| The Asian citrus psyllid(ACP),Diaphorina citri Kuwayama(Hemiptera:Psyllidae),is an important pest in citrus production.Both nymphs and adults of ACP feed on leaf sap and secrete a large amount of honeydew to reduce photosynthesis and cause citrus fuliginous.Furthermore,ACP is the vector of Candidatus Liberibacter spp.,which causes Citrus Huanglongbing(HLB or citrus greening),a destructive disease of citrus,and currently there is no treatment for it.Therefore,the most practical way to control HLB is to prevent the spread of the ACP.At present,the application of insecticides is considered to be effective in reducing the ACP populations in many areas,however,the abuse of chemical pesticides has led to "3R"(resistance,residue,resurgence).Therefore,novel and environmentally friendly management strategies to control the ACP are urgently needed.Push-pull strategy: a control method that uses attractive odours or repellent odours to interfere with pest behaviour.Push-pull strategy is environmental-friendly as the compounds are generally non-toxic.As push-pull strategy mainly depends on the olfactory perception of pests,the study of olfactory mechanism of the ACP can provide theoretical basis and technical reference for the new control strategy.Insects recognize the odor in the surrounding environment through the chemical sensors on the antennae.In these sensors,there are peripheral olfactory neurons and a large number of proteins involved in olfactory perception,among these proteins,odorant-binding proteins(OBPs),odorant receptors(ORS),and ionotropic receptor(IRS)are considered to be the important target to interfere with insect olfactory perception.Some of the previous studies on olfactory genes of the ACP have not clearly revealed the recognition function of olfactory proteins to specific odours.In addition,neuropeptides and their receptors play an important role in the regulation of insect olfactory perception,but there is no relevant research.Therefore,the molecular mechanism of olfactory perception of the ACP was studied as follows: 1)The genes that involved olfactory perception of the ACP were identified based on 12 transcriptome data of adult ACP and genome data.2)The expression patterns of these olfactory genes in different tissues and different developmental stages were analyzed.3)The OBPs and CSPs of the ACP were expressed and purified by prokaryotic expression technology.4)The binding characteristics of the purified OBPs and CSPs with compounds were studied by fluorescence competitive binding technology,and the binding state of OBP and the compound molecules was demonstrated by simulating molecular docking.5)The function of Dcit OBP6 in perception of an important host volatiles β-caryophyllene was verified by RNA interference,EAG and behavior test.(6)In order to find out the neuropeptides or receptors that may be involved in regulating olfactory perception of the ACP,the neuropeptides and their receptors were identified and their expression characteristics were analyzed.The results are as follows:(1)The antennae,head,thorax,leg,abdomen,male genitalia and female ovipositor of male and female adult ACPs were used for RNA-seq,297614 unigenes were obtained.The genes that involved olfactory perception of the ACP were identified based on the RNA-seq and genome data.A total of 106 olfactory genes,including 9 OBPs,13 CSPs,52 ORs,32 IRs and 1 SNMP were identified,and all these genes were submitted to the NCBI Gen Bank.Phylogenetic analysis showed the evolutionary relationship of these proteins among different species.(2)The expression profiles of these olfactory genes in different tissues and developmental stages of adult ACPs were studied by FPKM value and q RT-PCR.The results showed that Dcit OBP2,Dcit OBP7,Dcit OBP8,Dcit OBP9,Dcit CSP12 and Dcit Orco were all highly or specifically expressed in antennae.Two OBPs,Dcit OBP3 and Dcit OBP6 were highly expressed not only in the antennae of females and males,but also in the ovipositors of females.These results suggest that these proteins may be involved in olfactory perception in the ACP.(3)The recombinant proteins of Dcit OBPs and Dcit CSPs were expressed using a bacterial system,four proteins,Dcit OBP3,Dcit OBP6,Dcit OBP8 and Dcit CSP12,were successfully expressed.SDS-PAGE showed that these proteins existed in the supernatant.The target proteins were obtained after digestion and re-purified.(4)The fluorescence competitive binding assay was performed to investigate the binding properties of the four proteins to ligands.Five host plant volatiles(Methyl salicylate,Linalool,(1S)-(-)-α-pinene,(1R)-(+)-α-pinene and β-caryophyllene),two putative sex pheromone(Acetic acid and Dodecanoic acid)and one repellent odorant(Dimethyl trisulfide)were selected for the competitive binding assay.The results showed that β-caryophyllene show higher affinities to Dcit OBP3,Dcit OBP6 and Dcit CSP12.Furthermore,Dcit OBP6 exhibited the highest affinity towards β-caryophyllene.The three-dimensional model of Dcit OBP6 shows that hydrophobic amino acid residues form a hydrophobic binding pocket.Docking results showed that ten amino acid residues Lys11,Ile67,Thr70,Ser71,Met74,Leu75,Val83,Val110,Phe118 and Lys119 of Dcit OBP6 interact with β-caryophyllene and suggests that these amino acid residues may play an important role in the binding of Dcit OBP6 to β-caryophyllene.(5)RNA interference was used to study the function of Dicit OBP6 in the perception ofβ-caryophyllene.After the female adults of D.citri were injected with ds RNA or RNase-free water,the expression level of Dcit OBP6 in ds Dcit OBP6 treated psyllids show a significant reduction compared with ds GFP and RNase-free water treatments.To detect the role of Dcit OBP6 in response to β-caryophyllene,the ds Dcit OBP6 injection significantly reduced antennae EAG activity in response to β-caryophyllene and the preference of ds Dcit OBP6 treated females to β-caryophyllene was significantly reduced compared to ds GFP and RNase-free water treated insects.The results strongly suggest that Dcit OBP6 is involved in the perception of β-caryophyllene.(6)The neuropeptides and their receptors of the ACP were identified via adult tissues transcriptome analysis.40 neuropeptide precursor genes and 42 neuropeptide receptor genes were found.The present transcriptional sequences of D.citri appear to contain most of the genes for insect neuropeptides and GPCRs except for several genes.The expression patterns of these genes in tissues and developmental stages were analyzed,the results show that neuropeptides ASTA,CCH1,s NPF,SIF,TK,and the neuropeptide receptors Dc A24(TK)and Dc A14(CCH1)were highly expressed in the head and antennae of adults,indicating that these neuropeptides or receptors may be involved in the regulation of olfactory perception of the ACP. |
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