasteur pipettes (VWR, Milan, Italy), for a total amount of 10.0 μg of compound per stimulus. To minimize possible effects from the solvent, pipettes were left at least 10 minutes after preparation under the fume hood for solvent evaporation. Puffing provided an additional 2.5 mL of air through the pipette for 0.5 seconds by inserting the pipette within a side hole of the glass tube, directing the humidified air flow to the antennae. To characterize the intensity of the response, spike frequency was calculated as in Lebreton et al. (2017) by subtracting ab3A spikes counted for 0.5 seconds before the stimulus from the number of spikes counted for 0.5 seconds after the stimulus to calculate spike frequency in terms of ∆spikes/0.5sec. Responses to compounds of the panel were compared for five replicates, using a single insect as a replicate. Before validating significant differences in spike counting, tests of normality with the IBM SPSS Statistics software 29.0 (https://www.ibm.com/) unveiled that for some ligands, data were not normally distributed (Kolmogorov-Smirnova/Shapiro-Wilk test p<0.05, Supplementary Data File 3). Using the same software, spike frequencies of each compound were compared with respective values from the solvent (hexane) by the non-parametric Wilcoxon Signed Rank Test (p<0.05). For box-plot analysis, ∆spikes/0.5sec of each recording was normalized to the averaged ab3A firing rate for the specific insect replicate, as done in our previous studies(Cattaneo et al. 2023).

Gas chromatography coupled with single sensillum recordings (GC-SSR)

GC-SSR was performed with the same GC equipment in our labs that interfaced with the SSR rig we used in our previous investigation (Cattaneo et al. 2023). In brief, samples were injected on a 7890 GC-system (Agilent Technologies Inc., Santa Clara, CA, USA) provided with a 30 m x 0.32 mm fused silica capillary column (Agilent Technologies Inc.), coated with HP-5, df = 0.25 µm, programmed from 30°C (hold 3 min) at 8°C/min to 250°C (hold 5 min) (software: GC-SSR-1 - Agilent.OpenLab, Agilent Technologies). The outlet split from the GC column was a 1:1 ratio between the flame ionization detector and the mounted antenna, according to instrument settings. A humidified flow of 3.5-4.0 L/min charcoal-filtered air was directed into a 90-degree-angled glass tube with a hole on the angle where part of the column exiting from the transfer line was accessed. Glass-tubing was adjusted to a length of 17 cm, and ab3 sensilla was tested following the same optimization to 1.0 nanogram of active compound that we have adopted in Cattaneo et al (2023). The recording window was set to 35 min upon preliminary observation of retention times for the injected compounds. By GC-SSR we tested 1.0 ng and 10.0 ng aliquots of nonanal and (Z)-6-undecenal that we have chosen from our SSR-screening based on their effects (Table 1). Compounds were diluted in hexane between 0.001 and 0.010 μg/μL depending on the experiment condition, injecting 2.0 μL dilutions into the gas-chromatograph. Parallel experiments tested volatile collections from apple headspace (Hoplomalus and Malus) already available in our labs. To test headspace collections by GC-SSR, aliquots of 4.0 μL were injected into the gas chromatograph.

The headspace components that invoked responses were validated by recording in the GC-SSR using a DB-wax column. In brief, 2.0 μL of headspace was injected in the GC (7890) with an injector temperature of 225°C in splitless mode. The GC was fitted with a silica capillary column coated with DB-wax column (Agilent Technologies Inc., df = 0.25 μm) and temperature programmed from 30°C (hold time 3 min) until 225 °C (hold time 8 min) at 8 °C/min.

Hydrogen gas was used as a mobile phase at 7.5 mL/min. The GC effluent was carried onto the antennal preparation through a Gerstel ODP-2 transfer line connected to a glass tubing as described above. Retention times associated with neuronal activation were collected from the chromatograms exported from Chemstation B.03.02 (Agilent Technologies, Santa Clara, CA-USA) or from Autospike. To estimate neuronal activation, spikes were counted within 5 seconds from the emission of their respective GC peaks or the beginning of the ab3A effect, depending on the case. These counted spike numbers were subtracted from spikes five seconds antecedent to the effect and divided by 5 to calculate ∆spikes/second. Statistical analysis was performed as described above, including tests of normality (Supplementary Data File 3).

GC-MS analysis of active headspaces

The volatile samples were injected on a GC-MS (Agilent technologies, 7890B GC coupled with 5975 MSD) equipped with a DB-WAX capillary column (60 m x 250 μm x 0.25 μm). A volume of 2.0 μL samples were injected into the splitless injection port at 225°C in splitless mode. The carrier gas was helium, and the total column flow was 34.883 mL/min. The temperature program of the oven started at 30°C, which was held for 3 minutes and heated up at the rate of 8°C/min to 225°C, holding for 10 minutes the final temperature. To perform GC-MS, we used the mass spectrometer in electron ionization mode at 70 eV, and the detector scanned in the 29-400 mass-to-charge range.

The volatile samples were also injected on a GC-MS (Agilent technologies, 6890 GC coupled with 5977A MSD) equipped with an HP-5 capillary column (column: 60 m x 250 μm x 0.25 μm). The column, inlet, and mass spectrometer settings were the same as those described above. The oven temperature program started at 50°C, it was held for 2 minutes, and increased to 250°C at the rate of 8°C/min, holding the final temperature for 10 minutes.

GC-MS data were analyzed using Agilent Mass Hunter B.08.00. The area counts were calculated using manual integration. The volatile components were tentatively identified by comparing the experimental mass spectra to those found in MS Libraries (NIST11 and Wiley12) using the Nist MS Search v. 2.4 program. The spectrum-based identification was verified by calculating the Kovats retention indices (RI) of components and comparing those to Kovats retention indices found in the NIST WebBook (https://webbook.nist.gov/) or PubChem databases.

Cloning and heterologous expression of CpomOR22 in oocytes from Xenopus laevis

To further investigate the responsiveness of CpomOR22 to environmental volatiles, we expressed CpomOR22 in conjunction with the obligate coreceptor, C. pomonella Orco (CpomOrco), in unfertilized, defolliculated X. laevis oocytes. The main objectives of the Xenopus experiments were to validate positive findings from the Drosophila empty neuron system and expand the odor space/set of odorants used to test OR22, beyond those used in the SSR and GC-SSR experiments.

Using the two-electrode voltage clamp technique, we recorded responses of the odorant receptor complex, observed by the change in inward current, to 16 different odorant blends. These blends together contain 138 compounds, including 15 that were screened in Drosophila, grouped by chemical classes based on structure (Supplementary Table 2). The reason we have chosen this method was to make use of parallel heterologous systems, allowing us to test a broader range of ligands within blends. Among these ligands, we kept nonanal as a reference, based on our observed SSR responses from this ligand being one the most active, which was also validated in the headspace from apple. The individual compounds, each at a concentration of 10^-4 M in the blends, were applied to the oocyte with buffer perfusion.

CpomOrco and CpomOR22 templates were synthesized by Twist Bioscience (South San Francisco, CA, USA) and delivered in pENTR vector, and then subcloned into the X. laevis compatible destination vector pSP64t (Gateway™ LR Clonase™ II Enzyme Mix, Invitrogen Corp., Carlsbad, CA, USA). Plasmids were purified using GeneJET Plasmid Miniprep Kit (ThermoFisher Scientific, Waltham, MA, USA) and verified using bidirectional Sanger sequencing (Psomagen, Rockville, MD, USA). 5,000 ng of each plasmid was linearized using XbaI (FastDigest XbaI, ThermoFisher Scientific, Waltham, MA, USA). The linearized plasmids were purified (GeneJET Gel Extraction Kit, ThermoFisher Scientific) and checked for concentration using the Nanodrop (NanoDrop™ One UV-Vis Spectrophotometer, Witec AG, Sursee, Switzerland). cRNA was synthesized from the linearized plasmids (mMESSAGE mMACHINE™ SP6 Transcription Kit, ThemoFisher Scientific, Waltham, MA, USA) and incubated at 37°C for 12 hours. 1.0 uL of RNase inhibitor was added to each reaction (RNaseOUT™ Recombinant Ribonuclease Inhibitor, ThermoFisher Scientific, Waltham, MA, USA). Synthesis was verified using gel electrophoresis, and concentration was checked using the Nanodrop (ThermoFisher Scientific).

Stage V-VII X. laevis defolliculated oocytes were ordered from Xenopus1 (Dexter, MI, USA) and incubated in ND96 incubation media (96 mM NaCl, 2 mM KCl, 5 mM HEPES, 1.8 mM CaCl₂, 1 mM MgCl₂, pH 7.6) augmented with 5% horse serum (ThermoFisher Scientific, Waltham, MA, USA), 50 μg/mL tetracycline, 100 μg/mL streptomycin, 100 μg/mL penicillin, and 550 μg/mL sodium pyruvate. Oocytes were injected with 30 nL of RNA (30 ng of each cRNA) using the Nanoliter 2010 injector (World Precision Instruments, Inc., Sarasota, FL, USA). The resting membrane potential and odorant-induced changes of oocytes expressing odorant receptor cRNAs were recorded 72 hours post-injection using the 2-microelectrode voltage-clamp technique (TEVC). The OC-725C oocyte clamp (Warner Instruments, LLC, Hamden, CT, USA) held a −80 mV holding potential. The effect of the blends (Supplementary Data File 4) on odorant receptor complexes was determined by perfusing 10⁻⁴ M concentration blends across individual oocytes. The membrane current was permitted to return to baseline between blend introductions. Data was recorded with the Digidata 1550 B digitizer and pCLAMP10 software (Molecular Devices, Sunnyvale, CA, USA). Raw data was collected and normalized according to the blend or compound producing the greatest response (Supplementary Data 3). Following initial screenings, unitary compounds comprising activating blends were perfused to establish tuning curves. Concentration responses for gamma-undecalactone and undecanal were determined by challenging oocytes with half-log concentrations between 10⁻⁷ M and 10⁻⁴ M for 10 s. The current was allowed to return to baseline between compound administrations. Blends and unitary compounds were perfused on 8-10 oocytes per trial. GraphPad Prism 8 (GraphPad Software, Inc., La Jolla, CA, USA) was utilized for data analyses.

Expression analysis of CpomOR22 in C. pomonella

Our qRT-PCR study confirmed the previously non-replicated transcriptomic studies that indicated female bias for OR22, named OR15 in Bengtsson et al.(2012), and afterward changed to its current nomenclature in Walker et al.(2016). CpomOR22 was expressed in female antennae at a 44.28-fold higher level than male antennae (p = 0.0019; Fig. 1, Supplemental Data File 1). As a point of comparison, the odorant receptor coreceptor (CpomOrco) was observed to be expressed in female antennae at a 1.61-fold lower level than in male antennae without a statistically significant difference (p = 0.078).

Functional characterization of CpomOR22 in transgenic Drosophila

Testing a panel of possible ligands based on previous knowledge of compounds active on ORs/OSNs of C. pomonella (Table 1), SSR screening unveiled the aldehydes nonanal, (Z)-4-undecenal and (Z)-6-undecenal as the most active (Fig. 2A). In parallel, the Wilcoxon Signed Rank Test revealed slight activating differences from the effect of the solvent when testing other compounds, including ethyl-(E,Z)-2,4-decadienoate and the corresponding E, E-isomeric aldehyde, (E,E)-2,4-decadienal (7.40 ± 4.46 and 14.60 ± 3.78 spikes/0.5sec respectively). Conversely, the sesquiterpenoid alcohol (E,E)-α-farnesol (-0.1 ± 0.52 spikes/0.5sec) and its corresponding sesquiterpene (E,E)-α-farnesene (-0.1 ± 0.43 spikes/0.5sec) were associated with an apparent decrement in the spiking effect, being most likely inhibitory (Table 1).

To test the validity of the response to the aldehydes, we injected GC-SSR with the minimal dosages expected from the sensitivity of our optimized equipment ([1.0-10.0] ng (Cattaneo et al. 2023)). For both the saturated (nonanal) and the most active unsaturated [(Z)-6-undecenal] aldehydes, for both dosages tested, an increment of associated neuronal spike frequency was observed (Fig. 2B).

C. pomonella OR22 responds to gamma-undecalactone and undecanal in the Xenopus oocyte system.

Oocytes expressing the combination of CpomOR22 & CpomOrco were stimulated with blends of compounds, organized by chemical class, to increase the number of compounds tested (Supplementary Table 2). Oocytes responded best to a blend of Lactones, while also responding to blends of Aldehydes and Carboxylic Acids (Fig. 3A,B). Subsequently, we tested each component from the three blends that produced the highest receptor complex activation. Individual compounds that elicited the highest activation were undecanal and decanal from the Aldehyde blend, gamma-undecalactone and delta-dodecalactone from the Lactone blend, and undecanoic and decanoic acids from the Carboxylic Acid blend #2 (Fig. 3C). It is interesting to note that Lactones produced a higher normalized response in blends than aldehydes, yet individual aldehydes, e.g., undecanal and decanal, elicited higher normalized responses than lactones (Fig. 3B,C). One possible explanation for this apparent contradiction is that lower efficacy aldehydes in the blend, such as heptanal and octanol, compete for the same ligand binding site in CpomOR22 as undecanal and decanal, thus reducing the overall response magnitude in the context of the blend (Rospars et al. 2008, Singh et al. 2019). This blend effect may be less pronounced in the lactones. The similar carbon chain lengths of the most efficacious compounds from each blend suggest a conserved activation mechanism. Next, we examined CpomOR22 dose responsiveness to undecanal and gamma-undecalactone across five orders of magnitude and calculated the effective concentrations at the half-maximal response, EC₅₀, which were 16.7 μM 8.4 μM, respectively (Fig. 3D).

Identification of active ligands from apple headspace

The Hoplomalus and Malus volatile extracts were injected on both polar (DB-WAX) and non-polar (HP-5) capillary columns and in both cases, four components elicited consistent electrophysiological responses. The calculated Kováts retention indices of these components were 1393, 1498, 1606 and 1713 using the DB-WAX capillary column and 1105, 1206, 1307 and 1409 using HP-5 capillary column. We have analyzed the volatile extracts using GC-MS on both polar (DB-WAX) and non-polar (HP-5) capillary columns and identified active components based on their mass spectra and Kovats indices, which represented four saturated aldehydes: nonanal [DB-wax: RT: ~11.50 min; kovats ~1395; HP-5: RT: ~11.48 min; kovats ~1105], decanal [DB-wax: RT: ~13,15 min; kovats ~1499; HP-5: RT: ~13,32 min; kovats ~1207], undecanal [DB-wax: RT: ~14.78 min; kovats ~1608; HP-5: RT: ~15.04 min; kovats ~1308] and dodecanal [DB-wax: RT: ~16.30 min; kovats ~1716; HP-5: RT: ~16.66 min; kovats ~1410] (Fig. 2CD, Fig. 4). In addition, one component that was only visible on the non-polar (HP-5) column with retention time proximal to 11.66 min (Kováts retention index 1114) was also active on CpomOR22 (6,15 ± 1,21 spikes/sec), but it remained unidentified due to low peak abundance and coelution of multiple peaks (Fig. 2 C, D; Table 2).

Among the saturated aldehydes, decanal was the most active (25.87± 4.66 spikes/sec), followed by dodecanal and undecanal, with similar values (16.18 ± 3.77 spikes/sec and 16.10 ± 3.55 spikes/sec respectively) and nonanal (14.28 ± 2.89 spikes/sec). Performing a Wilcoxon Signed Rank Test comparison with the effect elicited by the solvent demonstrated all these ligands were significantly active when they reached D. melanogaster ab3A neurons expressing CpomOR22, with the lowest p-value for dodecanal (p = 0.002).

While much focus has deservedly been placed on studying the function of male-specific or male-biased candidate PRs of Lepidoptera, more attention should be given to the female-specific and female-biased ORs. In this study, we isolated, heterologously expressed, and functionally characterized CpomOR22, previously reported as a candidate pheromone receptor and having a clear female-antenna expression bias(Walker et al. 2016). Functional characterization performed using SSR and GC-SSR on transgenic D. melanogaster demonstrated binding of this OR to a few ligands, including aldehydes emitted from apple headspace. An in vitro approach using oocytes from Xenopus confirmed binding to these aldehydes and added evidence of responses to lactone structural analogues.

Our study demonstrates binding of the candidate pheromone receptor CpomOR22 to both unsaturated and saturated aldehydes and it adds to the knowledge of volatile compounds known for being emitted by hosts and active on the olfactory chemoreceptor systems of C. pomonella, such as pear ester ethyl-(E,Z)-2,4-decadienoate (Bengtsson et al. 2014, Cattaneo et al. 2017) (Fig. 2A).

While we recognize the importance to verify the impact on live-insects of ligands, such as apple-emitted aldehydes, that we have found active on CpomOR22, behavioral experiments are beyond the scope of this project at the present time. Moreover, previous chemical ecology research on Cydia pomonella has already investigated some of these aspects by demonstrating the role of aldehydes on live moths. Indeed, aldehydes like nonanal and decanal, have been part of comprehensive electrophysiological and behavioral investigations on C. pomonella, previously reported to generate maximal electroantennal responses in both sexes of this insect (Casado et al. 2006). Furthermore, nonanal was shown to increase oviposition in behavioral bioassays comparing synthetic plant volatiles to green apples(Witzgall et al. 2005). Other studies, however, using olfactometers with two selection Y-tube assays, identified nonanal among the repellent odors, the repellency of which was overcome when this compound was part of a more complex mixture of apple volatiles(Vallat et al. 2005). Conversely, the addition of aldehydes to pear ester did not increase the number of codling moth trap catches, for neither males or females, in walnut and apple orchards (Light and Knight 2005). Collectively, these studies highlight the complexities of insect chemical ecology as it relates to single compounds versus blends, and disparate roles in mediating different behaviors. Apart from their effects on adult codling moths, aldehydes are also among the minor components of the bouquet of larval aggregation pheromones (Jumean et al. 2005a). Beyond codling moth, aldehydes are also part of a wide range of host semiochemicals that stimulate attraction to hosts and oviposition for gravid females of various insects from different taxa (Gonzalez 2007).

We recognize that in SSR-experiments, responses to pear ester are weaker than the responses to aldehydes for CpomOR22 (Fig. 2A). However, it deserves to be noted that the effects enhanced by this ligand are significantly different than spiking from the solvent (raw spiking 7,40 ± 4,46 spikes/0.5sec; p = 0,043, Table 1). Interestingly, our SSR analysis unveiled inhibitory effects of the sesquiterpene (E,E)-α-farnesene and its analogue alcohol, (E,E)-α-farnesol (-0.10 ± 0.42 and -0.10 ± 0.52 spikes/0.5sec, p = 0.043; Supplementary Data File 3). (E,E)-α-farnesene is the most abundant compound in apple headspace and it is among the most potent elicitors of antennal and behavioral responses of codling moth females (Wearing et al. 1973, Light et al. 2001, Ansebo et al. 2004, Coracini et al. 2003). Conversely, (E,E)-α-farnesol, which has been identified only in trace quantities from Granny Smith apples (Matich et al. 1996), has been reported to enhance the upwind flight of codling moth males (Ansebo et al. 2005, Coracini et al. 2003), consistent with findings that specific neuronal receptors responding to this ligand have been found only on male antennae (Ansebo et al. 2005). Collectively, these studies, together with our findings on CpomOR22, suggest that other antennal chemoreceptors of C. pomonella may be involved in sensing (E,E)-α-farnesene, while our evidence of OR22-inhibition by (E,E)-α-farnesol is in accordance with the absence of the reported evidence of its attractivity for females.

Among the unsaturated aldehydes, we observed significant effects on CpomOR22 from (E,E)-2,4-decadienal (p = 0.043, Supplementary Data File 3), despite not being outstanding (raw spiking 14.60 ± 3,78 spikes/0.5sec, p = 0,043; Table 1). Together with pear ester, this compound was reported among the ligands enhancing the highest electrophysiological responses when tested on female antennae, while males were repelled when (E,E)-2,4-decadienal was added in blends with other active compounds (Ansebo et al. 2004). The same study also revealed that (Z)-3-hexenol and (R/S)-linalool enhanced strong responses in female antennae. However, when tested by SSR on D. melanogaster ab3A neurons expressing CpomOR22, neither of these compounds elicited a significant response (p>0.05, Supplementary Data File 3). Most likely, other CpomORs are involved in binding these ligands, and further heterologous studies are needed to search for candidates aimed at decrypting female-related mechanisms to detect the main apple-emitted semiochemicals. However, despite that averages of normalized responses to (E,E)-2,4-decadienal and pear ester are negligible when compared to the effects from the other aldehydes that we have tested, our reported CpomOR22 activation by these ligands is interesting because of their common effects on both males and females (Ansebo et al. 2004, 2005, Coracini et al. 2003). Indeed, while pear ester is attractive for both sexes of the codling moth, and has been used for decades primarily for main monitoring programs (Light et al. 2001), (E,E)-2,4-decadienal is electroantennographically active on females and behaviorally repulsive on males, despite that behavioral effects on females have yet to be tested (Ansebo et al. 2004). In addition, both ligands are renowned semiochemicals capable of interfering with the chemosensory systems of other insects, belonging or not to the order of Lepidoptera (Anfora et al. 2014, Wenda-Piesik et al. 2018). Evidence of CpomOR22 activation by ethyl-(E,Z)-2,4-decadienoate and (E,E)-2,4-decadienal may add to future functional characterization efforts for orthologues of this receptor from other species.

As mentioned, unsaturated aldehydes like (Z)-4-undecenal showed a more evident effect (Fig. 2 A, B). (Z)-4-undecenal is renowned among the aldehydes emitted from the autoxidation of Drosophila's cuticular hydrocarbons, and it has been part of our previous pharmacological investigations on the OR69a-subunits of D. melanogaster (Lebreton et al. 2017)and Drosophila suzukii (Cattaneo et al. 2023). While the activation of CpomOR22 as a candidate pheromone receptor with a female bias (Walker et al. 2016), by aldehydes like (Z)-4-undecenal is intriguing, no ecological roles for (Z)-4-undecenal are yet known in association with codling moth. CpomOR22 activation by this ligand may result from a mere pharmacological effect due to its structural similarity with other aldehydes. Indeed, aldehydes are volatile compounds emitted by diverse sources, including plants, animals, microbes, and raw meat (Cui et al. 2023). Our experimental records demonstrated CpomOR22 versatility in response to various saturated and unsaturated aldehydes, including undecanal (Fig. 2D). Although hypothetically, unsaturated aldehydes from also nonanal, decanal, and dodecanal may represent possible ligands for CpomOR22, and future projects may attempt further deorphanization assays to resolve this. For instance, the unsaturated (E)-2-nonenal has been found to elicit similar behavioral responses as nonanal in larvae of C. pomonella (Jumean et al. 2005a); 2-decenal has been found in the diethyl-ether apple frass extract from caterpillars of C. pomonella, which was found to be attractive for the parasitic wasp Hyssopus pallidus (Gandolfi et al. 2003); the codlemone aldehyde (E, E)-8-10-dodecadienal is part of the female odor bouquet, and it was reported to attract males to field traps (Greenway 1984, Witzgall et al. 1993). The evidence in this report of the response to aldehydes by CpomOR22 opens the door to future experiments validating binding of this sensor to the codlemone aldehyde. Despite being hypothetical, such binding would indicate the role of CpomOR22 in female aggregation, or in facilitating host finding. We previously examined this concept in the context of activation of another C. pomonella pheromone receptor by other odorants that are part of the bouquet emitted by codling moth females (Cattaneo et al. 2017).

In addition to our main findings (Fig. 2 A, B), we conducted supplementary experiments testing effects from the same transgenic Drosophila with doses of (Z)-6-undecenal ranging from 1 ng to 150 μg. We observed CpomOR22-associated SSR spiking to (Z)-6-undecenal with a sensitivity of 2.903 ± 1.709 µg (EC50), Fmax of 0.8772 ± 0.1129, Hill coefficient of 0.8466 ± 0.3356 and a saturation at 50 µg dose (Supplementary Figure 1, Supplementary Table 3). Interestingly, together with nonanal-to-dodecanal, 6-undecenal has been found among the main constituents from the essential oils of coriander (Coriandrum sativum), though it was not specified if it was present either in the E or the Z isomer (Parthasarathy et al. 2008, Rajeshwari and Andallu 2011). Although limited research has been conducted on this topic, recent experiments testing essential oils of coriander demonstrated their repulsive effects on Tricogrammatiddae cacoeciae (Cointe 2020), which is a renowned egg parasitoid of C. pomonella (Mansour 2019). Other studies also reported that nonanal and decanal are required in a minimal blend to attract another pupal parasitoid of the codling moth, Mastrus ridibundus (Jumean et al. 2005b).

Although behavioral studies are necessary to demonstrate whether the aldehydes that we have found active on CpomOR22 are attractants or repellents for codling moth females and the natural enemies of this insect, evidence of their emission by host apple fruits and their reported identification in coriander is compelling. If on one side we may hypothesize that the identified aldehyde kairomone active on CpomOR22 may direct codling moth females on apples or being somehow responsible for the insects’ aggregation, the same ligands may also modulate egg or pupal parasitization of C. pomonella being either repulsive or (possibly) attractive on the natural enemies of this insect. Indeed, in their experiments, Gandolfi et al. (2003) demonstrated that H. pallidus parasitoids are attracted to both the frass extracts from larvae fed with apple food and the same apples’ extracts, validating that both samples shared content of four main aldehydes that are commonly emitted from apple (2-heptenal, nonanal, decanal, 2,4-decadienal). Among these aldehydes, nonanal, decanal and 2,4-decadienal are active on CpomOR22 (Fig. 2). To test whether these aldehydes may be sensed by the parasitoids of C. pomonella, electrophysiological and behavioral trials are needed on these insects to investigate their chemosensory systems. This would unveil the possible, though speculative, roles of aldehyde ligands in more complex ecological interactions.

The reported ecological complexity behind the interaction of aldehydes with the codling moth may involve additional mechanisms for their detection. While we cannot exclude their activation of other chemosensors with an antennal male bias, which may be part of future projects, in a recent investigation, we have decrypted the chemosensory receptors repertoire of the female abdomen tip of the codling moth (Walker et al. 2023). Conducting an RT-PCR analysis from RNA extracts of the ovipositor and pheromone glands, we have identified traces of CpomOR22, suggesting possible roles for this receptor in guiding oviposition behaviors or having possible roles in courtship (Roscoe et al. 2016), though to our knowledge, no male courtship pheromones have been identified for C. pomonella.

Aside from our findings on nonanal and decanal, to the best of our knowledge, ours is the first study reporting activation of the C. pomonella olfactory system by undecanal and dodecanal (Fig. 2 D). Undecanal and dodecanal together with nonanal and decanal have been shown to be emitted by both infested and non-infested apples (Hern and Dorn 2002) (Fig. 2 D, Table 2) Given that essential oils from coriander, which contain these two aldehydes among the others, are repellent to egg-parasitoids of the codling moth (Cointe 2020, Rajeshwari and Andallu 2011), evidence of the CpomOR22-sensing of undecanal and dodecanal suggests broader ecological importance for parasitoid interactions with C. pomonella that deserves further investigation. Furthermore, in the apple headspace that we have analyzed, we have identified another activating ligand represented by a retention time proximal to 11.60 min (Kovats Retention Index 1114) in the HP-5 column, which, by GC-MS analysis, we were not able to characterize (Fig. 2 CD, Table 2). When conducting GC-SSR experiments setting the gas-chromatograph with a DB-wax column, this ligand was not released (Table 2), possibly because of polar incompatibility, as discussed in our recent investigation performing similar trials with the HP-5 column (Cattaneo et al. 2023). On the other side, it might be as well that this compound was present as a contaminant rather within the HP-5 column or within the inlet of the GC. Independent of any possible scenario, since activation of CpomOR22 by this unknown ligand is compelling and may hide additional aspects of the C. pomonella molecular ecology, possible trials to attempt its characterization are warranted and may lead to a better understanding of the ecological importance of the broad tuning we have so far demonstrated for this C. pomonella subunit.

Using the Xenopus laevis heterologous expression system, we confirmed CpomOR22 response to nonanal, decanal, and undecanal. Among other tested ligands, from lactones to carboxylic acids, we observed higher responses to γ-undecalactone. Like undecanal, γ-undecalactone is an 11-carbon compound that is found in fruit and fruit product odors (Pérez-Olivero et al. 2014, Liu 2022, Lasekan et al. 2018, Hijaz et al. 2016)and it has previously been identified as a sensory receptor agonist (Tobita et al. 2021, Bezerra-Silva et al. 2016), as well as an arthropod behavioral regulator(Phelan et al. 1986, Weeks et al. 2020, Leyrer and Monroe 1973). Although it is also possible that the response of CpomOR22 to γ-undecalactone may be related to the similarity of its structure with the carbon chain of the undecanal and the presence of a double bond to oxygen (Fig. 3) instead of any ecological relevance for codling moth.

Using SSR and GC-SSR recordings, this project deorphanized a candidate pheromone receptor of the codling moth that we heterologously expressed in vivo using empty neurons of transgenic Drosophila. Combining oocytes from Xenopus, we demonstrated the binding of the identical ligands in vitro and unveiled activation by γ-undecalactone. The subsequent identification of lactones as ligands for CpomOR22 deserves further investigation: additional research may shed light on whether these lactones would merely represent structural analogues to the ecologically relevant aldehydes or if they are novel semiochemicals not previously identified among the extensive body of research done on codling moth chemical ecology. In case of the latter, identification of sources for their emission would become necessary. Although it must be emphasized that the lactones we have found active may merely represent structural analogues, given their common absence from apple volatile headspace (Bengtsson et al. 2002). The potential of structural chemistry/analogs in establishing novel ligands was reported in other species (Douglass et al. 1993, Bengtsson et al. 1990, Gonzalez et al. 2015). Given the evidence that the aldehyde and lactone from our studies share a structural backbone and both activate CpomOR22, a structural analysis investigation may help prediction for novel ligands in the phase of studying insect chemoreceptors, as it has recently been demonstrated for other ORs from C. pomonella (Gonzalez et al. 2015) and from another moth, S. littoralis (de Fouchier et al. 2017, Caballero-Vidal et al. 2021).

Despite our working hypothesis based on the phylogenetic analysis investigated CpomOR22 as a candidate pheromone receptor (Walker et al. 2016), evidence from our study suggested this subunit as a typical female-biased expressed OR, apparently not binding to sex pheromones (Figure 2A, Table 1), but rather responding to various apple volatile kairomones. The potential ecological relevance of these kairomones for the codling moth requires future investigations to validate their influence on the behavior of C. pomonella and their potential as semiochemicals for integrated push-pull strategies.

Used

Ethics statement

Experiments from this study on D. melanogaster were performed in compliance with European law and the SLU institutional guidelines. These animals are insects from the genus Drosophila, and there are no ethical issues or particular restrictions requiring committee-based approval to conduct experiments.

Funding Sources

SSR/GC-SSR studies were performed within the frame of the FORMAS Swedish Research Council project number 2016-01281, title: “Control of apple pest insects with fruit and yeast odorants" and the homonymous Crafoordska Stiftelsen project number 20180954. Part of running costs have been covered by the Lars Hiertas Minne Grant N. FO2018-0209, title: “Control of apple pest insects with fruit- and yeast-odorants”. Parts of this study were also supported by The Swedish Research Council Formas, project numbers 2021-00834 and 2018-00891.

Author Contribution Statement

Conceptualization, W.B.W.III. and A.M.C.; Methodology, A.M.C., R.J.P., and W.B.W.III,; Validation, A.M.C., C.A.K., H.L.-P., A.L.E., R.J.P., and W.B.W.III.; Formal Analysis, A.M.C., C.A.K., H.L-P., A.L.E., R.J.P, and W.B.W.III.; Investigation, A.M.C., C.A.K., H.L-P., and W.B.W.III.; Resources, W.B.W.III and R.J.P.; Data Curation, A.M.C., C.A.K., and W.B.W.III; Writing—Original Draft Preparation, A.M.C., C.A.K., H.L-P., A.L.E., and W.B.W.III.; Writing—Review & Editing, AMC, CAK, H.L-P., A.L.E., R.J.P., and W.B.W.III.; Visualization, A.M.C., H.L-P., and W.B.W.III.; Supervision, W.B.W.III and R.J.P; Project Administration, W.B.W.III; Funding Acquisition, W.B.W.III and R.J.P. All authors have read and agreed to the published version of the manuscript.

Anderson AR, Wanner KW, Trowell SC, Warr CG, Jaquin-Joly E, Zagatti P, Robertson H, Newcomb RD (2009) Molecular basis of female-specific odorant responses in Bombyx mori. Insect Biochem Mol Biol 39(3):189-197. https://doi.org/10.1016/j.ibmb.2008.11.002
Anfora G, Vitagliano S, Larsson MC, Witzgall P, Tasin M, Germinara GS, De Cristofaro A (2014) Disruption of Phthorimaea operculella (Lepidoptera: Gelechiidae) oviposition by the application of host plant volatiles. Pest Manag Sci 70(4):628-635. https://doi.org/10.1002/ps.3597
Ansebo L, Coracini MDA, Bengtsson M, Liblikas I, Ramírez M, Borg-Karlson AK, Tasin M, Witzgall P (2004) Antennal and behavioural response of codling moth Cydia pomonella to plant volatiles. J Appl Entomol 128(7):488-493. https://doi.org/10.1111/j.1439-0418.2004.00878.x
Ansebo L, Ignell R, Löfqvist I, Hansson BS (2005) Responses to sex pheromone and plant odours by olfactory receptor neurons housed in sensilla auricillica of the codling moth, Cydia pomonella (Lepidoptera: Tortricidae). J Insect Physiol 51(10):1066-1074. https://doi.org/10.1016/j.jinsphys.2005.05.003
Bastin-Heline L, de Fouchier A, Cao S, Koutroumpa F, Caballero-Vidal G, Robakiewicz S, Monsempes C, Francois MC, Ribeyre T, Maria A, Cheremps T, de Cian A, Walker III WB, Wang G, Jacquin-Joly E, Montagné N (2019) A novel lineage of candidate pheromone receptors for sex communication in moths. Elife 8:e49826. https://doi.org/10.7554/eLife.49826
Bengtsson JM, Trona F, Montagné N, Anfora G, Ignell R, Witzgall P, Jacquin-Joly E (2012) Putative chemosensory receptors of the codling moth, Cydia pomonella, identified by antennal transcriptome analysis. PLoS One 2012, 7(2): e31620. https://doi.org/10.1371/journal.pone.0031620
Bengtsson, JM, Gonzalez F, Cattaneo AM, Montagné N, Walker III WB, Bengtsson M, Anfora G, Ignell R, Jacquin-Joly E, Witzgall P (2014) A predicted sex pheromone receptor of codling moth Cydia pomonella detects the plant volatile pear ester. Front Ecol Evol 2:33. https://doi.org/10.3389/fevo.2014.00033
Bengtsson M, Liljefors T, Hansson BS, Löfstedt C, Copaja SV (1990) Structure-activity relationships for chain-shortened analogs of (Z)-5-decenyl acetate, a pheromone component of the turnip moth, Agrotis segetum. J Chem Ecol 16:667-684. https://doi.org/10.1007/BF01016478
Bengtsson M, Bäckman AC, Liblikas I, Ramirez MI, Borg-Karlson AK, Ansebo L, Anderson P, Löfqvist J, Witzgall P (2001) Plant odor analysis of apple: antennal response of codling moth females to apple volatiles during phenological development. J Agric Food Chem 49(8):3736–3741. https://doi.org/10.1021/jf0100548
Bezerra-Silva PC, Dutra KA, Santos GKN, Silva RCS, Iulek J, Milet-Pinheiro P, Navarro DMAF (2016). Evaluation of the activity of the essential oil from an ornamental flower against Aedes aegypti: electrophysiology, molecular dynamics and behavioral assays. PLoS One 11(2):e0150008. https://doi.org/10.1371/journal.pone.0150008
Butenandt A, Beckmann R, Stamm D, Hecker E (1959) Uber den Sexuall-ockstoff des seidenspinners Bombyx mori. Reindarstellung Und Konstitution. Z Naturforschg 14:283.
Bäckman AC, Anderson P, Bengtsson M, Löfqvist J, Unelius CR, Witzgall P (2000) Antennal response of codling moth males, Cydia pomonella L. (Lepidoptera: Tortricidae), to the geometric isomers of codlemone and codlemone acetate. J Comp Physiol A 186(6):513–519. https://doi.org/10.1007/s003590000101
Caballero-Vidal G, Bouysset C, Gévar J, Mbouzid H, Nara C, Delaroche J, Golebiowski J, Montagné N, Fiorucci S, Jacquin-Joly E (2021) Reverse chemical ecology in a moth: machine learning on odorant receptors identifies new behaviorally active agonists. Cell Mol Life Sci 78(19-20):6593–6603. https://doi.org/10.1007/s00018-021-03919-2
Casado D, Gemeno C, Avilla J, Riba M (2006) Day-night and phenological variation of apple tree volatiles and electroantennogram responses in Cydia pomonella (Lepidoptera: Tortricidae). Environ Entomol 35(2):258–267. https://doi.org/10.1603/0046-225X-35.2.258
Carde RT, Minks AK (1995) Control of moth pests by mating disruption: successes and constraints. Annu Rev Entomol 40(1):559-585. https://doi.org/10.1146/annurev.en.40.010195.003015
Cattaneo AM (2018) Current status on the functional characterization of chemosensory receptors of Cydia pomonella (Lepidoptera: Tortricidae). Front Behav Neurosci 12:189. https://doi.org/10.3389/fnbeh.2018.00189
Cattaneo AM, Gonzalez F, Bengtsson JM, Corey EA, Jacquin-Joly E, Montagné N, Salvagnin U, Walker III WB, Witzgall P, Anfora G, Bobkov YV (2017) Candidate pheromone receptors of codling moth Cydia pomonella respond to pheromones and kairomones. Sci Rep 7(1):41105. https://doi.org/10.1038/srep41105 (2017).
Cattaneo AM, Witzgall P, Kwadha CA, Becher PG, Walker III WB (2023) Heterologous expression and functional characterization of Drosophila suzukii OR69a transcript variants unveiled response to kairomones and to a candidate pheromone. J Pest Sci 96(3):1149–1171. https://doi.org/10.1007/s10340-022-01585-2
Charmillot PJ, Hofer D, Pasquier D (2000) Attract and kill: a new method for control of the codling moth Cydia pomonella. Entomol Exp Appl 94(2):211-216. https://doi.org/10.1046/j.1570-7458.2000.00621.x
Cointe M (2020) Unintentional effects of biopesticides on biocontrol agents. Life Sciences [q-bio] dumas-02986694. https://dumas.ccsd.cnrs.fr/dumas-02986694/document. Accessed 02 October 2024
Coracini M, Bengtsson M, Liblikas I, Witzgall P (2003) Attraction of codling moth males to apple volatiles. Entomol Exp Appl 110(1):1–10. https://doi.org/10.1111/j.0013-8703.2004.00124.x
Corcoran JA, Jordan MD, Thrimawithana AH, Crowhurst RN, Newcomb RD (2015) The peripheral olfactory repertoire of the lightbrown apple moth, Epiphyas postvittana. PLoS One 10(5):e0128596. https://doi.org/10.1371/journal.pone.0128596
Crava CM, Bobkov YV, Sollai G, Anfora G, Crnjar R, and Cattaneo AM (2022) Chemosensory receptors in the larval maxilla of Papilio hospiton. Front. Ecol. Evol. 9:795994. doi:10.3389/fevo.2021.795994
Cui H, Wang Y, Liu X, Wang Y, Zhang L, Chen Y, Jia Y, Zhao G, Wen, J (2023) Identification of common aroma contributors and the regulated metabolites of different kinds of meat. LWT 181: 114737. https://doi.org/10.1016/j.lwt.2023.114737
de Fouchier A Walker III WB, Montagné N, Steiner C, Binyameen M, Schlyter F, Chertemps T, Maria A, François MC, Monsempes C, Anderson P, Hansson BS, Larsson MC, Jacquin-Joly E (2017) Functional evolution of Lepidoptera olfactory receptors revealed by deorphanization of a moth repertoire. Nat Commun 8(1):15709. https://doi.org/10.1038/ncomms15709
Dobritsa AA, van der Goes van Naters W, Warr CG, Steinbrecht RA, Carlson JR (2003) Integrating the molecular and cellular basis of odor coding in the Drosophila antenna. Neuron 37(5):827-841. https://doi.org/10.1016/S0896-6273(03)00094-1
Douglass SK, Juvik JA, Pyun HJ, Coates RM (1993) Structure-activity relationships for analogs of (+)-(E)-endo-β-bergamoten-12-oic acid, an oviposition stimulant of Helicoverpa zea (Boddie). J Chem Ecol 19:11-27. https://doi.org/10.1007/BF00987467
Fleischer J, Pregitzer P, Breer H, Krieger J (2018) Access to the odor world: olfactory receptors and their role for signal transduction in insects. Cell Mol Life Sci 75:485–508. https://doi.org/10.1007/s00018-017-2627-5
Galizia CG, Münch D, Strauch M, Nissler A, Ma S (2010) Integrating heterogeneous odor response data into a common response model: A DoOR to the complete olfactome. Chem Senses 35(7):551–563. https://doi.org/10.1093/chemse/bjq042
Gandolfi M, Mattiacci L, Dorn S (2003) Mechanisms of behavioral alterations of parasitoids reared in artificial systems. J Chem Ecol 29:1871-1887. https://doi.org/10.1023/A:1024854312528
Gonzalez DC (2007) Cydia pomonella behavior and responses to host volatiles. Dissertation, Universidad Lleida, Departament de Quimica
Gonzalez F, Bengtsson JM, Walker WB, Sousa MF, Cattaneo AM, Montagné N, de Fouchier A, Anfora G, Jacquin-Joly E, Witzgall P (2015) A conserved odorant receptor detects the same 1-indanone analogs in a tortricid and a noctuid moth. Front Ecol Evol 3:131. https://doi.org/10.3389/fevo.2015.00131
Gonzalez F, Witzgall P, Walker III WB (2016) Protocol for heterologous expression of insect odourant receptors in Drosophila. Front Ecol Evol 4:24. https://doi.org/10.3389/fevo.2016.00024
Greenway A (1984) Sex pheromone of the pea moth, Cydia nigricana (F.) (Lepidoptera: Olethreutidae). J Chem Ecol 10:973–982. https://doi.org/10.1007/BF00987506
Gregg PC, Del Socorro AP, Landolt PJ (2018) Advances in attract-and-kill for agricultural pests: beyond pheromones. Annu Rev Entomol 63:453-470. https://doi.org/10.1146/annurev-ento-031616-035040
Hallem EA, Ho M, Carlson JR (2004) The molecular basis of odor coding in the Drosophila antenna. Cell 117(7):965–980. https://doi.org/10.1016/j.cell.2004.05.012
Hern A, Dorn S (2002) Induction of volatile emissions from ripening apple fruits infested with Cydia pomonella and the attraction of adult females. Entomol Exp Appl 102(2):145-151. https://doi.org/10.1046/j.1570-7458.2002.00934.x
Hern A, Dorn S (2004) A female-specific attractant for the codling moth, Cydia pomonella, from apple fruit volatiles. Naturwissenschaften 91(2):77–80. https://doi.org/10.1007/s00114-003-0484-6
Heinbockel T, Kaissling KE (1996) Variability of olfactory receptor neuron responses of female silkmoths (Bombyx mori L.) to benzoic acid and (±)-linalool. J Insect Physiol 42(6):565-578. https://doi.org/10.1016/0022-1910(95)00133-6
Hijaz F, Nehela Y, Killiny N (2016) Possible role of plant volatiles in tolerance against Huanglongbing in citrus. Plant Signal Behav 11(3): e1138193. https://doi.org/10.1080/15592324.2016.1138193
Jennings WG, Sevenants MR (1964) Volatile esters of Bartlett pear III. J Food Sci 29(2):123-240. https://doi.org/10.1111/j.1365-2621.1964.tb01711.x
Jumean Z, Gries R, Unruh T, Rowland E, Gries G (2005a) Identification of the larval aggregation pheromone of codling moth, Cydia pomonella. J Chem Ecol 31:911–924. https://doi.org/10.1007/s10886-005-3552-x
Jumean Z, Unruh T, Gries R, Gries G (2005b) Mastrus ridibundus parasitoids eavesdrop on cocoon-spinning codling moth, Cydia pomonella, larvae. Naturwissenschaften 92:20–25. https://doi.org/10.1007/s00114-004-0581-1
Knight A, Light D, Chebny V (2012) Evaluating dispensers loaded with codlemone and pear ester for disruption of codling moth (Lepidoptera: Tortricidae). Environ Entomol 4(2):399-406. https://doi.org/10.1603/EN11309
Knight AL, Preti M, Basoalto E, Mujica V, Favaro R, Angeli S (2022) Combining female removal with mating disruption for management of Cydia pomonella in apple. Entomol Gen 42(2):309-321. https://doi.org/10.1603/EN11309
Koenig C, Hirsh A, Bucks S, Klinner C, Vogel H, Shukla A, Mansfield JH, Morton B, Hansson BS, Grosse-Wilde E (2015) A reference gene set for chemosensory receptor genes of Manduca sexta. Insect Biochem Mol Biol 66:51-63. https://doi.org/10.1016/j.ibmb.2015.09.007
Krieger J, Grosse-Wilde E, Gohl T, Dewer YM, Raming K, Breer H (2004) Genes encoding candidate pheromone receptors in a moth (Heliothis virescens). Proc Natl Acad Sci 101(32):11845-50. https://doi.org/10.1073/pnas.0403052101
Krupke C, Roitberg B, Judd G (2002) Field and laboratory responses of male codling moth (Lepidoptera: Tortricidae) to a pheromone-based attract-and-kill strategy. Environ Entomol 31(2):189-197. https://doi.org/10.1603/0046-225X-31.2.189
Kutinkova H, Subchev M, Light D, Lingren B (2005) Interactive effects of ethyl (2E, 4Z)-2, 4-decadienoate and sex pheromone lures to codling moth: apple orchard investigations in Bulgaria. J Plant Prot Res 49-52.
Landolt P, Suckling D, Judd G (2007) Positive interaction of a feeding attractant and a host kairomone for trapping the codling moth, Cydia pomonella (L.). J Chem Ecol 33:2236-2244. https://doi.org/10.1007/s10886-007-9391-1
Landolt PJ, Guédot C (2008) Field attraction of codling moths (Lepidoptera: Tortricidae) to apple and pear fruit, and quantitation of kairomones from attractive fruit. Ann Entom Soc Am 101(3):675-681. https://doi.org/10.1603/0013-8746(2008)101[675:FAOCML]2.0.CO;2
Lasekan O, Hussein FK (2018) Classification of different pineapple varieties grown in malaysia based on volatile fingerprinting and sensory analysis. Chem Cent J 12(1):1-12. https://doi.org/10.1186/s13065-018-0505-3
Lebreton S, Borrero-Echeverry F, Gonzalez F, Solum M, Wallin EA, Hedenström E, Hansson BS, Gustavsson AL, Bengtsson M, Birgersson GA, Walker III WB, Dweck, HKM, Becher PG, Witzgall, P (2017) Drosophila female pheromone elicits species-specific long-range attraction via an olfactory channel with dual specificity for sex and food. BMC Biol 15:1-14. https://doi.org/10.1186/s12915-017-0427-x
Liu ZQ (2022) What about the progress in the synthesis of flavonoid from 2020? Eur J Med Chem 243:114671. https://doi.org/10.1016/j.ejmech.2022.114671
Light DM, Knight A (2005) Specificity of codling moth (Lepidoptera: Tortricidae) for the host plant kairomone, ethyl (2E,4Z)-2,4-decadienoate: field bioassays with pome fruit volatiles, analogue, and isomeric compounds. J Agric Food Chem 53(10):4046–4053. https://doi.org/10.1021/jf040431r
Light DM, Knight AL, Henrick CA, Rajapaska D, Lingren B, Dickens JC, Reynolds KM, Buttery RG, Merrill G, Roitman J, Campbell BC (2001) A pear derived kairomone with pheromonal potency that attracts male and female codling moth, Cydia pomonella (L.). Naturwissenschaften 88(8):333–338. https://doi.org/10.1007/s001140100243
Leyrer RL, Monroe RE (1973) Isolation and identification of the scent of the moth, Galleria mellonella, and a revaluation of its sex pheromone. J Insect Physiol 19(11):2267–2271. https://doi.org/10.1016/0022-1910(73)90143-1
Löfstedt C, Wahlberg N, Millar JM (2016) Evolutionary patterns of pheromone diversity in Lepidoptera. In: Pheromone communication in moths: evolution, behavior and application. University of California Press, Berkley, pp 43-82. https://doi.org/10.1525/9780520964433-005
Lü J, Yang C, Zhang Y, Pan H (2018) Selection of reference genes for the normalization of RT-qPCR data in gene expression studies in insects: a systematic review. Front Physiol 9:1560. https://doi.org/10.3389/fphys.2018.01560
Mansour M (2019) Development and reproduction of Trichogramma cacoeciae Marchal, 1927 (Hymenoptera: Trichogrammatidae) on Cydia pomonella (Linnaeus, 1758) (Lepidoptera: Tortricidae) eggs. Pol J Entomol 88(1):25-39. https://doi.org/10.2478/pjen-2019-0003
Matich AJ, Rowan DD, Banks NH (1996) Solid phase microextraction for quantitative headspace sampling of apple volatiles. Anal Chem 68(23):4114–4118. https://doi.org/10.1021/ac9604548
Miller JR, Gut LJ (2015) Mating disruption for the 21st century: matching technology with mechanism. Environ Entomol 44(3):427-453. https://doi.org/10.1093/ee/nvv052
Montagné N, Wanner K, Jacquin-Joly E (2021) Olfactory genomics within the Lepidoptera. In Insect pheromone biochemistry and molecular biology 469-505. Academic Press. https://doi.org/10.1016/B978-0-12-819628-1.00015-8
Münch D, Galizia CG (2011) DoOR: The database of odorant responses. ChemoSense 13(4):1-6.
Parthasarathy VA, Chempakam B, Zachariah TJ (2008) Chemistry of spices 2008, CABI.
Pérez-Olivero SJ, Pérez-Pont ML, Conde JE, Pérez-Trujillo JP (2014) Determination of lactones in wines by headspace solid-phase microextraction and gas chromatography coupled with mass spectrometry. J Anal Methods Chem 1:863019. https://doi.org/10.1155/2014/863019
Phelan PL, Silk PJ, Northcott CJ, Tan SH, Baker TC (1986) Chemical identification and behavioral characterization of male wing pheromone of Ephestia elutella (Pyralidae). J Chem Ecol 12(1):135–146. https://doi.org/10.1007/BF01045597
Poivet E, Gallot A, Montagné N, Glaser N, Legeai F, Jacquin-Joly E (2013) A comparison of the olfactory gene repertoires of adults and larvae in the noctuid moth Spodoptera littoralis. PLoS one 8(4):e60263. https://doi.org/10.1371/journal.pone.0060263
Preti M, Knight AL, Mujica MV, Basoalto E Larsson-Herrera S, Tasin M, Angeli S (2021) Development of multi-component non-sex pheromone blends to monitor both sexes of Cydia pomonella (Lepidoptera: Tortricidae) J Appl Entomol 145(8):822–830. https://doi.org/10.1111/jen.12898
Pophof B (1997) Olfactory responses recorded from sensilla coeloconica of the silkmoth Bombyx mori. Physiol Entomol 22(3):239-248. https://doi.org/10.1111/j.1365-3032.1997.tb01164.x
Rajeshwari U, Andallu B (2011) Medicinal benefits of coriander (Coriandrum Sativum L). Spatula DD 1(1):51-58. https://doi.or/10.5455/spatula.20110106123153
Reed H, Landolt P (2002) Attraction of mated female codling moths (Lepidoptera: Tortricidae) to apples and apple odor in a flight tunnel. Fla Entomologist 324-329.
Roscoe LE, Silk P, Eveleigh ES (2016) Evidence of male hair pencil pheromone in Choristoneura fumiferana (Lepidoptera: Tortricidae). J Insect Sci 16(1):27. https://doi.org/10.1093/jisesa/iew010
Rospars JP, Lansky P, Chaput M, Duchamp-Viret P (2008). Competitive and noncompetitive odorant interactions in the early neural coding of odorant mixtures. J Neurosci 28(10):2659-2666. https://doi.org/10.1523/JNEUROSCI.4670-07.2008
Singh V, Murphy NR, Balasubramanian V, Mainland JD (2019) Competitive binding predicts nonlinear responses of olfactory receptors to complex mixtures. Proc Natl Acad Sciences 116(19):9598-9603. https://doi.org/10.1073/pnas.1813230116
Steinwender B, Thrimawithana AH, Crowhurst RN, Newcomb RD (2015) Pheromone receptor evolution in the cryptic leafroller species, Ctenopseustis obliquana and C. herana. J Mol Evol 80:42-56. https://doi.org/10.1007/s00239-014-9650-z
Thwaite WG, Mooney AM, Eslick MA, Nicol HI (2004) Evaluating pear-derived kairomone lures for monitoring 'Cydia pomonella' (L.)(Lepidoptera: Tortricidae) in Granny Smith apples under mating disruption. Gen Appl Entomol: J of Entomol Soc New South Wales 33:55-60.
Tobita N, Makino M, Fujita R, Jyotaki M, Shinohara Y, Yamamoto T (2021) Sweet scent lactones activate hot capsaicin receptor, TRPV1. Biochem Biophys Res Commun 534:547–552. https://doi.org/10.1016/j.bbrc.2020.11.046
Tom MT, Cortés Llorca L, Bucks S, Bisch-Knaden S, Hansson BS (2022) Sex-and tissue-specific expression of chemosensory receptor genes in a hawkmoth. Front Ecol Evol 10:976521. https://doi.org/10.3389/fevo.2022.976521
Vallat A, Dorn S (2005) Changes in volatile emissions from apple trees and associated response of adult female codling moths over the fruit-growing season. J Agric Food Chem 53(10):4083-4090. https://doi.org/10.1021/jf048499u
Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3(7):1-12. https://doi.org/10.1186/gb-2002-3-7-research0034
Walker III WB, Gonzalez F, Garczynski SF, Witzgall P (2016) The chemosensory receptors of codling moth Cydia pomonella – expression in larvae and adults. Sci Rep 6(1):23518. https://doi.org/10.1038/srep23518
Walker III WB, Roy A, Anderson P, Schlyter F, Hansson BS, Larsson MC (2019) Transcriptome analysis of gene families involved in chemosensory function in Spodoptera littoralis (Lepidoptera: Noctuidae). BMC Genom 20:428. https://doi.org/10.1186/s12864-019-5815-x.
Walker III WB, Cattaneo AM, Stout JL, Evans ML, Garczynski SF (2023) Chemosensory receptor expression in the abdomen tip of the female codling moth, Cydia pomonella L. (Lepidoptera: Tortricidae). Insects 14(12):948. https://doi.org/10.3390/insects14120948
Wan F, Yin C, Tang R, Chen M, Wu Q, Huang C et al (2019) A chromosome-level genome assembly of Cydia pomonella provides insights into chemical ecology and insecticide resistance. Nat Commun 10(1):4237. https://doi.org/10.1038/s41467-019-12175-9
Wanner KW, Anderson AR, Trowell SC, Theilmann D, Robertson HM, Newcomb RD (2007) Female‐biased expression of odourant receptor genes in the adult antennae of the silkworm, Bombyx mori. Insect Mol Biol 16(1):107-119. https://doi.org/10.1111/j.1365-2583.2007.00708.x
Wearing CH Hutchins RFN (1973) α-Farnesene, a naturally occurring oviposition stimulant for the codling moth, Laspeyresia pomonella. J Insect Physiol 19(6):1251–1256. https://doi.org/10.1016/0022-1910(73)90208-4
Weeks ENI, Logan JG, Birkett MA, Caulfield JC, Gezan SA, Welham SJ, Brugman VA, Pickett JA, Cameron MM (2020) Electrophysiologically and behaviourally active semiochemicals identified from bed bug refuge substrate. Sci Rep 10(1):4590. doi.org/10.1038/s41598-020-61368-6
Wenda-Piesik A, Piesik D, Krasińska A (2018) Response of mated insects of both sexes of granary weevil to blends of volatiles – short communication. Plant Prot Sci 54(3):190–193. https://10.17221/136/2017-PPS
Witzgall P, Bengtsson M, Unelius CR, Löfqvist J (1993) Attraction of pea moth Cydia nigricana F.(Lepidoptera: Tortricidae) to female sex pheromone (E, E)-8, 10-dodecadien-1-yl acetate, is inhibited by geometric isomers E, Z, Z, E, and Z, Z. J Chem Ecol 19:1917–1928. https://doi.org/10.1007/BF00983796
Witzgall P, Ansebo L, Yang Z, Angeli G, Sauphanor B, Bengtsson, M (2005) Plant volatiles affect oviposition by codling moths. Chemoecology 15:77-83. https://doi.org/10.1007/s00049-005-0295-7
Witzgall P, Stelinski L, Gut L, Thomson D (2008) Codling moth management and chemical ecology. Annu Rev Entomol 53:503-522. https://doi.org/10.1146/annurev.ento.53.103106.093323
Witzgall P, Kirsch P, Cork A (2010) Sex pheromones and their impact on pest management. J Chem Ecol 36:80-100. https://doi.org/10.1007/s10886-009-9737-y
Witzgall P, Proffit M, Rozpedowska E, Becher PG, Andreadis S, Coracini M, Lindblom TUT, Ream LJ, Hagman A, Bengtsson M, Kurtzman CP, Piskur J, Knight A (2012) “This is not an Apple”–Yeast Mutualism in Codling Moth. J Chem Ecol 38:949–957. https://doi.org/10.1007/s10886-012-0158-y
Yan F, Bengtsson M, Witzgall P (1999) Behavioral response of female codling moths, Cydia pomonella, to apple volatiles. J Chem Ecol 25:1343-1351. https://doi.org/10.1023/A:1020978826346
Zhang DD, Löfstedt C (2015) Moth pheromone receptors: gene sequences, function, and evolution. Front Ecol Evol 3:105. https://doi.org/10.3389/fevo.2015.00105

Table 1. Compounds screened by SSR on D. melanogaster expressing CpomOR22. Compound classes, compound purity, source, CAS numbers, and physicochemical properties are reported, including averages and standard deviation of the response to each ligand, based on five replicates. Sign.: asterisks denote significant effects (Wilcoxon Signed Rank Test, p < 0.05, Supplementary Data File 3); if “i”: significant inhibition. For compound purity, “unknown” indicates information is not available from the compound producer, while “-” indicates purity not presently identified on source used in this study. Sources, from which the compounds have been initially found, and the respective studies, are indicated in the table. Letter on apices denote references as it follows: ^ABäckman et al. 2000; ^BBengtsson et al. 2001; ^CCattaneo et al. 2023; ^DHern and Dorn 2004; ^ELebreton et al. 2017; ^FWitzgall et al. 2012.

Compound class	Compound name	Source	CAS	Purity (%)	Mol. weight (g/mol)	Vapor pressure (mmHg @ 25°C)	Raw spiking		Normalized		p-val.	Sign.
Compound class	Compound name	Source	CAS	Purity (%)	Mol. weight (g/mol)	Vapor pressure (mmHg @ 25°C)	Average	St.Dv.	Average	St.Dv.	p-val.	Sign.
Terpene alcohol	(R)-linalool	Malus domestica cv. Discovery^A	126-91-0	97	154,2527	0,09100	-1,60	5,04	-0,79	0,77	0,465
Terpene alcohol	(S)-linalool	Malus domestica cv. Discovery^A	78-70-6	97	154,2527	0,01600	5,00	5,10	-0,23	0,75	0,345
Aromatic alcohol	2-phenylethanol	Metschnikowia ssp^B	8/12/1960	99	122,1669	0,08680	3,20	6,89	-0,54	0,86	0,715
Aliphatic alcohol	3-octanol	Our collection^C	589-98-0	95	130,2307	0,51200	1,40	6,19	-0,15	0,48	0,588
Aliphatic alcohol	(E,E)-alpha-farnesol	Malus domestica Cv. Granny Smith^D	106-28-5	95	222,3714	0,00037	4,20	4,42	-0,10	0,52	0,043	i
Aliphatic alcohol	(Z)-3-hexenol	Malus domestica cv. Discovery^A	928-96-1	98	100,1608	1,03900	5,40	4,75	-0,13	0,76	0,345
Aliphatic alcohol	(E,E)-8,10-dodecadien-1-ol	C. pomonella^E	33956-49-9	-	182,3065	0,00100	4,40	5,16	-0,29	0,80	0,500
Saturated aldehyde	nonanal	Malus domestica cv. Discovery^A; Metschnikowia ssp^B	124-19-6	95	142,2417	0,53200	92,60	8,45	9,30	2,53	0,043	*
Monoinsaturated aldehyde	(Z)-4-undecenal	Our collection^C	68820-32-6	-	168,2796	0,04500	62,80	12,21	5,78	1,23	0,043	*
Monoinsaturated aldehyde	(Z)-6-undecenal	Novel	60671-73-0	-	168,2796	0,04540	93,00	14,25	8,96	2,15	0,043	*
Polyinsaturated aldehyde	(E,E)-2,4-decadienal	Our collection^C	25152-84-5	-	152,2367	0,03000	14,60	3,78	1,31	0,42	0,043	*
Terpenoid aldehyde	citral	Our collection^C	5392-40-5	95	152,2367	0,20000	6,80	7,12	-0,36	1,17	0,500
Terpenoid aldehyde	β-cyclocitral	Our collection^C	432-25-7	-	152,2370	0,17600	-1,80	2,08	-0,36	0,40	0,500
Aliphatic chetone	2-heptanone	Metschnikowia ssp^B	110-43-0	98	114,1878	4,73200	-6,00	4,80	-0,95	0,58	0,345
Fatty acid ester	ethyl acetate	Our collection^C	141-78-6	99.5	88,1062	111,71600	-5,40	4,81	-0,96	0,59	0,225
Fatty acid ester	ethyl hexanoate	Malus domestica Cv. Granny Smith^D	123-66-0	98	144,2139	1,66500	8,60	6,89	0,04	0,83	0,225
Fatty acid ester	ethyl-(E,Z)-2,4-decadienoate	Pyrus communis Cv. Williams^F	3025-30-7	85	196,2898	0,01000	7,40	4,46	0,15	0,51	0,043	*
Fatty acid ester	methyl-(E,Z)-2,4-decadienoate	Pyrus communis Cv. Williams^F	4493-42-9	unknown	182,2629	0,02800	3,60	5,92	-0,46	1,16	0,500
Fatty acid ester	hexyl 2-methyl-butanoate	Malus domestica cv. Discovery^A	10032-15-2	95	186,2947	0,15800	10,20	7,28	0,04	0,89	0,345
Aromatic ester	Methyl salicylate	Malus domestica cv. Discovery^A	119-36-8	99	152,1494	0,03430	-3,60	3,92	-0,75	0,51	0,273
Acetate	(E,E)-8,10-dodecadien-1-yl acetate	C. pomonella^E	53880-51-6	unknown	224,3437	0,00100	3,80	5,43	-0,33	1,00	0,345
Acetate	(Z,Z)-3,13-Octadecadien-1-yl acetate	Novel: main pheromone of Synanthedon myopaeformis	53120-27-7	-	308,5053	Unknown	6,20	6,54	-0,20	1,11	0,345
Sesquiterpene	(E)-beta-farnesene	Malus domestica cv. Discovery^A	18794-84-8	90	204,3563	0,01000	2,80	4,47	-0,26	0,56	0,068
Sesquiterpene	(E,E)-α-farnesene	Malus domestica cv. Discovery^A	502-61-4	unknown	204,3563	0,01000	3,60	4,52	-0,10	0,43	0,043	i
Sesquiterpene	(E)-beta-caryophyllene	Malus domestica cv. Discovery^A	87-44-5	98.5	204,3563	0,01300	0,80	4,81	-0,49	0,64	0,893
Cyanide	2-phenylacetonitrile	Novel	140-29-4	99	204,3563	0,01300	6,40	8,26	-0,46	1,15	0,686
Acyclic monoterpenoids	(E)-4,8–dimethyl–1,3,7-nonatriene	Malus domestica cv. Discovery^A	26049-69-4	unknown	254,27	0,00032	2,80	4,55	-0,35	0,72	0,500
Solvent	hexane	-	110-54-3	97	86,1776	151,00000	-1,60	3,31	-0,52	0,50	-

Table 2. GC-SSR spike counting for responses to Hoplomalus (Hopl) and Malus (Mal) headspace. Raw (Spikes/5.0 sec), spike counting (spikes/sec), and parameters from GC-SSR runs (retention times, RT and Kovats index, Kov.) testing Hoplomalus and Malus headspace on DB-wax (Rep1-4) and HP-5 columns (Rep 5-12) are available in Supplementary Data File 3.

Spike counting (Spikes/sec)
Headspace	Hopl 583			Mal 590	Hopl 566	Hopl 560	Hopl583	Hopl500	Mal 561		Mal 559	Mal 603	Wilcoxon Signed Rank Test
GC-MS	R1	R2	R3	R4	R5	R6	R7	R8	R9	R10	R11	R12	Average	St.Er.	*p-val.*
hexane	3	6	3	13	1	0,2	0,2	-0,2	2,8	-1,6	-0,8	2,6	2,43	1,14	-
nonanal	16,8	2,8	19	27,4	36	8,4	17,6	3,2	12,8	13	3,6	10,8	14,28	2,89	0,003
unknown	-	-	-	-	12,8	5,2	8	2,6	8,2	5,8	3,8	2,8	6,15	1,21	0,012
decanal	19,2	22,8	22	42,4	53,8	16,8	51	-2,6	14,6	24	17,2	29,2	25,87	4,66	0,003
undecanal	33	9,8	8,8	21	43,6	11	22,6	-1,2	9,6	10,6	8	16,4	16,10	3,55	0,003
dodecanal	28,6	11,2	16,6	25,4	46	9,4	27,4	3	7,6	5,2	4,4	9,4	16,18	3,77	0,002
Column	DB-wax				HP-5

No competing interests reported.

SupplementaryDataFile1qRTPCRdatafile.xlsx
Supporting information descriptions Supplementary Data File 1. qRT-PCR data file: raw CT values and relative quantity (RQ) values with target gene values normalized to the geometric mean of RQ values control genes; normalized gene expression values for each biological sample calibrated to average of gene expression values from male antennae samples; bar plot for normalized and calibrated relative gene expression fold-change is shown in Fig. 1 (XLSX).
SupplementaryDataFile2OR22SequenceInfo.docx
Supplementary Data File 2. Open reading frame sequence information for OR22, including non-codon optimized nucleotide sequence (from the transcriptome of Walker et al. (2016)) used in Xenopusoocyte experiments, codon-optimized nucleotide sequence, optimized for D. melanogaster codon usage, used in Drosophila empty neuron SSR/GC-SSR experiments, and complete odorant receptor protein sequence (DOC).
SupplementaryDataFile3updatedv3.xlsx
Supplementary Data File 3. SSR-sheet: raw and normalized spike counting of compounds tested on CpomOR22 (Table 1), statistical analysis, including tests of normality, from normalized spike counting. GC-SSR sheet: raw and normalized spike counting of characterized retention time tested by GC-SSR on CpomOR22 (Table 2) and statistical analysis, including tests of normality. (XLSX). Note: p-values lower than 0,05 are highlighted in yellow.
SupplementaryDataFile4revised.xlsx
Supplementary Data File 4. Effects of the blends on odorant receptor complexes expressed in oocytes from Xenopus laevis. Raw data was collected, and normalized according to the blend or compound producing the greatest response (XLSX).
SupplementaryTable1qPCRPrimers.docx
Supplementary Table 1. Primers used for qRT-PCR analysis (DOC).
SupplementaryTable2revised.xlsx
Supplementary Table 2. List of compounds tested on oocytes from Xenopus. The compounds are organized into blends according to chemical class. Table includes supplier, compound purity, and CAS numbers (XLSX).
SupplementaryTable3DoseResponseCpomOR22toZ6undecenal.xlsx
Supplementary Table 3. Raw and normalized data of dose response effects testing CpomOR22 to (Z)-6-undecenal (XLSX).
Supplementaryfigure1CpomOR22toZ611Al.pdf
Supplementary Figure 1.Dose-response characteristics of CpomOR22-effects to (Z)-6-undecenal. The ab3A neurons expressing CpomOR22 generated spiking in response to the application of the compounds. The effects were concentration-dependent and reversible. Concentration dependences of the compounds are expressed as a function of normalized spike frequency [(Δspikes/s)/spikes] versus μg-doses. Responses were individually normalized to their saturating concentrations (50 µg). Error bars represent the standard error of the mean. Data were fit with the Hill equation (solid blue line). Below: spike trains elicited by doses ranging from 0.5 to 100.0 μg extracted from replicate 6 (Supplementary Table 3). Note: at saturating doses, we observed initial bleaching of the response effect. Red bar: stimulus (PDF).

Functional characterization of a female-biased chemoreceptor of the codling moth (Cydia pomonella) responding to aldehydes and other volatile compounds

Status:

Version 1

Abstract

Figures

Key messages

Introduction

Material and Methods

Results

Discussion

Abbreviations

Declarations

References

Tables

Additional Declarations

Supplementary Files

Status:

Version 1