Odorant receptors (ORs) are essential for plant-insect interactions. However, despite the global impacts of Lepidoptera (moths and butterflies) as major herbivores and pollinators, little functional data is available about Lepidoptera ORs involved in plant volatile detection. Here, we initially characterized the plant-volatile-sensing function(s) of 44 ORs from the cotton bollworm Helicoverpa armigera, and subsequently conducted a large-scale comparative analysis that establishes how most orthologous ORs have functionally diverged among closely related species whereas some rare ORs are functionally conserved. Specifically, our systematic analysis of H. armigera ORs catalogued the wide functional scope of the H. armigera OR repertoire, and also showed that HarmOR42 and its Spodoptera littoralis orthologue are functionally conserved. Pursuing this, we characterized the HarmOR42-orthologous ORs from 11 species across the Glossata suborder and confirmed the HarmOR42 orthologues form a unique OR lineage that has undergone strong purifying selection in Glossata species and whose members are tuned with strong specificity to phenylacetaldehyde, a floral scent component common to most angiosperms. In vivo studies via HarmOR42 knockout support that HarmOR42-related ORs are essential for host-detection by sensing phenylacetaldehyde. Our work also supports that these ORs co-evolved with the tube-like proboscis, and has maintained functional stability throughout the long-term coexistence of Lepidoptera with angiosperms. Thus, beyond providing a rich empirical resource for delineating the precise functions of H. armigera ORs, our results enable a comparative analysis of insect ORs that have apparently facilitated and currently sustain the intimate adaptations and ecological interactions among nectar feeding insects and flowering plants.

Grapholita molesta is one of the most destructive fruit pests distributed worldwide. Odorant receptors (ORs) located on the dendritic membrane of chemosensory neurons are deemed to be key molecules for sensing exogenous chemical signals. In this study, GmolOR9, a general OR from G. molesta, was functionally characterized. Quantitative real-time polymerase chain reaction revealed that GmolOR9 was more highly expressed in adults than in other stages, including eggs, larvae, and pupae. GmolOR9 expression was highly significantly more in the antennae of females than in those of males, and the highest level occurred in the antennae of 3-day-old female adults. GmolOR9 was broadly tuned to eight of 47 odorant components tested, including (Z)-3-hexenyl acetate, butyl propionate, ethyl hexanoate, ethyl heptanoate, 1-hexanol, (Z)-3-hexenol, 2-ethyl-1-hexanol, and linalool, by in vitro heterologous expression. Furthermore, electroantennogram responses indicated that the effects of dsOR9-injected females to (Z)-3-hexenyl acetate dramatically decreased. These results suggested that GmolOR9 might be involved in detecting hostplant volatiles. Moreover, (Z)-3-hexenyl acetate might serve as a potential attractant for the biological control of G. molesta.

Grapholita molesta is a global pest of stone and pome fruits. The sensitive olfactory system plays a crucial role in regulating key behavioral activities of insects and G. molesta relies heavily on general odorant receptors (ORs) to detect host-plant volatiles. In this study, three general OR genes from G. molesta (GmolOR12, GmolOR20, and GmolOR21) were identified. Quantitative polymerase chain reaction revealed that GmolORs expression was considerably higher in adults and adult antennae than in any other life stages and body parts, respectively. Moreover, the expression of GmolORs was significantly higher in the antennae of females than in those of males, with a peak in the antennae of 3-days-old adult females. GmolOR20 and GmolOR21 displayed no responses to any of the odorant compounds tested in the Xenopus oocyte system. GmolOR12 was tuned mainly to 5 of the 47 odorant components tested (including decanol, heptanal, octanal, nonanal, and decanal), and the response to aldehydes among the 5 components was the highest. Additionally, they all elicited female and male antennae electroantennogram responses, and the aldehydes elicited the highest response among the 5 components. These results suggested that GmolOR12 in the G. molesta olfactory system plays an important role in sensing aldehydes and that GmolOR12 is involved in sensing host-plant volatiles. These findings provide insight into the possibility of using host-plant volatiles for the control of G. molesta.

Sex pheromones serve a critical role in Lepidopterans finding mates. Male moths perceive and react to sex pheromones emitted by conspecific females through a delicate pheromone communication system. Pheromone receptors (PRs) are the key sensory elements at the beginning of that process. The codling moth (Cydia pomnonella) is an important pome fruit pest globally and a serious invasive species in China. Pheromonebased techniques have been used successfully in monitoring and controlling this species. We conducted ribonucleic acid sequencing analysis of the codling moth antennal transcriptome and identified 66 odorant receptors (ORs) in a population from Xinjiang province, China, of which 14 were PRs, including two novel PRs (CpomOR2e and CpomOR73). Four PRs that contain full-length open reading frames (CpomOR1, OR2a, OR5, OR7) and four PRs with ligands that have not been reported previously (CpomOR1, OR2a, OR5, OR7) were selected to deorphanize in the heterologous Xenopus oocyte expression system. Specifically, we found that CpomOR2a and CpomOR5 responded to (E, E)-8, 10-dodecadien-1-yl acetate (codlemone acetate). Furthermore, CpomOR5 (EC 50 = 1.379 × 10 −8 mol/L) was much more sensitive to codlemone acetate than CpomOR2a (EC 50 = 1.663 × 10 −6 mol/L). Since codlemone acetate is an important component of C. pomonella sex pheromone, our results improve the current understanding of pheromone communication in codling moths and will be helpful for the development of pest management strategies.

The oriental fruit moth, Grapholita molesta, is a worldwide pest that damages Rosaceae fruit trees. Sex pheromones play an important role in controlling this pest; however, the corresponding chemosensation mechanism is currently unknown. In this study, 60 candidate odorant receptors, including eight pheromone receptors (PRs), were identified by antennal transcriptome analysis. Expression profiles indicated that most PRs were highly expressed in the males, except GmolOR21 and GmolOR22, which were specifically expressed in the females. Among them, GmolOR2 was identified in response to the main sex pheromone Z8-12:OAc and E8-12:OAc, and its in vivo function was confirmed by RNA interference analysis. Electrophysiological analysis showed that the males had a significantly reduced sensitivity to the main pheromones after the knockdown of GmolOR2. Our research makes a better understanding of pheromone chemoreception and provides a theoretical basis to developing novel, efficient, and environmentally friendly insect attractants.