| Ectropis obliqua prout and Ectropis griscens Warren are two closely related tea geometrid species with similar morphology,low degree of genetic differentiation and similar feeding habits.It mainly harms tea plants in the larval stage.Larvae mainly host plants through the chemoreceptors in the head,and then feed on leaves.Based on the successful construction of the transcriptome Library of Etropis obliqua and Ectropis griscens in the early stage of the laboratory,the chemosensory proteins(CSPs)of two closely related tea geometrid species,Ectropis obliqua and Ectropis grisescens were identified by bioinformatics analysis,and the identified homologous genes were analyzed by sequence alignment and phylogenetic analysis.The open reading frame(ORF)of Eobl CSP10 gene of Ectropis obliquawas used to design specific primers to clone and identify the potential homologous gene Egri CSP10 of Ectropis griscens.The evolutionary relationship of Eobl CSP8 and Eobl CSP10 and Egri CSP8 and Egri CSP10 in Lepidoptera was studied by sequence alignment and phylogenetic tree construction analysis.Then,was studied by using the real-time quantitative PCR experiment detailed Eobl CSP8 and Eobl CSP10,Egri CSP8 and Egri CSP10 homologous genes in between Ectropis obliqua and Ectropis grisescens.In addition,the effects of volatile induction and Wolbachia on the expression and distribution of this gene were discussed,which provided a theoretical basis for the research and development of target functional gene mediated larval behavior regulation mechanism of two closely related tea geometrid species and related green control technologies.The results are as follows:1.The sequence alignment and evolutionary analysis showed that there were18 pairs of homologous genes of chemosensory protein between Ectropis obliqua and Ectropis grisescen,which were highly similar and conserved.Full-length c DNA sequences of Egri CSP10 genes were cloned successfully,the amino acid sequences,physicochemical properties,secondary and tertiary structures of CSP8 and CSP10 were analyzed.The results show that they conform to the basic characteristics of CSPs,which contain 4 cysteine residues and can form 2 disulfide bonds.The secondary structure has six α-helices,and the three-dimensional structure is spherical.After removing the same amino acid sequence of signal peptide Eobl CSP8 and Egri CSP8,it was named Eobl-Egri CSP8.The functional amino acid sequences of Eobl CSP10 and Egri CSP10 were different in 4 sites,which were H-L,V-T,M-I and A-G,respectively.2.The expression patterns of Eobl CSP8 、 Eobl CSP10 and Egri CSP8 、Egri CSP10 in two closely related tea geometrid species,Ectropis obliqua and Ectropis grisescens were analyzed by using the real-time quantitative PCR experiment.It was found that Eobl CSP8、Eobl CSP10 and Egri CSP8、Egri CSP10 were highly expressed in the third instar larvae of Ectropis obliqua and Ectropis grisescens.Furthermore,the expression profiles of Eobl CSP8、Eobl CSP10 and Egri CSP8、Egri CSP10 in the third instar larvae of Ectropis obliqua and Ectropis grisescens were analyzed.Eobl CSP8、Egri CSP8 was highly expressed in the head of the third instar larvae of Ectropis obliqua and Ectropis grisescens,followed by the epidermis.Eobl CSP10 and Egri CSP10 was highly expressed in the head of the3 rd instar larvae of Ectropis obliqua and Ectropis grisescens.3.Using tetracycline to treat Ectropis grisescens to obtain a population without Wolbachia(Ectropis grisescens without Wolbachia)The expression profiles of Egri CSP8 and Egri CSP10 in Ectropis grisescens without Wolbachia were analyzed,and it was found that Egri CSP8 and Egri CSP10 were highly expressed in the third instar larvae of Ectropis grisescens without Wolbachia.The effect of endosymbiotic strain Wolbachia on the expression of CSP8 and CSP10 in the third instar larvae of two closely related tea geometrid species,Ectropis obliqua and Ectropis grisescens were further studied by fluorescence quantitative q PCR,the results showed that the expression levels of CSP8 and CSP10 in Ectropis obliqua were significantly higher than Ectropis grisescens,and they were also significantly higher than Ectropis grisescens without Wolbachia;the expression levels of CSP8 and CSP10 in Ectropis grisescens were significantly higher than Ectropis grisescens without Wolbachia.Another comparative analysis on the expression of the third instar larvae of Ectropis obliqua、Ectropis grisescens and Ectropis grisescens without Wolbachia.The results showed that the expression levels of CSP8 and CSP10 were the highest in Ectropis obliqua;The second was the expression of CSP8 and CSP10 in Ectropis grisescens,which indicated that Wolbachia down regulated the expression of CSP8 and CSP10.4.The effects of plant volatiles cis-3-hexenyl acetate and cis-3-hexenyl acetate on host selection of the 3rd instar larvae of Ectropis obliqua、Ectropis grisescens and Ectropis grisescens without Wolbachia were studied,the results showed that the plant volatiles cis-3-hexenyl acetate and cis-3-hexenyl acetate could attract the 3rd instar larvae.q RT-PCR was used to analyze the induced expression of CSP8 and CSP10 genes of Ectropis obliqua and Ectropis grisescens by plant volatile cis-3-hexenol,the results showed that there was no significant difference in the transcription level of Eobl CSP8 between cis-3-hexenol treatment group and control group at different time points of cis-3-hexenol treatment;there was no significant change at 4h and 8h in the treatment group,but the transcription level increased at12 h,but the difference was not significant;the expression levels of Egri CSP8 and Egri CSP10 in the 3rd instar larvae of Ectropis grisescens treated with cis-3-hexenol for 4 h and 8 h were not significantly different from those in the control group,and there was no significant difference after 12 h.5.Homology modeling was carried out by using amino acid sequences of EoblEgri CSP8,Eobl CSP10 and Egri CSP10 to analyze the binding characteristics of sex pheromone components Z3Z9-6,7-epo-18 H,Z3Z6Z9-18 H and Z3,Z9-6,7-epo-19 H,plant volatiles(Z)-3-hexen-1-ol,(Z)-3-Hexenyl acetate and(Z)-3-hexenyl hexanoate and Eobl-Egri CSP8,Eobl CSP10 and Egri CSP10,and the functional sites were predicted to provide theoretical basis for site directed mutagenesis.The energy of Thr68,Arg71,Gln111 and Leu115 was significantly higher than that of other amino acid residues in the binding of Eobl-Egri CSP8 with the sex pheromone.Leu115 may play an important role in the binding of Eobl-Egri CSP8 with the six ligands.Gln58,Leu39,Asn61 and other sites were more important in the binding of Eobl CSP10 to six ligands.Tyr6,Ser8,Asp9,Gln39,Asp46,Glu57,and Gln62 provide more energy when binding to three types of ligands.Tyr6,Asp46,Leu49,Ala50,Glu57,and Cys58 provide more energy when binding to three types of ligands.In addition,mutation of Tyr6,Asp46,Glu57,and Cys58 may affect the protein function of Egri CSP10. 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