| The hazelnut weevil Curculio dieckmanni Faust.(Coleoptera: Curculionidae)is a pest that harms hazelnut nuts in economic forests.In recent years,it has commonly occurred in hazelnut orchards in our country.The larvae,which causes huge economic losses,mainly uses hazelnut nuts as food.Herbivorous insects recognize host plant volatile active components through the olfactory system to complete host positioning,feeding and egg-laying site selection.On the basis of revealing the molecular mechanism of olfactory recognition of the hazelnut weevil,it is a new way to explore the development of green and safe attractants and the use of RNAi interference technology to control Hazel weevil.In this study,we observed and described the type,distribution and structure of antennal sensilla in male and female adults of the hazelnut weevil,and analyzed their differences and potential functions between males and females.The volatile components of the host hazel tree with electrophysiological and olfactory behavioral activities on the hazelnut weevil were screened.The difference of antennal transcriptome between male and female adults of Hazelnut weevil and the metabolic pathway of their related genes were analyzed.The genes involved in olfactory recognition and their functional characteristics were explored and clarified.Therefore,the host recognition mechanism of the hazelnut weevil was analyzed from the three aspects of morphology,behavior and molecular biology,in order to use RNAi technology,develop attractants and other efficient chemical communication regulation technology,and then realize the green safety and sustainable management of the hazelnut weevil provide important theoretical basis and technical support.Thus,the molecular mechanism of olfactory recognition of Hazelnut weevil was clarified from three aspects of morphology,behavior and molecular biology.It provides important theoretical basis and technical support for further realizing the green and sustainable management of hazelnut weevil by using RNAi technology,developing attractant and other efficient chemical communication control technologies.(1)Types,distribution and ultrastructure of antennal sensilla on adult hazelnut weevil.Studies have shown that the antennae of the hazelnut weevil are knee-shaped,and a total of 10 types of sensilla are found on the antennae,including Sensilla basiconica,Sensilla trichoid,Sensilla chaetica,Sensilla styloconica,Sensilla falciform,Cuticular pore,Sensilla sporangia,Sensilla squamiformia,Sensilla coeloconica and B?hm bristles.Among them,Sensilla sporangia was first found in Weevils.The total length of the antennae of female adults was significantly longer than that of male adults,and the total number of sensilla in the antennae distribution of female adults was significantly larger than that of male adults.The types of sensilla on the antennae of male and female increased gradually from scape segment to pedicel segment to flagllum segment.The terminal club segment had the most abundant types of sensilla,and the number of sensilla was most distributed.It was speculated that the club segment was the most important part of the hazelnut weevil to recognize odor molecules.(2)Screening of effective components of the hazelnut weevil with electrophysiological and behavioral responses on host plant volatiles.The results showed that cis-3-hexenol,cisocimene,4-hexen-1-ol,anisole,methyl crotonate,myrcene,cis-3-hexenyl acetate,(+)-dihydrocarveol,ethyl tiglate,linalool and cis-3-Hexenyl n-valerate,these eleven hazel tree volatiles can have electrophysiological activity in weevil male and female.Further EAG showed that cis-ocimene,myrcene,(+)-dihydrocarveol could make weevil male and female adults have significant EAG responses,and cis-3-Hexenyl n-valerate,cis-3-Hexenyl acetate,4-hexen-1-ol,and linalool also showed a certain EAG response,and linalool and cis-3-Hexenyl n-valerate made the EAG response of male weevil adults significantly greater than that of females,suggesting that these two compounds may affect weevil male olfactory sense and help locate host plants.The EAG response values of female adults of weevil to cis-3-hexenyl acetate and(+)-dihydrocarveol were significantly higher than those of male adults,indicating that these two volatiles may be involved in the process of host localization of weevil female adults It plays a different role in the spawning position,such as the positioning of the spawning position.The olfactory behavioral experiments showed that myrcene,cis-ocimene,(+)-dihydrocarveol and linalool had significant attracting effects on male and female adult males and females.Cis-3-hexenol showed a weaker attracting effect on the male and female weevil,and anisole had a more obvious repelling effect on the male and female weevil.(3)Transcriptome and differential analysis of male and female antennae of the hazelnut weevil.A total of 42,971 and 43,190 Unigenes were obtained from the male and female antennae transcripts,respectively.A total of 44,024 different Unigenes were obtained by filtering,splicing and assembling the male and female antennae transcripts,and 21,803 were successfully annotated in the Nr,Swiss Prot,KEGG and KOG protein databases.8027 unigenes were annotated by GO functional classification,and 7502 unigennes were annotated by KEGG pathway.A total of 355 genes were differentially expressed in male and female antennae in the transcriptome of male and female antennae of the hazelnut weevil,of which 69 genes were significantly(up-regulated)expressed in male antennae and 286 genes were significantly(upregulated)expressed in male antennae.We annotated differential genes to GO and KEGG for enrichment classification,188 differential genes were annotated to GO functional classification,and 150 differential genes were annotated to KEGG pathway.(4)Identification of olfactory genes.A total of 87 olfactory genes were identified from the antennal transcriptomes of male and female adults of the hazelnut weevil,including 23 Cdie OBPs,15 Cdie CSPs,15 Cdie ORs,9 Cdie GRs,23 Cdie IRs and 2 Cdie SNMPs.(5)Analysis of olfactory gene expression profiles.The expression levels of RPKM in the transcriptome of male and female antennae showed that 6 genes were preferentially expressed in the transcriptome of male and female antennae.Cdie OBP9,Cdie OBP10,Cdie OBP19,Cdie IR22 and Cdie GR4 were preferentially expressed in female antennae,and Cdie CSP15 was preferentially expressed in male antennae.The expression levels of RPKM in other olfactory gene transcriptomes were not significantly different between males and females.Further Realtime q PCR showed that Cdie OBP1,Cdie OBP2,Cdie OBP5,Cdie OBP9,Cdie OBP19,Cdie OBP20,Cdie OBP21,Cdie CSP15,Cdie OR13,Cdie OR15,Cdie GR4,Cdie IR8,Cdie IR20,Cdie IR22 and Cdie SNMP1 were preferentially expressed on female antennae.Cdie GR5 and Cdie IR1 are preferentially expressed in male antennae.Cdie OBP1,Cdie OBP2,Cdie OBP5,Cdie OBP9,Cdie OBP10,Cdie OBP11,Cdie OBP14,Cdie OBP16,Cdie OBP17,Cdie OBP19,Cdie OBP20,Cdie OBP21,Cdie CSP6,Cdie CSP10,Cdie CSP15,Cdie OR10,Cdie OR15,Cdie GR4,Cdie GR5,Cdie IR19,Cdie IR20,Cdie IR22 and Cdie SNMP2 were highly expressed in both male and female antennae.At the same time,it was found that the expression level of most olfactory genes in the antennae was significantly higher than that in other tissues,indicating that the antennae was the main organ of olfactory recognition in the hazelnut weevil.Some genes also showed preferential expression,high expression,low expression or no expression in other tissues,and this difference in expression was speculated to be closely related to their different functional roles in the process of olfactory recognition.To sum up,this study clarified the olfactory mechanism of host location of Hazelnut weevil from the aspects of morphology,behavior and molecular biology,and analyzed the expression profile of host location olfactory related genes,which laid a solid foundation for further research on the function of host location olfactory related genes of Hazelnut weevil. |
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