Functional Characterization of a Female-Biased Chemoreceptor of the Codling Moth (Cydia pomonella) Responding to Aldehydes and Other Volatile Compounds

Abstract

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 and revealed lactones (γ-undecalactone and δ-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.

Keywords: Cydia pomonella chemoreceptors; Xenopus oocytes; Gas-chromatography-coupled SSR (GC-SSR); Heterologous expression; Single sensillum recording (SSR); Transgenic Drosophila melanogaster.

Fig. 1

qRT-PCR expression fold change of CpomOR22 and CpomOrco in female and male antennae. Relative gene expression fold-change values are shown on a log10 scale, normalized to reference genes (ActR2 and HSP40), with average values of three biological replicates for each gene and tissue type shown. For each gene and biological sample, ∆(CT) values were calculated against the average CT of all male samples. Hence, values greater than one indicate higher expression in females, and values less than one indicate higher expression in males. Statistical assessments of differences in values between log2 transformed male and female values were conducted with Student's t-test (a two-tailed distribution with two-sample equal variance); "**" indicates a p-value less than 0.002. “n.s.” indicates “not significant” with a p-value greater than 0.05. Error bars are standard error values. Raw and normalized data are shown in Supplementary Data File 1

Fig. 2

SSR and GC-SSR effects from ab3A neurons of transgenic Drosophila expressing CpomOR22. A. Boxplot of normalized ab3A spiking from transgenic D. melanogaster expressing CpomOR22, tested with the compound library reported in Table 1. Colors depict ligands belonging to different compound classes (see legend and Table 1). Bold-font compounds were significantly active on CpomOR22, with slight activators indicated with Magenta. Raw and normalized data from spike counting are shown in Supplementary Data File 3. B. GC-SSR experiment conducted by injecting GC-system provided with capillary column coated with HP-5, using 10.0 ng and 1.0 ng of nonanal [CAS: 124–19-6] and (Z)−6-undecenal [CAS: 60,671–73-0]. Note: frequency plots (above) summarize effects associated with 10 ng and 1.0 ng, while the gas-chromatogram shown below indicates peaks related to only 10.0 ng. C. GC-SSR experiment conducted by injecting the apple headspace of Hoplomalus 583 (Table 2, replicate 7); above, frequency plot associated with active components: 1 – nonanal/unidentified, 2 – decanal, 3 – undecanal, 4 – dodecanal; below, retention times (red/blue, min) identified in the GC-track. D. Magnification of the frequency plot (above), spiking effect (middle), and GC-FID (below) of each Section (1 to 4) selected from C. Retention times (min) are indicated as in C, and if not active, are shown in black

Fig. 3

Functional characterization of CpomOR22/CpomOrco heterologously expressed in oocytes from X. laevis. Oocytes from Xenopus injected with CpomOR22/CpomOrco respond to environmentally relevant compounds. A. Trace from the TEVC software shows the changes in a single oocyte internal membrane potential. B. The averaged, normalized responses of 10 individual oocytes to exposure of the 16 compound blends. C. The averaged, normalized response of 10 individual oocytes to exposure of the individual components of the relevant compound blends. Note: the two most active lactones (gamma-undecalactone and delta-dodecalactone) and two other lactones, among the less active (delta-nonalactone and gamma-octalactone) were analyzed by GC–MS following standards indicated in methods confirming the absence of aldehydes contaminants and validating lactones purities ranging between 98 and 99%. D. The averaged, normalized responses of 10 individual oocytes to exposure of half log concentrations of undecanal (top) and γ-undecalactone (bottom) ranging from 10^–7 to 10^–4 M

Fig. 4

Total ion chromatogram of H. testudinea infested apple volatile collections separated on DB-WAX capillary column. X-axis: retention time (min); Y-axis: total ion abundance. The tentatively identified sensillum-active components are nonanal (1), decanal (2), undecanal (3), dodecanal (4)