Olfactory Mechanism Of Β-ocimene Recognition In Hyphantria Cunea And Its Application

β-ocimene is an herbivore-induced plant volatile(HIPVs)released by mulberry trees induced induced through larval feeding of Hyphantria cunea(H.cunea).Contrary to the repellent behavioral responses to HIPVs observed in other insects,H.cunea males were significantly attracted to β-ocimene and β-ocimene could promote mating and oviposition in females.At present,the electrophysiological and behavioral responses of H.cunea to β-ocimene have been fully researched,but the neural and molecular olfactory mechanisms of β-ocimene recognition in H.cunea are unclear.In this study,the neural and molecular mechanisms of β-ocimene recognition by H.cunea were studied and applied with the techniques of "Y" tube assay,single sensillum recording(SSR),transcriptome sequencing technology,real-time quantitative PCR(RT-q PCR),transgenic Drosophila SSR assays,homologous modeling,molecular docking technology and biosensor technology,which provided new ideas for monitoring the occurrence of H.cunea and addressed the application value of insect olfactory mechanisms.The main findings are as follows:1.Electrophysiological and behavioral responses of H.cunea to β-ocimeneThe behavioral selection responses of adult H.cunea to β-ocimene was tested by "Y" tube assay,and the results showed that β-ocimene could attract H.cunea females and males at a concentration of 10 μg significantly.Females were more sensitive to low doses of β-ocimene.The electroantennogram(EAG)responses of adult H.cunea to β-ocimene and 14 host volatiles were compared,and it was found that the responses of females and males to β-ocimene at a concentration of 100 μg were significantly stronger than 7 volatiles,indicating that the EAG responses of H.cunea to β-ocimene were strong.The SSR responses of H.cunea females to β-ocimene were tested,and the results showed that H.cunea females recognized β-ocimene through short trichoid sensilla mainly,and the short trichoid sensilla were mainly distributed in the 13 th,14th and 17 th flagellar segments of the flagellum,and female adults could produce a SSR response to β-ocimene with a concentration as low as 10 ng.2.Antennal transcriptome analysis of male and female adults of H.cuneaSecond-generation transcriptome sequencing was performed on the female and male antennae tissues of H.cunea,and a total of 222 olfactory-related genes with complete open reading frames were identified,including 26 odorant receptors(ORs),78 odorant-binding proteins,46 chemosensory proteins,3 gustatory receptors,40 ionotropic receptors,11 sensory neuron membrane proteins,and 18 odorant degrading enzymes.A total of 96 sex-specific olfactory genes were identified,indicating that there was a difference in the sense of smell in male and female adults of H.cunea.The relative expression levels of 21 general odorant receptors in male and female antennae were tested by RT-q PCR,Hcun OR4 and Hcun OR5 were female-biased,and Hcun OR48,Hcun OR49 and Hcun OR50 were male-biased,which were consistent with the transcriptome difference analysis results.3.Olfactory molecular mechanism of β-ocimene recognition in H.cuneaThe ORs of H.cunea were analyzed by evolutionary tree,and the odorant receptor gene Hcun OR31 for recognizing β-ocimene was screened according to the principle of similar ligand of receptor proteins.The relative expression levels of the target gene in six tissues including antennae,head and leg were analyzed by RT-q PCR and the results showed that Hcun OR31 was mainly expressed in the antennae of adults.And there was no significant difference in male and female antennae.The function of Hcun OR31 was characterized by the transgenic Drosophila SSR assays combined with SSR,and the results showed that Hcun OR31 was the odorant receptor of H.cunea for recognizing β-ocimene.The experiments showed that Hcun OR31 was specific for β-ocimene and could recognize β-ocimene at 1 μg.4.The odorant receptor-derived peptide(ORP)sensors for detecting β-ocimeneThe transmembrane domains(TMDs)of Hcun OR31 were predicted by TOPCONS,and the results showed that Hcun OR31 had 7 TMDs.The TMDs of Hcun OR31 were modeled using SWISS-MODEL,and molecular docking was performed by Discovery Studio 2016.The ligand binding sites of Hcun OR31 for recognizing β-ocimene were Ala79,Ile82,Ile83,Ala209,Ile310,Ile313,Ala314,and Phe317.According to the binding sites,three segments of polypeptide fragments containing the binding sites(ORP)were designed and synthetized.The ORPs were connected to the single walled carbon nanotubes modified with carboxyl groups and interdigital electrode by native chemical ligation method and physisorption sequentially,the sensors were defined as ORP-sensor.Two ORP-sensors that could recognize gaseous β-ocimene were fabricated.The experiments showed that the ORP3-sensor could detect gaseousβ-ocimene as low as 1ppb and had a good selectivity.The responses to β-ocimene released from mulberry trees feeding on H.cunea were further tested,and the results showed that ORP3-sensor could detect as low as one 3-instar H.cunea larva feeding on mulberry trees.In summary,this study revealed the olfactory molecular mechanism of β-ocimene recognition in H.cunea and applied its molecular mechanism to fabricate two odor receptorderived peptide sensors that can monitor gaseous β-ocimene.The ORP-sensors had the potential for early monitoring and early warning of the occurrence of H.cunea larvae in the form of the Internet of Things.The results of this study provided innovative strategies and ideas for the development of dynamic technologies for monitoring pest occurrence.