Midge Resistance Detection Of Target Gene Screening, Identification And Application Field

The mosquito-borne disease is one of the world’s most important humaninfectious diseases, including malaria; dengue fever. It is seriously harmful for publichealth. Chemical metheod could effecitvely control the spread of mosquito-bornediseases. However, insecticide resistance deveplped with excessive and continuousapplication of insecticides, which has become a major obstacle to control ofmosquito-borne diseases. The premise of the mosquito-borne resistance managementis early detection of insecticide resistance and monitoring of resistance status.Exploring resistance targets molecule and establishing a method for field detectionfor insecticide resistance has been the key topics of the mosquito-borne diseasecontrol.This paper focus mainly on the widely spread Culex pipiens pallens in China. anddeveloped a small-scale microarray containing25genes putatively involved inmetabolically based insecticide resistance in the Culex mosquito.An oligonucleotidemicroarray was designed for totally8P450genes and2GST genes along with othergenes which were previously confirmed with pyrethroids resistance to detect theexpression profile of thesegenes in different populations (laboratory populations and the Nanjing field populations) between the resistant strains and the susceptible strains. To confirm the changes inthe transcriptional activity observed for genes in the microarray experiments,qRT-PCR was used to determine relative levels of mRNA accumulation. Candidatetarget genes which can be used as resistance testing were screened and preliminaryidentified. Furthermore theses candidate target genes were confirmed in multiple fieldpopulation. Finally the target genes were identified. Ultimately, the data generatedfrom these experiments should lead to the identification of metabolic targets for newsynergists to block insecticide resistance and to simple molecular tools to detect insecticide resistant alleles in field populations, an important requirement foreffective insecticide resistance management strategies.1Susceptible strain (Ex/S strain) and resistant strain (Ex/R strain) of experimentpopulation were established with larval dipping method (WHO). Cx. pipiens pallens,which were collected from Nanjing field population, were tested for susceptibility todeltamethrin, using the standard WHO bottle bioassay with standard0.05%deltamethrin-treated papers. Susceptible strain (NJ/S strain) and resistant strain (NJ/Rstrain) were established.2Expression microarrays of deltamethrin resistance-related genes from Culexpipiens pallens were successfully constructed. RNA QC report showed A260/A280≧1.8; A260/A230≧1.5; Total RNA amount>6μg. AminoAllyl aRNA and labeling QCresult showed Labeling Efficiency>10. Pearson correlation tables (R values) for eachtechnical repeats and biological repeats were calculated. R value ranged from0.951to1.000.These results indicated that sample data were credible and repeatable.3We used this microarray to monitor the expression of the resistance genes ofresistant strains and susceptible strains in different populations. By statistical analysis,8P450family members showed various gene expression profiles between theexperiment population and Nanjing field population. These results indicated that inthe metabolitic resistance, P450s family members play different roles.GSTT2was overexpressed2.75-fold (p<0.05) in NJ/R strain. The present studyalso found higher expression of GSTT2in the EX/R strain, but the1.28-foldoverexpression was below our previously defined cutoff value of1.5-fold. The role ofGSTT2in deltamethrin resistance in mosquitoes warrants further investigation.There were more genes overexpressed in NJ/R compared to Ex/R. We speculatedthat different environmental factors may affect the expression of the resistance genes,on the other hand,. the various strains between resistance levels may vary greatly in WHO bottle bioassays, which showed different dose-effect relationship. Althoughthese genes have been confirmed as insecticide resistance genes, it seemed that thesegenes were not suitable for detecting insecticide resistant in field populations as targetgenes.Comprehensive results of the study,11candidate genes as candidate target geneswere preliminary identified, including chymotrypsin-2、CYP6A1、CYP6Z10、GSTT2、LDH、MLC-2、opsin-1、PSMB6、RPP、RPS29and trypsin-2.4To confirm the changes in the transcriptional activity of the11candidate genes,qRT-PCR was used to determine relative levels of mRNA accumulation in Nanjingfield population. The results indicated that the expression trends inCYP6Z10and PSMB6were consistent.Either in experiment population or in field population,CYP6Z10is overexpressed in the resistance strain compared to the correspondingsusceptible strain. It was overexpressed3.60-fold (p<0.05),5.60-fold (p<0.05)respectively. Results of qRT-PCR experiments were consistent with the microarraydata, CYP6Z10is overexpressed.2.56-fold (p<0.01) in NJ/R strain. The resultsindicated that CYP6Z10is involved in the metabolism of pyrethroids. Komagata et al.reported CYP6Z10showed no stage-dependent pattern in Culexquinquefasciatus.The mosquitoes we tested were adult female mosquitoes afteremergence3days.These results indicated that CYP6Z10were suitable for detectingeither adults or larvae resistance to insecticides in field as target gene.Either in experiment population or in field population,PSMB6is overexpressed in the resistance strain compared to the susceptible strain.Itwas overexpressed8.40-fold (p<0.05),2.26-fold (p<0.05). The results of qRT-PCRwere consistent with the microarray data, PSMB6is overexpressed3.57-fold (p<0.05)in NJ/R strain. Previous study in our lab showed that the transcriptional level of PSMB6in DM-resistant larvae was1.41folds as high as that in DM-susceptiblelarvae (p<0.05).These results indicated that the PSMB6was not only overexpressedamong cell, larvae and aduls in mRNA level, but also overexpressed in protein level.The results above demonstrated that PSMB6is associated with DM resistance inmosquitoes. PSMB6were suitable for detecting insecticide resistance in fieldpopulations as target gene.The results of the bivariate correlation analysis showed that both CYP6Z10and PSMB6exhibited strong correlation with the resistance phenotype.The R valuewere0.933(p<0.01) and0.787(p<0.01), respectively.Based on the above results, CYP6Z10and PSMB6were identified forfield resistance detection as target genes.5The specimens of Cx. pipiens pallens collected from6field populations inShandong were tested for susceptibility to deltamethrin, using the standard WHObottle bioassay with standard0.05%deltamethrin-treated papers. Susceptible strain(SD/S strain) and resistant strain (SD/R strain) were established. Applying CYP6Z10as target gene to detect the resistant population in Shandong, the detection rates wereup to100%in Bincheng and Pingyin, while the detection rates were range from66.7%to83.3%in the rest districts, the average detection rate being86.1%(155/180).Applying PSMB6as target gene to detect the resistant population in Shandong, thedetection rates were up to100%in Dongpin, Gudao and Huimin,while the detectionrates were83.3%in the rest districts,the average detection rate being92.0%(165/180).Applying both CYP6Z10and PSMB6, the detection rates were up to100%in any oneof the6districts. We recommend that PSMB6should be an optimal target gene if onetarget gene was used to detect the resistant population, and application of bothCYP6Z10and PSMB6should be as right as the conditions permit.ThePSMB6and CYP6Z10synchronous detection method is worthy of further studying.The results in this paper show the new evidence for clarifying the molecularmechanisms of insecticide resistance and a new resistance testing methods wasdeveloped. It has an important theoretical value and potentially practical application.