An odorant receptor mediates the avoidance of Plutella xylostella against parasitoid

Abstract

Background: Ecosystems are brimming with myriad compounds, including some at very low concentrations that are indispensable for insect survival and reproduction. Screening strategies for identifying active compounds are typically based on bioassay-guided approaches.

Results: Here, we selected two candidate odorant receptors from a major pest of cruciferous plants-the diamondback moth Plutella xylostella-as targets to screen for active semiochemicals. One of these ORs, PxylOR16, exhibited a specific, sensitive response to heptanal, with both larvae and adult P. xylostella displaying heptanal avoidance behavior. Gene knockout studies based on CRISPR/Cas9 experimentally confirmed that PxylOR16 mediates this avoidance. Intriguingly, rather than being involved in P. xylostella-host plant interaction, we discovered that P. xylostella recognizes heptanal from the cuticular volatiles of the parasitoid wasp Cotesia vestalis, possibly to avoid parasitization.

Conclusions: Our study thus showcases how the deorphanization of odorant receptors can drive discoveries about their complex functions in mediating insect survival. We also demonstrate that the use of odorant receptors as a screening platform could be efficient in identifying new behavioral regulators for application in pest management.

Keywords: Plutella xylostella; Avoidance behavior; Heptanal; Odorant receptor; Parasitoid wasp.

Fig. 1

PxylOR16 is expressed in all the larval and adult stages of Plutella xylostella and is specifically tuned to heptanal. A Tissue expression patterns of P. xylostella OR genes. The cDNA templates for PCR analyses were from larval heads (first, second, third female, third male, fourth female, and fourth male instar larvae) and adult antennae (male adults: MA, female adults: FA). W water control. Among the 54 P. xylostella ORs, only PxylOR16 and PxylOR27 were detected in all larval stages and in adults. Actin was used for cDNA quality control. B Inward current responses of PxylOR16/PxylOrco Xenopus oocytes to plant volatile compounds (10^–4 M). C No ligand was identified for PxylOR27/PxylOrco. D Inward current responses of PxylOR16/PxylOrco Xenopus oocytes stimulated with a range of heptanal concentrations. E Response profile of PxylOR16/PxylOrco Xenopus oocytes to a panel of 71 odorants (n = 6). F Dose–response curve of PxylOR16/PxylOrco Xenopus oocyte responses to heptanal. Heptanal EC₅₀ = 1.757 × 10^–5 M. Error bars indicate SEM (n = 6)

Fig. 2

PxylOR16 knockout mutants show impaired electrophysiological responses to heptanal. A Schematic diagram of the sgRNA target in Exon II of PxylOR16. The target sequence is shown in blue, the PAM sequence is marked in red, and the non-homologous insertion in the genome in yellow. Deleted bases are represented by dashes. PxylOR16 mutants show impaired electrophysiological responses to heptanal. B Electrophysiological responses measured as electroantennograms of Plutella xylostella antennae to heptanal and trans-2-hexen-1-ol in wild-type (WT) animals and in PxylOR16 knockout mutants generated by CRISPR/Cas9 (PxylOR16^−/−). Left, dose-dependent electroantennographic (EAG) responses of female and male moths. Heptanal was used at doses ranging from 10 ng to 10 μg. WT female and male antennae exhibited dose-dependent EAG responses to heptanal, with responses increasing with increasing heptanal doses. EAG responses of female and male PxylOR16^−/− moths to heptanal were far lower than those of WT female and male moths at all heptanal doses. Right, as a control, we tested the responses of female and male moths to trans-2-hexen-1-ol, which is not a ligand of PxylOR16. As expected, we observed no differences in the responses to this compound between PxylOR16^−/− moths and WT moths. Error bars indicate SEM (n = 10). Different letters indicate significant differences among insects (two-way ANOVA followed by Tukey’s pairwise test; P < 0.05)

Fig. 3

Heptanal elicits obvious avoidance behaviors in both Plutella xylostella larvae and adults. Preference index of P. xylostella wild-type (WT) and CRISPR/Cas9 PxylOR16 knockout (PxylOR16^−/−) adults and larvae for heptanal. Preference indexes = (number of choices at treatment – number of choices at control) / number of total choices. A Preference index of WT and PxylOR16^−/− mutant female and male moths for control (filter paper vs filter paper) and for filter paper + paraffin oil vs filter paper + heptanal in a Y-tube olfactometer (n = 16). WT male moths exhibited significant avoidance to heptanal at doses of 100 ng, 1 μg, and 10 μg (left). Avoidance was also observed for WT female moths, but only at doses of 1 μg and 10 μg (Welch’s t-test; NS, no significant difference, P > 0.05; *** P < 0.001) (right). PxylOR16^−/− mutant female and male moths did not show a preference for one side of the device (Welch’s t-test; NS, no significant difference, P > 0.05). B Preference index of WT and PxylOR16^−/− mutant female and male moths for blank (one side blank vs a piece of Brassica pekinensis), for control (a piece of B. pekinensis vs a piece of B. pekinensis), and for a piece of B. pekinensis + paraffin oil vs a piece of B. pekinensis + heptanal in a Y-tube olfactometer (n = 16). WT and PxylOR16^−/− mutant female and male moths were significantly attracted to B. pekinensis, compared with blank. WT male moths were significantly less attracted to B. pekinensis + heptanal at doses of 100 ng, 1 μg, and 10 μg than to B. pekinensis alone (with paraffin oil) (left). Female moths were also less attracted to B. pekinensis + heptanal than to B. pekinensis alone but only at heptanal doses of 1 μg and 10 μg (Welch’s t-test; NS, no significant difference; P > 0.05; *** P < 0.001) (right). The PxylOR16^−/− mutant female and male moths lost their avoidance responses to heptanal significantly, compared with WT adult female and male moths (Welch’s t-test; NS, no significant difference; P > 0.05). C Preference indexes of 10 WT and mutant female and male third instar larvae for blank (one side blank vs a piece of B. pekinensis), for control (a piece of B. pekinensis vs a piece of B. pekinensis), and for a piece of B. pekinensis + paraffin oil vs a piece of B. pekinensis + heptanal in a 10-cm diameter plastic Petri dish (n = 28). Both sexes of WT and mutant larvae were attracted to B. pekinensis. Significantly more female and male third instar larvae were located in the control area (B. pekinensis + paraffin oil) than in the heptanal-supplemented area (B. pekinensis + heptanal) at all heptanal doses (100 ng, 1 μg, and 10 μg on filter paper) except for 10 ng (Welch’s t-test; NS, no significant difference, P > 0.05; *** P < 0.001). As shown for PxylOR16^−/− adults, detection of heptanal was abolished in PxylOR16^−/− larvae (Welch’s t-test; NS, no significant difference, P > 0.05)

Fig. 4

Heptanal does not participate in the direct interaction between Plutella xylostella and its host plant. Preference index of P. xylostella wild-type (WT) and PxylOR16-knockout (PxylOR16^−/−) adults and larvae to heptanal. A Preference index of WT and PxylOR16^−/− mutant female and male moths for blank (one side blank vs an intact healthy Brassica parachinensis plant), for control (an intact healthy B. parachinensis plant vs an intact healthy B. parachinensis plant), and for an intact healthy B. parachinensis plant + paraffin oil vs an intact healthy B. parachinensis plant + heptanal in a Y-tube olfactometer (n = 16). WT and PxylOR16^−/− mutant female and male moths were significantly attracted to healthy B. parachinensis plant, compared with blank. Three doses (100 ng, 1 μg, and 10 μg) and two doses (1 μg and 10 μg) of heptanal elicited avoidance behavior in WT female and male P. xylostella adults, (Welch’s t-test; NS, no significant difference, P > 0.05; *** P < 0.001). The numbers of PxylOR16^−/− adults on each side of the Y-tube olfactometer were not significantly different at all heptanal doses tested, when comparing B. parachinensis + heptanal with B. parachinensis plant + paraffin oil (Welch’s t-test; NS, no significant difference, P > 0.05). B Preference indexes of 10 WT and mutant female and male third instar larvae for blank (one side blank vs an intact healthy B. parachinensis plant), for control (an intact healthy B. parachinensis plant vs an intact healthy B. parachinensis plant), and for an intact healthy B. parachinensis plant + paraffin oil vs an intact healthy B. parachinensis plant + heptanal in a screening device (n = 10). Both sexes of WT and mutant larvae were attracted to the B. parachinensis plant. WT female and male larvae (third instar larvae) preferred the healthy plant with paraffin oil to a healthy plant with heptanal at three doses (100 ng, 1 μg, and 10 μg), unlike PxylOR16^−/− larvae (Welch’s t-test; NS, no significant difference, P > 0.05; *** P < 0.001). C GC–MS analysis of the volatiles produced by larval-infested plants and healthy plants; heptanal was not detected. Larval-infested B. parachinensis (top), healthy B. parachinensis (middle), and synthetic heptanal (bottom). D Preference index of WT and PxylOR16^−/− mutant adults for control (intact healthy B. parachinensis plant vs intact healthy B. parachinensis plant) and for an intact healthy B. parachinensis plant vs a larval-infected B. parachinensis plant in a Y-tube olfactometer (n = 16). There was no difference in selection between the WT and PxylOR16^−/− mutant; both preferred the larval-infected plant (Welch’s t-test; NS, no significant difference, P > 0.05; *** P < 0.001). E Preference index of WT and PxylOR16^−/− mutant larvae for control (intact healthy B. parachinensis plant vs intact healthy B. parachinensis plant) and for an intact healthy B. parachinensis plant vs a larval-infected B. parachinensis plant in a screening device (n = 10). Like adults, WT and PxylOR16^−/− mutant larvae both preferred the larval-infected plant (Welch’s t-test; NS, no significant difference, P > 0.05; *** P < 0.001)

Fig. 5

Plutella xylostella larvae and adult detect and avoid Cotesia vestalis odorants. A GC/MS analysis of the volatiles produced by C. vestalis. Heptanal could be detected in extracts of C. vestalis. IS internal standard. B Synthetic heptanal. C Preference index of WT and PxylOR16^−/− P. xylostella mutant adults (left) and larvae (right) for filter paper + dichloromethane vs filter paper + C. vestalis body wash in a Y-tube olfactometer and a 10-cm diameter plastic Petri dish, respectively (n = 16, adults; n = 28, larvae). Both sexes of WT larvae and adults of P. xylostella had avoidance responses to a C. vestalis body wash, but the PxylOR16^−/− mutant did not (Welch’s t-test; NS, no significant difference; P > 0.05; * P < 0.05; *** P < 0.001). D Preference index of WT and PxylOR16^−/− mutant adults (left) and larvae (right) for air + 20 live C. vestalis vs air in a Y-tube olfactometer and a screening device, respectively (n = 16, adults; n = 10, larvae). Both sexes of WT larvae and adults of P. xylostella showed avoidance of C. vestalis, but the PxylOR16^−/− mutants did not (Welch’s t-test; NS, no significant difference; P > 0.05; * P < 0.05; ** P < 0.01)