| Sogatella furcifera(Horváth)(Hemiptera: Delphacidae),the white-backed planthopper,is one of the most serious migratory pests on rice crops.Because of the special karst geographical location,environment,and climate make Guizhou province an epicenter of S.furcifera populations during the long distance migration.In recent years,the occurrence of S.furcifera in Guizhou has increased significantly.Due to the control of S.furcifera primarily depends on the use of chemical insecticides in a long period,the increase of resistance level to conventional insecticides has been a serious problem in rice production.However,the present knowledge about the insecticide resistance of S.furcifera in Guizhou were still ambiguous since the previous resistance detection were mostly not compared with the relatively susceptibility baseline.This means that there is a high risk in resistance developing to these insecticides.Nitenpyram has been extensively used to control rice planthoppers in China,and cycloxaprid has been considered a substitute for imidacloprid for the control of important pests in agricultural production in China because it has performed well in controlling a broad spectrum of insect pests.Along with the two insecticides' application frequently and extensively,the resistance would be an issue.At present,it is little known about the molecular mechanism of resistance to the two chemicals in S.furcifera.Therefore,the purpose of this thesis are monitoring continuously the insecticide resistance development of S.furcifera in Guizhou and exploring the molecular response of S.furcifera induced by nitenpyram and cycloxaprid,respectively,which could provide valuable data for expounding the resistance mechanism and making practicable strategies of the integrated resistance management.1 Analysis of insecticide resistance status in S.furcifera from Guizhou ProvinceThe resistance levels to 7 traditional insecticides were evaluated in 8 geographic populations of S.furcifera collected from paddy fields in Guizhou in Jun-Jul 2015.Adults or nymphs were collected from paddy fields and reared for one generation in laboratory,and the third-instar nymphs were used for bioassays.By the rice-stem dipping method,the concentration-responses of S.furcifera to different insecticides were measured and compared with the corresponding relative susceptibility baseline.7traditional insecticides,chlorpyrifos,isoprocarb,imidacloprid,buprofezin,pymetrozine,thiamethoxam and nitenpyram.The results showed that the resistance of S.furcifera to buprofezin(11.4-167.6 fold)and pymetrozine(20.8-84.7 fold)had reached to a moderate to high resistance level.The resistance to chlorpyrifos and imidacloprid had been at moderate level.S.furcifera is still susceptible to isoprocarb,thiamethoxam and nitenpyram.2 The susceptibility monitoring to 12 insecticides in field populations of S.furcifera in GuizhouThe susceptibility change trend to 12 insecticides in six geographic populations of S.furcifera from Guizhou Province was monitored yearly using the rice-stem dipping method in 2012-2015 to precisely estimate development trends of insecticide susceptibility in S.furcifera in Guizhou.12 traditional insecticides detected were chlorpyrifos,dichlorvos,isoprocarb,carbaryl,beta-cypermethrin,deltamethrin,imidacloprid,buprofezin,pymetrozine,thiamethoxam,nitenpyram and cycloxaprid.Through 4 years monitoring,the results suggest that chlorpyrifos resistance of most populations had reached to moderate level(63.64%).So,S.furcifera in Guizhou has the potential to further develop resistance to chlorpyrifos in the field.All populations collected in the 4 years remained sensitive to isoprocarb.The most populations(90.91%)reached to moderate resistance level to buprofezin andmore than half populations(59.09%)were high resistance level to this chemical.To pymetrozine,95.45% populations had developed moderate resistance and 54.55% populations reached to high resistance in the 4 years.So,the resistance level to buprofezin and pymetrozine in S.furcifera had developed medium to high resistance in Guizhou Province.36.36% populations had developed medium resistance to imidacloprid,hence,imidacloprid could be still used to manage S.furcifera in Guizhou.The RRs of all populations were lower than 10-fold to thiamethoxam and nitenpyram,and90.91% populations were at low resistance level to thiamethoxam,as well as 85.71% populations remained sensitive to nitenpyram.So,nitenpyram and thiamethoxam would be better choices for effective management of S.furcifera at the present.As to dichlorvos,the susceptibility had an increasing trend in Pingba County,Bozhou District of Zunyi City,Qianxi County and Pingtang County during 2012-2015.However,in Huaxi District of Guiyang city and Yuping county,the susceptibility appeared a decreasing trend.All populations had a decreasing trend in susceptibility to carbaryl.For beta-cypermethrin,the populations from Huaxi,Pingba and Qianxi had a rising trend in susceptibility,while in those of Bozhou,Pingtang and Yuping,the susceptibility had a decreasing trend.The LC50 value of S.furcifera to deltamethrin increased in all populations year by year,which meant that the susceptibility had a decreasing trend.The susceptibility had an increasing trend to cycloxaprid in the populations of Huaxi,Bozhou,Qianxi and Pingtang,while the susceptibility of Pingba population was opposite.3 Selection of formulation combining insecticides to delay resistance development of S.furciferaThe toxicities of single-agent insecticides and their combined insecticides to 3rd instar nymph of S.furcifera were detected under the laboratory condition with rice stem-dipping method.The detected single-agent insecticides were chlorpyrifos,dichlorvos,isoprocarb,pymetrozine,imidacloprid,thiamethoxam,nitenpyram and cycloxaprid.From forty combinations,ratios of 16 groups were selected due to their values of cotoxicity factor were more than 20 qualitatively.Then,from the 16 groups,5 combinations with synergistic effect were determined using cotoxicity coefficient method.The ratios of the 5 combinations were as follows: nitenpyram : chlorpyrifos,1 : 30;imidacloprid :isoprocarb,7 : 40,7 : 200,7 : 400,7 : 1000.The cotoxicity coefficients of the 5 combinations were112.5,242.1,212.4,638.1 and 417.7 respectively.Moreover,the combination of imidacloprid and isoprocarb at the ratio of 7 : 400 showed the most significant synergetic effect.4 Analysis of transcriptome and resistance-related genes of S.furcifera induced by nitenpyramThe 5th instar nymph of S.furcifera were treated by concentration LC10(=0.14mg·L-1)of nitenpyram.After 48 h,the transcriptome of emerging female adult of treated and untreated S.furcifera were sequenced using the Illumina sequencing,that of the untreated was as control.We constructed two transcriptomes of S.furcifera and profiled the alternation of gene expression in response to nitenpyram(Accession number: SRR5277484 and SRR5277485).Over 162,337,759 nucleotides and103,793 different unigenes were generated from the nitenpyram-treated and the untreated,and 44,248 sequences were annotated in at least one database,accounting for 42.63% in total unigenes.The number of Coding DNA Sequence(CDS)were 33,023 and that of the amino acid sequence of coding region were 19,106.17,310 simple sequence repeats(SSRs)were also predicted,and among them,there were 7,802 mononucleotide repeats,accounting for 45.07% in total SSRs and(T)n was the most frequent motif in treated S.furcifera.There were 300 up-regulated genes and 243 down-regulated in total 543 differentially expressed genes(DEGs).We identified 31 unigenes,including Cytochrome P450,Glutathione-s transferase(GST),Acid phosphatase(ACP),Cadherin,ATP Synthase,etc.,associated with insecticide-resistance,5 Analysis of transcriptome and resistance-related genes of S.furcifera induced by cycloxapridThe 5th instar nymph of S.furcifera were treated by concentration LC10(=0.58mg·L-1)of cycloxaprid,After 48 h,and we sequenced the transcriptome of emerging female adult of treated and untreated(control)S.furcifera.The two transcriptomes of S.furcifera and profiled the alternation of gene expression in response to cycloxaprid were constructed(Accession number: SRR4294200 and SRR4294203).157,906,456 nucleotides and 131,601 unigenes were obtained and a total of 38,534 unigenes matched known proteins in at least one database,accounting for 29.28% in total unigenes.The number of CDS were 28,546 and that of the amino acid sequence of coding region were 22,299.A total 15,868 SSRs were identified.The trinucleotide repeats accounted for 45.1%(7,157)in total SSRs and the(AAG/CTT)n was the most frequent motif.There were 359 DEGs that might be induced by cycloxaprid.There were 131 genes up-regulated and 228 down-regulated.22 unigenes may take participate in the resistance to cycloxaprid,such as cytochrome P450,GST,ACP,cadherin,glycosyltransferase and decarboxylase/dehydrogenase.6 Expression differences of resistance-related genes induced by cycloxaprid using qRT-PCR in S.furciferaThe 5th instar nymphs of S.furcifera were treated by cycloxaprid 1×,2×,4×,8×LC10concentrations,with untreated as control,respectively,and sampled the emerging female adults at 24 h,48 h,72 h after treating.To clarify the relationship between these DEGs and the resistance of S.furcifera to cycloxaprid,we compared the expression profiles of 15 genes selected from of 22resistance-related ones,using qRT-PCR.The results showed that 5 genes have relatively lower expression level and no amplification;the other 5 genes have amplification with Ct value lower than 34,but showed more melting peaks and non-specific amplification.Another 5 genes,only,from 15 tested were validated successfully by qRT-PCR and annotated as P450 CYP4 family,CYP6 family,S-adenosylmethionine decarboxylase,6-phosphogluconate dehydrogenase,alpha-1,3-mannosyltransferase,respectively.In general,these studies have identified the resistance level and development trend to traditional insecticides used against S.furcifera in Guizhou Province at present.5 groups of combined insecticides with the cotoxicity coefficient more than 100 and higher synergetic effect.Furthermore,through comprehensive analysis on transcriptome of S.furcifera stressed with nitenpyram and cycloxaprid,resistance-related genes are determined with the expression status of genes.The results could be recognized as theoretical and practicable contributions to S.furcifera control with reference significance in making integrated insecticide-resistance management of S.furcifera. |
PDF Full Text Request