| Pagiophloeus tsushimanus is a new recorded wood borer in China,which was first reported in 2014.At present,Cinnamomum camphora forests in all administrative regions of Shanghai have been damaged by this weevil pest,with a trend of further spread.Because the larvae are"latent"in the trunk during the whole damage period,they are hidden,destructive and difficult to control,which seriously hinders the normal growth of C.camphora.There is no relevant research report on the effective control technology of this pest,especially the development of monitoring and control technology based on chemical attractants.In addition,the molecular mechanism of olfactory perception of C.camphora has not been reported.The mostly common host plant of this insect is C.camphora in the field.However,it can also complete its life cycle in C.chekiangensis and Phoebe chekiangensis in the laboratory.C.chekiangensis and P.chekiangensis are important tree species for urban greening in Shanghai.Therefore,the olfactory selectivity of adults to volatiles of three Lauraceae plants was studied.In addition,the theory of chemical ecology,insect ethology,insect physiology,antennal electrophysiology,analytical chemistry,and molecular biological techniques were used to isolate and identify the key components of volatiles from host plants and insects,and identify the behaviorally active components in the volatiles for intraspecific and interspecific chemical communication of P.tsushimanus.Finally,the active components of volatile semiochemicals were identified,which provided the basis for the development of trap lure for P.tsushimanus.At the same time,the tissue expression characteristics of odorant-binding protein genes(PtsuOBPs)and the function of key PtsuOBPs binding odorants were explored.The identification of volatile active components can be further verified by the results of the molecular biology studies.These results lays a foundation for further revealing the olfactory sensing mechanism of C.camphora.The main results are as follows:1.The results showed that female and male adults preferred to feed on the branches of C.camphora,and female adults preferred to oviposit on the branches of C.camphora.Both female and male adults were attractive to the volatiles from C.camphora,but repelled to the volatiles from C.chekiangensis and P.chekiangensis.In conclusion,the female and male adults had selective preference for the volatiles from C.camphora.The results can be used as a reference for the risk assessment of host shift in P.tsushimanus.2.Many volatiles emitted by C.camphora,C.chekiangensis and P.chekiangensis were identified by using dynamic headspace aeration,GC-MS,internal standard and retention index.D(+)-camphor was preliminarily identified as a key host volatile for host identification and location of P.tsushimanus adults,which was based on the techniques of GC-EAD,GC-MS,EAG and laboratory behavioral assays.In the volatiles of C.camphora,D(+)-Camphor and ocimene could induce GC-EAD reaction in both male and female adults.The EAG responses of female and male adults to D(+)-Camphor and ocimene were higher than other volatile compounds released from C.camphora.The results of bioassay in the laboratory and field showed that D(+)-Camphor had attractive activity to both male and female adults.In the field,a stronger attraction of this compound was obatined at a concentration of 5 mg/m L.There was no significant difference in the number of trapped female and male adults.Neither ocimene nor solvent control group(isopropanol)could attract adults,indicating that ocimene had no attractive activity.Moreover,ocimene had no synergistic effect on D(+)-camphor.Compared with other trapping devices,the self-made trapping bucket is more suitable for trapping this weevil pest in the field.In addition,the relative amount of D(+)-camphor in the volatile from C.camphora was significantly higher than that in C.chekiangensis and P.chekiangensis,but there was no significant difference in that of ocimene among the three Lauraceae plants,which further indicated that D(+)-camphor was one of the main chemicals orienting P.tsushimanus adults to C.camphora.Therefore,D(+)-camphor is a key olfactory signal chemical for adults to identify host plants.The host-volatile lure based on D(+)-camphor can be used to trap and monitor the adults of P.tsushimanus.3.Usingvariouschemicalecologicalmethods,(1?,3?,4?,6?)-4,7,7-trimethylbicyclo[4.1.0]heptane-3-ol was identified as a male-produced pheromone component of in P.tsushimanus.D(+)-camphor and D-borneol are released by adults,but are probably derived from host plants C.camphora and released from adult body cuticle or the faeces.The results showed that male and female adults had antennal responsed to(1?,3?,4?,6?)-4,7,7-trimethylbicyclo[4.1.0]heptane-3-ol,and the EAG reaction value was higher.In the field,a stronger attraction of this compound was obtained at a concentration of 0.5 mg/m L.There was no significant difference in the number of trapped female and male adults.Neither D-borneol nor solvent control group(isopropanol)could attract adults,indicating that ocimene had no attractive activity.Moreover,All the selected volatile components of C.camphora had no synergistic effect on the pheromone component.Therefore,(1?,3?,4?,6?)-4,7,7-trimethyl bis(4.1.0)heptane-3-alcohol is a male-produced aggregation pheromone in P.tsushimanus.The pheromone-based trap lure can be used to trap and monitor the adults of P.tsushimanus.4.Using Illumina Hiseq TM2000 technology platform to sequence the transcriptome of P.tsushimanus adults,58 088 unigenes were assembled and 29 824 unigenes were successfully annotated.Through homology comparison and bioinformatics analysis,39 odorant-binding protein genes(PtsuOBPs)were cloned and identified,named PtsuOBP1-39.Bioinformatics analysis showed that the coding sequences of PtsuOBPs had typical OBP structural characteristics,which contained 4-6 conserved cysteine sites.Phylogenetic analysis showed that there was a high degree of amino acid sequence conservation between PtsuOBPs and OBPs of other Coleoptera insects,indicating that PtsuOBPs were relatively conservative in evolution.q RT-PCR was used to analyze the expression characteristics of PtsuOBPs in P.tsushimanus adults.The results showed that the relative expression levels of most PtsuOBPs in female and male adults antennae were significantly higher than those in other tissues.Based on the screening of key PtsuOBPs induced by D(+)-Camphor and(1?,3?,4?,6?)-4,7,7-trimethylbicyclo[4.1.0]heptane-3-ol,D(+)-Camphor could induce up-regulation of the expression of five PtsuOBPs genes,including PtsuOBP7,PtsuOBP16,PtsuOBP24,PtsuOBP33and PtsuOBP39,while 1?,3?,4?,6?)-4,7,7-trimethylbicyclo[4.1.0]heptane-3-ol could induce up-regulation of the expression of PtsuOBP4,PtsuOBP11,PtsuOBP16,PtsuOBPP26 and PtsuOBP39.These key PtsuOBPs were screened to lay a foundation for the functional analysis of ptsuobps.5.Six key odorant-bingding proteins(PtsuOBPs),including PtsuOBP7,PtsuOBP11,PtsuOBP16,PtsuOBP24,PtsuOBP33 and PtsuOBP39,were obtained by prokaryotic expression and nickel ion resin gravity column purification.The binding ability of these PtsuOBPs to the identified host attractant D(+)-camphor and pheromone component(1?,3?,4?,6?)-4,7,7-trimethylbicyclo[4.1.0]heptane-3-ol was determined by fluorescence competitive binding method.The binding ability of PtsuOBP33(Ki=6.24?M)to D(+)-camphor was strong,and the binding ability of PtsuOBP39(Ki=1.11?M)to pheromone was medium.In addition,the main types and functional sites of PtsuOBP33 binding D(+)-camphor and PtsuOBP39 binding(1?,3?,4?,6?)-4,7,7-trimethylbicyclo[4.1.0]heptane-3-ol were verified by homologous modeling and molecular docking,combined with the point mutation technique of overlapping extended PCR and fluorescence competitive binding method.PtsuOBP33 mainly bound D(+)-camphor by the hydrogen bond of glutamine(Gln94)residue,and PtsuOBP39 bound(1?,3?,4?,6?)-4,7,7-trimethylbicyclo[4.1.0]heptane-3-ol mainly through hydrogen bonding and hydrophobic interaction.The hydrogen bonding site is lysine(Lys54)residue,and the hydrophobic interaction site is methionine(Met89)residue.The results showed that there were two odorant-binding proteins PtsuOBP33 and PtsuOBP39 with relatively strong binding ability to D(+)-camphor and(1?,3?,4?,6?)-4,7,7-trimethylbicyclo[4.1.0]heptane-3-ol,respectively,which further verified and supported the conclusion that D(+)-camphor is a key plant volatile for host recognization and location,and(1?,3?,4?,6?)-4,7,7-trimethylbicyclo[4.1.0]heptane-3-ol is a pheromone component,in P.tsushimanus. |
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