Studies On Target Resistance Mechanisms Of Culex Pipiens Pallens And Culex Pipiens Quinquefasciatus

Culex pipiens pallens and Culex pipiens quinquefasciatus all belong to the Culexpipiens complex. They are the major commensal species of China, and the importantmedium of filariasis and encephalitis. At present, chemical control is still one of theimportant measures of integrated control to mosquitoes. There are three kinds ofchemical insecticides are widely used in production and life. This has resulted in thegeneration and development of resistant in Cx. pipiens pallens and Cx. pipiensquinquefasciatus. There are many studied have demonstrated that Cx. pipiens pallensand Cx. pipiens quinquefasciatus in different areas of China have shaped differentdegrees of resistance to various insecticides. Therefore, there is an urgent need forin-depth study on the occurrence and development of insecticide resistance. In this study,field collection of Cx. pipiens pallens and Cx. pipiens quinquefasciatus populations asthe research object, determined the biological resistance, and the target geneacetylcholinesterase gene and sodium channel gene were studied. In addition, thecorrelation analysis between the gene frequency and resistance levels were conducted tounderstand the current situation of resistance gene mutation frequency in mosquitopopulation. Those studies could provide new targets for insect resistance management.The main results of this study are as follows:1. Resistance levels of Cx. pipiens pallens and Cx. quinquefasciatus populationsThe results of bioassay showed that the different geographic strains of mosquitoesall have developed a certain resistance to different insecticides. The resistance ratios offive strains of Cx. pipiens quinquefasciatus larvae to DDVP are2.8to17.6times; resistance to propoxur are from2.4to11.0times; the resistance ratios of eleven strainsCx. pipiens quinquefasciatus larvae to deltamethrin range from9to1019times, tocyhalothrin are from14to1291times. The resistance levels to deltamethrin andpermethrin of the adults of seven strains of Cx. pipiens pallens and four strains of Cx.pipiens quinquefasciatus all reach resistance levels.2. Mutations in Cx. pipiens quinquefasciatus target resistance genesThrough molecular cloning technology, five gene mutations in acetylcholinesteraseof natural populations of Cx. pipiens quinquefasciatus are found. They are V185M:GTG→ATG, G247S: GGC→AGC, A328S: GCC→TCC, A391T: GCC→ACC, T682A:ACA→GCA respectively. There are seven gene mutations in six gene positons insodium channel gene, which are V250M: GTG→ATG, R436K: AGG→AAG, M943V:ATG→GTG, I973T: ATC→ACA, L1035F: TTA→TTT, L1035S: TTA→TCA, E1901D:GAG→GAT respectively. In addition, eleven alternative splicings are found in thesodium channel. These alternative splicings may play a significant role in riching thediversity of sodium channel protein, but need to be further studied on the relationshipwith resistance of pyrethroids.3. Calculation of mutation frequency in Cx. pipiens quinquefasciatus targetresistance genesThrough gene sequencing, the mutation frequencies of each mutation are V185M:6.10%-25.0%, G247S:0.00%-18.1%, A328S:2.90%-47.2%, A391T:0.00%-54.3%,T682A:18.6%-51.6%in acetylcholinesterase gene; V250M:17.2%-33.9%, R436K:46.3%-50.0%, M943V:25.8%-41.9%, I973T:24.2%-35.5%, L1035F:28.8%-74.3%,L1035S:3.13%-21.2%, E1901D:8.33%-44.7%in sodium channel gene respectively.4. Correlation study of mutation frequency in Cx. pipiens quinquefasciatus targetresistance genes with resistanceCorrelation analysis shows that the RS frequency of V185M is correlatedsignificantly with propoxur resistance (P<0.05). G247S and A328S mutations are also associated with propoxur resistance. T682A mutation is significantly negative correlatedwith propoxur resistance (P<0.05). This indicates that these three mutations V185M、G247S and A328S are associated with propoxur resistance. L1035F/S mutation issignificantly or extremely significantly correlated with deltamethrin and cyhalothrinresistance. This indicates mutations in L1035position are associated with deltamethrinand cyhalothrin resistance. In addition, according to the analysis of correlation betweeninsecticide resistances, deltamethrin and cypermethrin exsit extremely significant crossresistance in Cx. pipiens quinquefasciatus (P<0.01).5. Hardy Weinberg equilibrium detection and linkage analysis of Cx. pipiensquinquefasciatus target resistance gene mutationsHardy Weinberg equilibrium test show that five mutations in acetylcholinesterasegene are all not deviating from the hardy Weinberg equilibrium, while there are four inthe six point mutations of sodium channel gene deviating from Hardy Weinbergequilibrium (R436K, M943V, I973T and L1035F/S), and the other two (V250M andE1901D) are not. The linkage disequilibrium analysis shows that V185M, A328S andA391T mutations in acetylcholinesterase gene exist significant linkage relationship;M943V and I973T mutations in sodium channel gene link extremnly significant.In summary, this study have found that the different field strains of Cx. pipienspallens and Cx. pipiens quinquefasciatus have developed a certain resistance to differentinsecticides, and classical (G247S and L1035F/S) and new (V185M and A328S) genemutations associated with resistance were determined. In addition, the resistancemutation detection and correlation analysis were created, which provide a theoreticalbasis for mosquito resistance management and a new approach for the detection ofresistance.