Resistance Monitoring To Thiametoxam And Imidacloprid And Functional Expression Of Cytochrome P450 Gene CYP9Ya In Leptinotarsa Decemlineata From Xinjiang

Colorado potato beetle（CPB）,Leptinotarsa decemlineata（Say）,belongs to Coleoptera,which is a highly destructive quarantine pest,causing great loss of potato yield and quality.As the fourth category of insecticides,neonicotinoids have become one of the main insecticides for Colorado potato beetle control due to their unique mechanism,high efficiency,low toxic and environmental safety.With the popularization and frequent use of neonicotinoid insecticides,it was found that the beetle had developed different levels of resistance to imidacloprid,thiamethoxam and other neonicotinoids.It is very important to elucidate the molecular mechanism of resistance to prevent resistance and prolong the service life of these medicament.This study monitored the resistance level of L.decemlineata adults collected from different regions of Xinjiang in 2019 to thiamethoxam and imidacloprid,measured the activities of three major detoxification enzymes in different populations and thiamethoxam treatment.The differentially expressed genes associated with resistance were analyzed by transcriptome analysis,and the expression levels of six P450 genes in seven field populations and by thiamethoxam LD₅₀ treated for 72 h were analyzed by RT-qPCR.In addition,CYP9Ya was successfully expressed by insect baculovirus expression system in vitro,and the metabolites of thiamethoxam and imidacloprid were further detected by UPLC-QTOF MS.The main research results are as follows:1.Monitoring on resistance to thiamethoxam and imidacloprid of L.decemlineata adults in XinjiangThe resistance level to thiamethoxam and imidacloprid was assayed using topical applications in seven field populations of L.decemlineata adults collected from Urumqi（URMQS,URMQT,URMQB）、Jimsar（JMS）、Qapqal（QPQL）、Yining（YNY,YNH）in Xinjiang.URMQS was considered as relative susceptible population with its LD₅₀ to thiamethoxam being the lowest(24.7 ng·adult^-1).URMQT and URMQB populations were sensitive to thiamethoxam with resistance ratio（RR）less than 3-fold.QPQL and YNH populations showed reduced susceptibility with RR of 3.4 and 4.5-fold,respectively.YNY and JMS populations had moderate level resistance to thiamethoxam with RR of 11.8 and14.0-fold,respectively.It can be found that L.decemlineata adults from Xinjiang were generally resistant to thiamethoxam with low to medium resistance level.As a relatively sensitive population,URMQB population had the lowest LD₅₀(38.3 ng·adult^-1)to imidacloprid.JMS,YNH and QPQL populations all showed reduced sensitivity to imidacloprid with RR of 3.1,3.3 and 4.3 times,respectively.This study revealed the sensitivity of field populations of L.decemlineata from Xinjiang to thiamethoxam and imidacloprid,which could provide a theoretical basis for rational application of thiamethoxam and imidacloprid to control L.decemlineata.2.Detoxification enzyme activities in different populations of L.decemlineata from Xinjiang and exposed to thiamethoxamIn this study,detoxification enzyme activities of seven field populations of L.decemlineata and four populations of adults treated by thiamethoxam LD₅₀ for 72 h were measured,to analyze the biochemical mechanism of resistance of L.decemlineata to thiamethoxam.Compared with URMQS,the GST（DCNB as substrate）activities of YNY and QPQL populations and EST activities of URMQB populations were 2.89,2.13 and1.34 times higher in the 4^th instar larvae,respectively.In adults,the activities of P450enzyme and GST（CDNB as substrate）in YNY population and GST（DCNB as substrate）in YNH population were significantly increased by 1.56,1.26 and 1.81 times,respectively.Treating adults with thiamethoxam LD₅₀ for 72 h significantly increased P450 activities of sensitive populations（URMQS and URMQT）and resistant populations（YNY and JMS）with 1.76,2.75 and 1.91,1.66 times as that in control,respectively.Based on the above results,it was suggested that P450 may be involved in the formation of thiamethoxam resistance in L.decemlineata.3.Transcriptome analysis and validation in susceptible and resistant populations of L.decemlineata in XinjiangRNA sequencing together with digital gene expression（DGE）libraries was used to sequence the transcriptome of L.decemlineata adults susceptible and resistant to thiamethoxam.The expression verification of three P450 genes were performed by real-time quantitative PCR（RT-qPCR）.Based on Illumina RNA sequencing,the raw reads（555 903 706 and 61 082 076）,clean reads（55 903 706 and 61 082 076）,clean bases（8.39and 9.16 G）and error of basic group（0.03%）were obtained for the thiamethoxam-susceptible sample and-resistant sample,respectively.There were 13 differentially expressed P450 genes,1 GST gene and 7 EST genes in the resistant population.No significantly differentially expressed GST and EST genes were found.Among them,CYP4C1 and CYP9e2 were significantly upregulated 13.45 and 4.44 times,respectively,and CYP305a1 was 3.6 times.The quantitative PCR results showed that CYP4C1,CYP9e2and CYP305a1 in resistant population were significantly up-regulated by 5.03,5.32 and8.01 times compared to those in susceptible population,respectively,which was basically consistent with the results of DEG sequencing.In addition,analysis of the expression levels of three overexpressed P450 genes,CYP9Ya,CYP9Yc and CYP9Z25,in the reported imidacloprid-resistant population from Long Island,USA,showed that CYP9Ya was significantly upregulated 1.68 times in the JMS population.4.Analysis of Cytochrome P450 genes expression in different populations of L.decemlineata from Xinjiang and beetles exposed to thiamethoxamIn this study,the expression of six cytochrome P450 genes CYP9Z25,CYP9Ya,CYP9Yc,CYP4C1,CYP9e2 and CYP305a1 was analyzed in different populations and beetles exposed to thiamethoxam by real-time quantitative PCR（RT-qPCR）to identify the P450genes related to neonicotinoid insecticide resistance.Compared with URMQS,the expression of CYP4C1 and CYP9Z25 in the 4^th instar larvae in YNH population was significantly increased by 6.74 and 4.15-fold,respectively.The expression levels of CYP9e2 and CYP305a1 in the JMS population were significantly increased by 5.35 and21.95 times,respectively.CYP9Ya and CYP9Yc in QPQL population were significantly up-regulated by 3.51 and 3.11 times,respectively.In adults,the expression of CYP4C1 and CYP9Yc in YNY population was significantly increased by 5.56 and 4.03 times,respectively.The expression of CYP305a1 in JMS population and CYP9Z25 in YNH population were significantly increased by 6.6 and 7.49 times,respectively.After 72 h treatment with thiamethoxam LD₅₀,CYP9Ya and CYP9Yc of the 4^th instar larvae and adults of UMRQS population were significantly upregulated by 1.5-3.0 times.In addition,correlation analysis showed that there was a significant correlation between resistance level to thiamethoxam against adults and CYP9Ya expression.The expression levels of CYP9Ya in different development stages of CPB were determined.The highest expression level was in the 4^th instar larvae,which was 6.03 times higher than that in eggs,this may be related to the important role of this gene in the 4^th instar larval stage.According to the above results,CYP9Ya may be involved in the development of thiametoxam resistance in L.decemlineata.5.Study on in vitro expression of Cytochrome P450 gene CYP9Ya and metabolism of thiamethoxam and imidaclopridIn this study,the insect baculovirus expression system was used to conduct in vitro protein expression of CYP9Ya,which is highly correlated with thiametoxam in L.decemlineata.The titer of p Fast Bac HTA,CPR and CYP9Ya P3 generation virus was obtained by plaque analysis.The titer of CYP9Ya was 4×10⁷ pfu·m L^-1,and that of Fast Bac-HTA and CPR was 7×10⁷ pfu·m L^-1.The content of P450 was detected by CO difference spectrometry,and the maximum absorption peak at 450 nm in the chromatogram indicated that the structure of P450 was successfully folded in vitro and kept intact.Then,according to the absorbance difference between 450 nm-490 nm,the content of P450 was calculated as 378.9 pmol·mg protein^-1.Using PNA and ECOD as substrates,the enzyme activity of CYP9Ya was 2.7 and 3.3 times higher than that of the control group.These results indicated that the recombinant protein could be further used to analyze the correlation between thiamethoxam metabolism and the recombinant protein.UPLC-QTOF-MS detection showed that CYP9Ya could metabolize thiamethoxam to clothianidin and imidacloprid to 5-hydroxy-imidacloprid.