(E)-?-Farnesene-Mediated Molecular And Olfactory Mechanism Of Aphid Localization In Eupeodes Corollae

(E)-?-farnesene(EBF)is an important chemical cue mediating the tritrophic interactions among plants,aphids,and hoverflies.On the one hand,EBF is an alarm pheromone component in most aphid species.The molecular mechanism of alarm pheromones detection in aphids has been elucidated in previous studies.On the other hand,EBF emitted by herbivore-induced plants can attract natural enemies.At present,little is known on the molecular mechanisms of EBF-mediated attraction of the natural enemies.In this study,a range of techniques,including behavioral assay,electrophysiological recording,scanning electron microscopy,transmission electron microscopy,real-time quantitative PCR(RT-q PCR),in vivo transgenic Drosophila system with single sensillum recording,and CRISPR/Cas9techniques,were used to study the molecular mechanisms of EBF-mediated aphid location in the predator Eupeodes corollae.These results provide a theoretical basis for efficiently screen behavioral chemoattractants of hoverflies and a new idea for sustainable control of aphids in agricultural ecosystems.The main results are as follows:1.Electroantennogram assay and behavioral choice to EBF in E.corollaeEBF with different dosages were used to electroantennogram(EAG)assay in E.corollae.The result showed that 0.2-2000?g EBF can elicit EAG responses of both male and female antenna of E.corollae.No significant differences between sexes were recorded by EBF with different dosages.The Y-tube olfactometer was used to determine the behavioral response of male and female E.corollae.The results showed that both sexes were extremely significantly attracted by EBF at the dose of 200?g and 2000?g(P<0.01).No response to EBF was observed from both sexes at the dose of 20?g.By compared behaviors of male and female adults to different doses of EBF in the wind tunnel,we found that 1000?g EBF could attract the hoverflies from long distances and significantly increase the retention ratio of the hoverflies.The behavioral choice of larvae to EBF was determined by the petri dish.The result suggested that larvae of E.corollae were significantly attracted by EBF at the dose of 20?g(P<0.05),while lower doses had no attractive effect.2.Identification of antennal sensilla in E.corollae adults and functional characterization of neurons to EBFThe morphology and ultrastructure of antennal sensilla of E.corollae adult were observed by scanning electron microscopy and transmission electron microscopy.A total of ten sensilla types were identified.B(?)hm bristles and sensilla chaetica(two subtypes)were distributed on the scape and pedicel of the antennae.The microtrichiae were widely distributed on the flagellum of antennae.Sensilla trichodea was the longest type of sensilla,was thick-walled and perforated by numerous pores.Cross-sections at the base of the sensilla showed one,two,or three dendrites in the lymph of the sensillum lumen.Sensilla basiconica(four subtypes)showed a thin,homogeneous cuticle through the hair shaft that was pierced by numerous,visible pores.There were 2 to 3 neurons.The outer dendritic segments in different subtypes may branch at somewhat different levels.Sensilla clavate(two subtypes)have a distal swelling with thin porous cuticular walls.The dendrites were highly lamellated in the sensilla lumen.Sensilla coeloconica was double-walled.There were two outer dendritic segments surrounded by a dendrite sheath,and which extended unbranched into the sensilla lumen.Sensilla styloconica,sensilla placodea,and sensory pit were also observed on the flagellum of antennae.The single sensillum recording result showed that only neuron A housed in subtype sensilla basiconica III can be activated by EBF and its structurally related chemicals,geranyl acetate and TMTT,while neuron B do not respond to any of these chemicals.3.Identification and functional characterization of odorant receptors detected by EBF in E.corollaeBase on the antennae transcriptome analysis of E.corollae and Episyrphus balteatus,fifteen odorant receptors(OR)of two hoverflies were screened in terms of sequence similarity(>80%).The expression levels of these odorant receptors in male and female antennae were determined by RT-q PCR.The result showed that the relative expression level of Ecor OR14 gene in female antennae was significantly higher than that of male antennae,while other 14 odorant receptor genes showed no significant difference between male and female antennae.In vitro functional studies found that Ecor OR3 can be activated by EBF,geranyl acetate and TMTT.The transgenic Drosophila Or67d^GAL4mutant system was used to further verify the function of Ecor OR3.The results showed that Ecor OR3-expressing neurons housed in at1 sensilla of Drosophila were strongly activated by EBF and its structurally related chemicals,which was consistent with that of Xenopus oocyte expression system.4.Gene knockout and functional characterization of Ecor OR3 in E.corollaeThe CRISPR/Cas9 technique was used to knocked out Ecor OR3 gene in E.corollae.Four genotypes in G0 generation were identified Then,hoverflies containing mutation genotype of Ecor OR3^-/-with413 bp deletion resulting in premature stop codon were screened through four generations and used in further tests.The results showed that the electrophysiological responses and attraction behaviors to EBF,geranyl acetate and TMTT were significantly reduced by Ecor OR3^-/-mutation in both sexes,while the response to the positive control(E)-2-hexenal was not affected.In another set of experiments,we compared the behaviour of Ecor OR3^-/-mutant hoverflies with that of WT in wind tunnel experiments.The results showed that Ecor OR3^-/-mutation adults lost the long-range detection of EBF.Thus,in similar experiments,we compared the behaviour response of WT larvae with that of Ecor OR3^-/-mutants.As verified with adults,Ecor OR3^-/-mutation larvae abolishes detection of EBF,GA and TMTT.Based on the behaviour responses of adults and larvae to different doses of EBF,and the preference of larvae to the pea aphid Acyrthosiphon pisum that released EBF,we confirmed that Ecor OR3 is the key odorant receptor for EBF recognition in adult and larvae of E.corollae and plays a vital role in the long-distance localization of adult and the larvae searching for aphids in close range.