| Flowering plants have evolved chemical strategies to attract pollinators.Floral scents,especially those aromatic compounds emitted by flower,can be accurately perceived and attracted to visit flowers.Syrphid fly is a group of insect pollinators in Diptera,and is also the natural enermy of aphids.However,little is known about how they detect aromatic compounds at molecular level.In this study,fourteen aromatic compounds were selected to Electroantennogram（EAG）assay.The putative aromatic-detecting ORs in E.corollae were predicted from full-function map of ORs in Drosophila melanogaster,and found orthologs of Dmel OR responding to aromatic volatiles.We then evaluated the tissue expression pattern of these two genes by Real-time Quantitative PCR（RT-q PCR）.We further characterized functions of candidate ORs in E.corollae using the Xenopus heterologous expression system.Finally,behavioural assays of both female and male E.corollae were performed using eugenol and methyl eugenol,that proved to be good ligands for OR25 E.corolla.Our study represents a first step in clarifying the molecular mechanisms underlying olfactory detection of aromatic volatiles in E.corolla,helps to improve pollination efficiency and provides a molecular basis for screening potential attractants of natural enemies.Main results were shown below.1.Fourteen aromatic compounds were used to Electroantennogram（EAG）assay in E.corollae.The results showed that all tested aromatic compounds at the concentration of 10μg/μL can elicit EAG responses of both male and female antenna of E.corolla.The lowest EAG value was elicited by 4-Methoxybenzyl alcohol for male,and highest EAG value was elicited by o-cresol for female and male.Female antennae were more sensitive than males’tested by o-cresol,phenol,p-anisaldehyde,benzyl alcohol,2-phenylethanol,methyl salicylate,4-methoxybenzyl alcohol（P<0.01）.No significant differences between sexes were recorded with eugenol,methyl eugenol,1,4-dimethoxybenzene,benzaldehyde and phenylacetaldehyde.2.In order to identify putative ORs tuned to aromatic compounds in E.corollae,we referred to full-function map of ORs in D.melanogaster and found orthologs of Dmel OR responding to aromatic volatiles.In total,we found 13 Dmel ORs tuned to aromatic volatiles.We found Ecor OR3,Ecor OR25 and Ecor OR28 from E.corolla are orthologs of13 Dmel ORs.On the basis of response profiles of Dmel ORs,we found that Dmel OR46a and Dmel OR71a,which are narrowly tuned to phenolic compounds and represent the orthologues of E.corollae OR25 and OR28,respectively.Taken together,the comprehensive analysis suggested that Ecor OR25 and Ecor OR28 may specifically respond to aromatic compounds.3.We therefore cloned full-length Ecor OR25 and Ecor OR28 genes from the mixed c DNAs of male and female antenna.They encode proteins of 388 and 384 amino acids,respectively,and possess 7 transmembrane domains（TMDs）.The real-time quantitative PCR（RT-q PCR）was implemented to evaluate the expression levels of Ecor OR25 and Ecor OR28 genes in antennae（A）,leg（L）,proboscis（P）,head without antennae and proboscis（H）,thorax（T）,abdomen without genitalia（Ab）and genitalia（G）of male and female E.corolla.The results indicate that both Ecor OR25 and Ecor OR28 are mainly expressed in antennae,more in males than in females（P<0.05）;very low expression was observed in other tissues,such as legs,proboscis,head without antennae and proboscis,thorax,abdomen,and genitalia.There had a significant difference between male and female antennae of two genes expression.4.The Xenopus oocytes expression system combined with two-electrode voltage clamp technique was used to characterize functions of Ecor OR25 and Ecor OR28 in E.corollae tested by 46 chemical compounds.The result shows that Ecor OR25/Orco is narrowly tuned to aromatic compounds.We recorded strong responses to 10-4M eugenol with an average current of 414.7±30.48 n A,followed by decreasing responses evoked by p-cresol,methyl eugenol,and o-cresol,etc.Thirty-two compounds included aliphatic alcohols,aldehydes,esters,terpenoids and heterocyclics were also tested,but none of these compounds elicited a response of Ecor OR25/Orco at the concentration of 10–4 M.In dose–response experiments,eugenol is still the strongest ligand with EC50 of 2.89×10-5M.Parallel experiments were performed for Ecor OR28/Orco.Ecor OR28/Orco only weakly responded to structurally related aromatic compounds p-anisaldehyde and4-methoxybenzyl alcohol at the concentration of 10–4 M.The results revealed that Ecor OR25 is one of the major receptors tuned to aromatic compounds.5.We selected two floral scent volatiles eugenol and methyl eugenol for behavioralassay of E.corolla.The result suggested that both male and female E.corolla were significantly attracted by eugenol and methyl eugenol at the concentration of 0.1 mol/L（P<0.05）,with eugenol being more effective on females（P<0.01）.No response to eugenol and methyl eugenol were observed for both male and female at concentration of 0.01mol/L. |
