Olfactory Mechanisms Of Host Location In Chinese Citrus Fly,Actrocera Minax Based On Omics Analysis

During the co-evolution of insects and plants,phytophagous insects possess highly advanced olfactory system which play pivotal roles in finding host,host location,mating and oviposition behavior.However,the underlying molecular mechanism of host location in phytophagous insects is not clear.As an oligophagous deleterious tephritid pest,Bactrocera minax female oviposits in citrus fruits and larvae develop inside,making a significant threat to citrus industry.How the females locate host fruits relying on olfactory system remians unknown.As such,we firstly conducted choice and no-choice assay to reveal the preferred host fruits of B.minax in this study,and then the potential volatile from the hosts were screened using Gas chromatography-mass spectrometry(GC-MS);Secondly,genome and transcriptome were constructed to screen key olfactory genes,tissue and time expression patterns were analyzed to predict the function of these target genes.Then the function of key OBPs and CSPs genes were identified using RNA interference(RNAi)as well as fluorescence competition combined experiments;Finally,the function of Bmin ORCO gene of B.minax was explored by combinging proteomic and transcriptomic analysis.The main results are as follows:1.The selection of B.minax toward different hosts and screening volatile substances from navel orangeWe chose three hosts,navel orange(Citrus sinensis Osbeck cv Newhall),mandarin orange(Fortunella japonica)and kumquat(Nobis tangerine),which were reported seriously damaged by Chinese citrus fly,to conduct choice and no-choice tests.The results showed that the number of eggs and oviposition wounds were significantly different among hosts in the choice experiments,B.minax preferred to oviposit on navel oranges,followed by mandarin oranges and kumquats.The no-choice experiments also suggested that the most suitable oviposition host of B.minax was navel orange.The headspace adsorption method was used to collect the volatiles of immature navel orange at different periods,and then the volatiles were analyzed by GC-MS.Totally 115 compounds were identified,among which four common volatiles,D-Limonene,1,4-dichloro-Benzene,β-Ocimene and(-)-trans-Caryophyllene,at different periods were selected for Y-tube olfactometer experiments.The results showed that D-limonene was highly attractive to sexually mature females of B.minax.2.Identification of olfactory genes in B.minax based on omics and expression pattern analyzitionWe constructed the genomes of Bactrocera minax and Bactrocera dorsalis and compared with the available genomes of tephritid pests published in NCBI database,Ceratitis capitata and Zeugodacus cucurbitae.Comparative genomes showed that the olfactory genes number is 230(B.dorsalis),195(C.capitata),192(Z.cucurbitae)and164(B.minax),respectively.Based on B.minax reference genome,we further constructed the head transcriptome of B.minax at different stages.Using q RT-PCR technology to analyze the tissue and time expression patterns,we found that Bmin CSP,Bmin OBP9,Bmin OBP13 and Bmin OBP21 were specifically expressed on female and male antennae,Bmin OR4 was specifically expressed on male and Bmin IR5 was specifically expressed on female antennae,whereas Bmin OBP8 was specifically expressed on the leg.3.Functional analysis of olfactory genes in B.minaxThe olfactory genes Bmin CSP and Bmin OBP21 which specifically expressed on adult antennae were selected as potential key olfactory genes.Compared with GFP-injected fly(control),ds Bmin OBP21-treated B.minax and ds Bmin CSP-treated B.minax had lower electrophysiological response to Dlimonene(attractant).The choice tests showed that the number of B.minax selecting navel orange were decreased when Bmin CSP and Bmin OBP21 were knock-down by RNAi.These results indicated that Bmin CSP and Bmin OBP21 are the key genes for seeking host and host localization in B.minax.4.Binding characteristics of Bmin CSP and Bmin OBP21 to volatiles in B.minaxThe binding characteristics of Bmin CSP and Bmin OBP21 proteins were analyzed after prokaryotic expression in vitro.The fluorescence competition binding experiments showed that Bmin CSP had binding ability with D-limonene,caryophyllene,and eicosane.Bmin OBP21 had a strong binding ability with heptadecane,and had binding ability with caryophyllene.This difference may be related to the tertiary structure of the proteins.Homology modeling and molecular docking of Bmin CSP and Bmin OBP21 were performed to predict their protein structure,the molecular docking of Bmin CSP and1-NPN showed that the 1-NPN molecule was surrounded by Thr5,Leu12,Asp38,Met41,Leu42,Lys60,Gln61 and Gly64,the molecular docking of Bmin OBP21 and 1-NPN showed that the 1-NPN molecule was surrounded by Leu4,Phe25,Lys31,Thr43,Leu46,Het47,His50,Leu67,Gln69 and Thr103.5.Functional analysis of Bmin ORCO gene in B.minax based on proteome and transcriptomeThe choice test showed that the number of B.minax adults selecting navel orange were significantly decreased when Bmin ORCO were knock-down,compared with control.We constructed B.minax proteomes and transcriptomes respectively,after RNAi silenced Bmin ORCO gene.In comparison with the control group,5 OBPs genes and 6 ORs genes were significantly down-regulated in comparative transcriptomes;5 OBPs genes were significantly down-regulated in comparative proteomes;combined analyses of proteomes and transcriptomes showed that there were 4 OBPs genes genes down-regulated.The tissue and time expression patterns of these 4 OBPs genes were analyzed by q RT-PCR,we found that Bmin OBP8 a was specifically expressed on newly emerged female antennae,while Bmin OBP19d2 was specifically expressed on mature male antennae.In summary,our study revealed that B.minax preferred to oviposit in navel orange,and D-limonene has strong attractive to sexually mature females.Based on B.minax genome and transcriptome as well as express pattern analysis,we screened key olfactory genes specifically expressed on female and male antennae.Silencing of Bmin CSP and Bmin OBP21 significantly influenced antennal response to D-limonene,and the number of B.minax selecting navel orange decreased.After Bmin ORCO were knock-down,combining proteomic and transcriptomic analysis,we found that Bmin OBP8 a,Bmin OBP19d2,Bmin OBP99a3 and Bmin OBP99c2 were down-regulated in RNA and protein levels.These results indicate that Bmin CSP,Bmin OBP21 and Bmin ORCO are of vital genes for seeking host and host location in B.minax,which broaden our knowledge regarding olfactory location in Tephritidae pests,and pave the way for screening potential target genes and developing plant attractants for pest management.