Functional Analysis Of Odorant-binding Proteins In The Oriental Fruit Fly,bactrocera Dorsalis

The oriental fruit fly,Bactrocera dorsalis(Hendel),is one of the most important pests the agriculture of tropical and subtropical countries.This species can damage over 250 species plants including economic fruits,such as orange,banana,mango,guava and melon.Due to its wide host-ranges,relatively wide ecological adaptation,high reproductive potential and dispersal capacity,this fruit fly has seriously hampered the development of the local fruit industry,and been listed as quarantine pest by many countries.Because of its biological characteristics,chemical insecticide applications are difficult to control effectively and led to contamination of the environment,as well as pesticide residues and the problems of food safety.In addition,male annihilation technique through methyl eugenol mass trapping could be ineffective.Thus,these issues have directed the search for novel,safety,effective and environment-friendly strategies to control B.dorsalis.Like other insects,the oriental fruit fly use its olfactory system to detect and respond to odors which are essential for foraging,mate recognition and oviposition.In insects,odorant-binding proteins(OBPs),chemosensory proteins(CSPs),odorant receptors(ORs),ionotropic receptors(IRs),and sensory neuron membrane proteins(SNMPs)are involved in peripheral olfactory processes,and as potential molecular targets,particularly OBP,OR and IR to develop novel control strategies that interfere with olfaction.Our previous transcriptome analysis of B.dorsalis(larvae,larvae,pupae and adult)provided a set of chemosensory genes and their expression profiles.This work have concluded that the genes involved in odorant reception and detection in B.dorsalis antennae.To insight into the OBPs response to attractant semiochemicals in B.dorsalis,the main contributions of this study are listed as follows:(1)B.dorsalis behavioral assay and electroantennogram(EAG)response to plant host volatiles,attractants and repellents was compared and analyzed in immaturity,sexual maturity and spawning periods.Also,comparative expression profile analysis of selected antennae-rich OBPs,pheromone binding protein related proteins(PBPRPs),and other olfactory gene families were analyzed in different physiological period.(2)Using fluorescence competitive binding assays and molecular docking(in silico),the binding affinities of the antennae-rich OBPs of B.dorsalis to attractive semiochemicals were measured(3)The gene function of the principal OBP(Bdor OBP83a-2)was studied,through RNA interference(RNAi)approach to inhibit the principal OBP(Bdor OBP83a-2)expression,and modulation of olfactory response phenotypes were compared using the EAG record and behavioral assay.1)To investigate the variation of olfactory attraction behavior in different physiological period such as sexual immaturity and sexual maturity,would been caused by olfactory peripheral nervous system.Firstly,the olfactory trap assay is used to measure olfactory attraction in sexual immaturity,sexual maturity or oviposition period of both sexes.The results show that almost the male and female were not responsible to previously published attractive compounds in the sexual immaturity.In contrast,both of male and female,were responsible to attractive compounds in the sexual maturity.Similar to the result of olfactory behavioral assay,in oviposition period,the female EAG responses to the majority of attractive compounds were significantly stronger than that in new emerged adults.In addition,male EAG responses to these compounds became stronger with a certain degree,especially when the male EAG responses to cedrol.Comparison of EAG responses to the attractive compounds in females,?-octalactone,?-nonanoic lactone,1-octen-3-ol,ethyl tiglate,Ethyl benzoate were higher than in any other compounds.In order to study whether both the behavior and EAG responses were determined by the transcription level of olfactory genes,q PCR was used to investigated the expression level of chemosensory genes from our previous transcriptome and their expression profiles,including the selected nine antenna-rich OBPs(included the PBPRPs,Bdor OBPlush,Bdor OBP19 a,Bdor OBP19d-1,Bdor OBP28 a,Bdor OBP56 h,Bdor OBP69 a,Bdor OBP83a-1,Bdor OBP83a-2,Bdor OBP84a-1),one antenna-specific CSP(Bdor CSP3),one ORCO(Bdor ORCO)and two antenna-specific SNMPs(Bdor SNMP1-1 and Bdor SNMP1-2)in sexual immaturity,sexual maturity or oviposition period.The result showed that 1)five OBPs in female were significantly upregulated in oviposition period when compared to sexual immaturity.In male,only Bdor OBP83a-2 was significantly upregulated in sexual immaturity,without other OBP members.2)Bdor SNMP1-1 were significantly down-regulated in oviposition period,and Bdor ORCO and Bdor SNMP1-2 had no significant changes.3).Eight OBPs(Bdor OBP19d-1,Bdor OBP56 h,Bdor OBP69 a,Bdor OBP83a-1,Bdor OBP83a-2,Bdor OBP84a-1,Bdor OBP84a-2,and Bdor OBPlush)were significantly upregulated in morning when compared to afternoon in mating period.In males,only Bdor OBP69 a was significantly upregulated.3)In addition,Bdor ORCO,Bdor SNMP1-1 and Bdor SNMP1-2,were significantly upregulated in morning when compared to afternoon in mating period.These olfactory genes upregulated in the particular physiological period such oviposition period and mating time,are suggested to play crucial roles in chemo-detection.2)In order to clarify the function and binding characteristics of OBP and CSP,five antenna-rich OBPs(Bdor OBP56 h,Bdor OBP83a-1,Bdor OBP83a-2,Bdor OBP84a-1,and Bdor OBP84a-2)and one antenna-rich CSP(Bdor CSP3),and their recombinant proteins was successfully heterologously expressed and purified,then the fluorescence competitive binding assays was used to measure the binding capacity of the OBP/CSP recombinant proteins with each of thirteen different compounds.The binding test results indicated that apart from Bdor CSP3 and Bdor OBP84a-2,all remaining OBPs can evidently bind to the different compounds examined.Two homologous OBP(Bdor OBP83a-1 and Bdor OBP83a-2)showed the higher affinity than the rest,and Bdor OBP83a-2 with the highest.Bdor OBP83a-2 showed the highest affinity to methyl eugenol and esters with the dissociation constant KD.The binding ffinity decreased followed the sequence of methyl eugenol,?-nonanoic lactone,?-octalactone,?-undecalactone,and ?-octalactone.Simultaneously,3D structure of 5 OBPs and 1 CSP were predicted and optimized,then thirteen compounds for their binding potential to 5 OBPs and 1 CSP using molecular docking(in silico)were screened.Combined the displacement binding data and molecular docking data,we conclude that Bdor OBP83a-2 could be a crucial OBP for detection of attractive semiochemicals.3)Previously,we had demonstrated that Bdor OBP83a-2 binds to attractive semiochemicals using the fluorescence competitive binding assays and molecular docking,but lacking direct functional evidence,so as to determine the role in B.dorsalis olfaction.Methyl eugenol and ?-octalactone were selected as attractive semiochemicals measured.Microinjection of ds RNA inhibits the expression of Bdor OBP83a-2 in antennae,then phenotypic effects included EAG response and attractant behavior of the phenotype were compared between RNAi treated B.dorsalis and its wild-type.Functional analysis indicated that reduction of Bdor OBP83a-2 transcript levels led to a significant decrease in EAG responses to methyl eugenol and ?-octalactone,as well as behavioral responses.These data suggest that Bdor OBP83a-2 mediate the behavioural response of attractive semiochemicals.