The Function And Mechanism Of Dastarcus Helophoroides OBPs In Recognizing And Responding To Herbivore-Induced Plant Volatiles Of Pinus Massoniana

In a variety of ecosystems such as forestry,semiochemicals play an important role for natural enemy insects in many physiological processes such as host localization,oviposition,and mating.Among which,herbivore-induced plant volatiles（HIPVs）,which were released after plants infested by herbivores,are considered as key semiochemicals in the host localization process of natural enemy insects.The perception of semiochemicals by insects relies on a variety of functional proteins in their olfactory system.And it is generally accepted that odorant molecules are mainly transported by odorant-binding proteins（OBPs）to be transported and cross the lymph to reach the vicinity of odorant receptors（ORs）,which are assisted by odorant receptor-coreceptor（ORco）to complete signal transduction and elicit behavioral responses.OBPs are considered as the first‘filter’in olfactory system and play an important role in the recognition processes of external odorants for insects.However,the physiological functions and mechanisms of insects OBPs are still in the fog.Dastarcus helophoroides（Fairmaire）is an effective parasitic natural enemy of important pest Monochamus alternatus Hope,which has been widely used in the pest control.Previous studies have shown that the olfaction played an important role in the host localization process of D.helophoroides.Compared with the healthy Pinus massoniana Lamb,D.helophoroides were significantly attracted by P.massoniana after infested by M.alternatus,which suggested that HIPVs emitted by P.massoniana might play an important role in the host localization process of D.helophoroides.To investigate the olfactory recognition mechanism of D.helophoroides,our group have identified 22 full-length sequences of DhelOBPs by transcriptome sequencing,however,the olfactory functions and mechanisms of DhelOBPs which are involved in the recognition process of HIPVs are not clear.Therefore,in this study,by D.helophoroides as the research object,we used chemical ecology,bioinformatics and molecular biology to systematically explore the olfactory functions of DhelOBP involved in the recognition process of HIPVs from P.massoniana at the transcriptional and protein levels,and analyzed the induced response patterns of DhelOBPs exposure to HIPVs,with the aim of providing a theoretical basis for exploring the functions and mechanisms of DhelOBPs in the chemical communication process of D.helophoroides.The main results were as follows:1.The primary structures and spatio-temporal expression characteristics of DhelOBPs in D.helophoroides were investigatedBased on the full-length sequences of 22 DhelOBPs identified by previous studies,2DhelOBPs were found to be misidentified,which meant D.helophoroides had 20 full-length sequences of DhelOBPs.The expression characteristics of 20 DhelOBPs were determined at different developmental stages and in different tissues of male and female adult of D.helophoroides using quantitative real-time PCR（q RT-PCR）.The results showed that the expression levels of DhelOBPs in the antennae varied significantly with adult development.Among which,the expression levels of 13 DhelOBPs increased significantly with adults’development,while the expression levels of 4 DhelOBPs significantly decreased.Different trends in expression levels of DhelOBPs might be related to their physiological functions in specific physiological states.The expression levels of DhelOBPs also showed significant tissue specificity.The results revealed that that 13 DhelOBPs were mainly expressed in adults’antennae,among which 8 DhelOBPs were specifically expressed in antennae;and compared with other DhelOBPs,the expression levels of DhelOBP4,DhelOBP5,DhelOBP9,DhelOBP13,DhelOBP14,DhelOBP18 and DhelOBP21 were significantly higher.The DhelOBPs which were specifically expressed or high expressed in the antennae might be involved in the olfactory recognition process of D.helophoroides.2.The binding characteristics of recombinant DhelOBPs with HIPVs and the olfactory function of DhelOBP4 were clarifiedBased on the principle of high sequence identity with the reported OBPs with clear olfactory functions and specifically expressed or high expressed in adults’antennae,DhelOBP4,DhelOBP5,DhelOBP6,DhelOBP14,DhelOBP18and DhelOBP20 were selected as target proteins for subsequent olfactory function studies.Based on the idea of reverse chemical ecology,the protein 3D models of the above six DhelOBPs were constructed by Alpha Fold Colab,and the molecular docking studies revealed that the above DhelOBPs have similar binding energy(-5.4～-7.4 kcal mol^-1)with HIPVs.The recombinant expression vectors of the above six DhelOBPs were constructed,and the corresponding recombinant pure proteins were obtained by prokaryotic expression and protein purification experiments.The binding characteristics of recombinant DhelOBPs with 8 HIPVs were measured by fluorescence competition binding assays.The results showed that only DhelOBP4 had broad binding affinities with 8 HIPVs,and 3 HIPVs with the strongest binding affinities wereα-Terpineol,γ-Terpinene and p-Cymene in order,with dissociation constants（K_i）of 9.6±0.1mM,10.1±0.3mM and 12.0±0.9mM（lower K_i values indicate stronger binding ability）,respectively.While the other 4 DhelOBPs did not show any binding affinities with 8 HIPVs（K_i>50mM）.Using Y-tube olfactometer,the behavioral responses of D.helophoroides to 3 HIPVs with the strongest binding affinities with DhelOBP4（α-Terpineol,γ-Terpinene and p-Cymene）were investigated.The results showed that both female and male adults were significantly attracted by 1/1000 v/vγ-Terpinene and p-Cymene,while 1/1000 v/vα-Terpineol did not elicit any behavioral response.After silencing of DhelOBP4 gene,the tendency behavior of males to 1/1000 v/vγ-Terpinene and p-Cymene turned to no significant behavioral response,while the tendency behavior of females to 1/1000 v/vγ-Terpinene and p-Cymene turned to significant avoidance behavior.DhelOBP4 plays an important role in the recognition processes ofγ-Terpinene and p-Cymene,but there may be different recognition mechanisms of its action in female and male adults.3.The effect of HIPVs exposure on the expression levels of DhelOBPs and Dhel ORs was exploredUsing Y-tube olfactometer,the behavioral responses of female and male adults of D.helophoroides to different concentrations（1/100 v/v and 1/1000 v/v）of 8 HIPVs were investigated（a total of 32 groups of behavioral experiments）.The results（including the results of above behavioral experiments）showed that female adults were significantly attracted by p-Cymene,γ-Terpinene,Terpinolene and（-）-Verbenone at 1/1000 v/v,and Terpinolene and Terpinen-4-ol at 1/100 v/v（active behavioral responses group）;and were significantly avoided by Fenchone,Camphor,and Terpinen-4-ol at 1/1000 v/v,andγ-Terpinene,Fenchone,α-Terpineol,and（-）-Verbenone at 1/100 v/v（active behavioral responses group）.The male adults were significantly attracted by p-Cymene andγ-Terpinene at 1/1000 v/v（active behavioral responses group）;and significantly avoided by Camphor and（-）-Verbenone at 1/1000 v/v,andα-Terpineol at 1/100 v/v（active behavioral responses group）.The other behavioral experiments of both male and female adults did not show significant behavioral response（no behavioral responses group）.The mRNA expression levels of DhelOBPs were determined by qRT-PCR after 1 h exposure to 8 HIPVs at different concentrations（1/100 v/v vs.1/1000 v/v）in both male and female adults of D.helophoroides.Compared with the control（exposure to solvent liquid paraffin）,the expression levels of DhelOBPs showed significant differences to adults’exposure to HIPVs.The expression levels of DhelOBPs mostly showed group responses,which meant that the expression levels of multiple DhelOBPs were up-or down-regulated after exposure to a single substance.The analysis revealed that the group responses of DhelOBPs was significantly correlated with the types of behavioral responses of D.helophoroides to different concentrations of HIPVs.The expression levels of most DhelOBPs in the no behavioral responses group showed different degrees of down-regulation,with DhelOBP5、DhelOBP9、DhelOBP11 and DhelOBP22 showing significant down-regulation（p<0.05）.The number of down-regulated DhelOBPs in the active behavioral responses group was less than that in the no behavioral responses group,and only DhelOBP5 showed significant down-regulation in all treatments of the active behavioral responses group;while the expression levels of 9 DhelOBPs（DhelOBP6、DhelOBP10、DhelOBP13、DhelOBP14、DhelOBP15、DhelOBP16、DhelOBP17、DhelOBP18 and DhelOBP19）were never significantly down-regulated（significant up-regulation or no significant change）.The group responses mechanisms of DhelOBPs suggested that the recognition of a single substance by D.helophoroides might be the result of multiple DhelOBPs acting together.A total of 39 Dhel ORs were identified by transcriptome sequencing of RNA from male and female antennae after exposure to p-Cymene,γ-Terpinene and（-）-Verbenone at 1/1000v/v.30 Dhel ORs were identified as complete open reading frames,encoding 303-431 amino acids and 4-7 transmembrane domains,respectively.The remaining 9 Dhel ORs had incomplete open reading frames,encoding 203-428 amino acids and 2-6 transmembrane domains,respectively.The TPM values of all Dhel ORs were calculated based on the transcriptome read counts value.The results revealed that several different Dhel ORs showed down-regulation trends after exposure to all 3 HIPVs mentioned above,while Dhel ORco also showed down-regulation trends after exposure.The down-regulation of Dhel ORs genes suggested that they might be involved in the recognition processes of corresponding HIPVs.In summary,based on the analysis of the primary structures and spatio-temporal expression characteristics of DhelOBPs,our study successfully identified DhelOBP4 is an important functional protein in the recognition processes of p-Cymene andγ-Terpinene by D.helophoroides,and proposed a model for the recognition mechanism of semiochemicals through DhelOBPs’group responses.The results of this study provide an important theoretical basis for further investigation of the biological functions of DhelOBPs in D.helophoroides,and also provide new ideas for further investigation of the olfactory recognition mechanisms of insect OBPs.