| Colorado Potato beetle [Leptinotarsa decemlineata (Say)], is the most important insect defoliator of potatoes and also one of main invasive species in China. The beetle has spread eastward and distributed widely in potato growing areas in northern regions of Xinjiangsince it invaded in 1993. Chemical control still remains the foundation of the beetle management. With misuse of insecticides, the beetle has developed different level of resistance to all kinds of insecticides, the Monitoring the resistance dynamic of L. decemlineata is the important basis for making the resistance managenment strategy. Imidacloprid and thiamethoxam are main insecticides used for controlling L. decemlineata in Xinjiang at present, which belong to neonicotinoid. Theit action target, nicotinic acetylcholine receptor (nAChRs) is still not unclearly, In this paper, the nAChRs were further studied using RNA interference from L. decemlineata. It’s of great significance to make clear the functions of nAChR subunits, resistance monitoring and management of harm pests, and developing new insecticides.Three full-length cDNAs encoded nAChR of L. decemlineata were cloned. The effects of the inhibited genes by RNAi on larva and adult were discussed to explore the functions of target genes. All results of the research were summarized as follows:1. Sensitivity to several neonicotinoid insecticides against L. decemlineata in northern XinjiangIn 2012, sensibility of field populations of Nalka, Urumchi, Fukang in northern Xinjiang to four neonicotinoid insecticides (acetaniprid, imidacloprid, thiacloprid, thiamethoxam) was conducted, with conventional insecticides (carbosulfan, azinphos-methyl, cyhalothrin, cyhalothrin) as control. Results showed that sensitivity to thiamethoxam of adult in Urumchi field was decreased; adult in Fukang had low level resistance to thiamethoxam; and the sensitivity of 4 instar larva in Fukang is in the stage of decreasing. Meanwhile, it was found that the beetles had high level of resistance to ordinary insecticides, such as carbosulfan and cyhalothrin.2. Cloning and expression analysis of three genes encoding nicotinic acetylcholine receptor α-subunit in L. decemlineataFull-length sequence of nAChR genes were cloned and their expression quantity in different development stages and parts of L. decemlineata were analyzed, which can provide foundation for the study of action mechanism of neonicotinoid insecticides and function of nAChR a-subunit. Based on transcriptome data analysis, the three full-length cDNAs of nAChR α-subunit genes were cloned using RACE clone technology. Genetic relationships of these three genes were analyzed by the base of their genetic similarity and evolutionary tree. Quantitative real-time PCR was employed to investigate the expression levels of the three α-subunit genes at various developmental stages and different body parts in adults. Three α-subunit gene fragments encoding nAChR subunit proteins were cloned, belonging to α4, α7 and α9 subunits rspectively, and full-length cDNAs of these genes were further obtained, correspondingly designated as Ldα4, Ldα7 and Ldα9 respectively. Sequence analysis shows that these genes shared high similarities with other insect homologous sequence, especially with those from the Coleoptera insect, Tribolium castaneum (87%,89% and 47% for Lda4, Lda7, Lda9, respectively).All tested genes, Ldα4, Ldα7, Ldα9 and Ldα10, exhibited the highest expression levels in the head and thorax of adults. These genes were expressed in different developmental stages (Pupa, larva and adult) with different express levels. Ldα4、Ldα7 had the highest expression level in adult and 3 instar larva respectively. Ldα9 and Ldα10 had the highest expression level in pupa. Ldα4、Ldα7 Ldα9 and Ldα10 possessed the typical structure characteristics of nAChR gene family and shared highest similarities with Tribolium castaneum. The analysis of phylogenetic tree indicated that the genes Lda4, Lda7 Lda9 and Ldα10 were clustered into same class with those in T.castaneum respectively, further confirming their closer evolutionary relationships. The expression levels of each gene in larvae, pupa and adult stages were different, suggesting that the nAChR subunit genes may play specific roles in different developmental stages of L. decemlineata.3. Establishment of RNAi system of nAChR genes and physiological toxicological effects on Colorado potato beetle.Preliminary test was conducted to establish RNA interference system of nAChR gene From L. decemlineata field population of Urumchi. Adult and 2ond instar larva were feed for 7 to 10 days with potato leaves soaked in E. coli solution containing dsRNA of target gene which has been diluted 25,50 and 100 times, respectively, then silencing efficiency of target gene was determined by using qRT-PCR. Results showed that relative expression quantity of target genes decreased significantly after feeding with dsRNA of nAChR gene compared with dsGSP and water,. But gene silence had little effects on mortality, phenotype, pupation and eclosion of larvae.4. Silence efficiency of RNA interference against nAChR genes and its effects on insecticide sensitivity of L. decemlineataBased on effective system of RNA interference of nAChR gene in L. decemlineata, adults and 2ond instar larvae were feed for 7 to 10 days with potato leaves soaked in E.coli solution that has been diluted 100 times, containing dsRNA of target genes. I Sensitivity to thiamethoxam after RNAi and silencing efficiency of target genes were investigated to make clear the relationships of neonicotinoid insecticides with nAchR. Results demonstrated that expression of all these target genes interfered in larva was significantly down regulated compared with control. Although nAChRa4-1 and nAChRa4-2 in adult are also down regulated compared with GFP, there is no significant differences, while other target genes showed obvious decreased trend. after treated by thiamethoxam, thhe motality of larvae apparently decreased through nAChRα9gene was interefered by RNAi.. Yet, death rate rose and sensitivity to thiamethoxam of adult both increased after nAChRα10-2 gene was interefered by RNAi. It suggests that action mechanism of thiamethoxam may be related to the subunits. |
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