The Role Of Cuticular Protein CPLCG5 In Resistance To Insecticide

Background:Mosquito-boren diseases,such as malaria,dengue,zika virus diseases,pose a big threat to human health.Chemical insecticides have massively reduced the prevalence of mosquito-borne diseases around the world.But extensively usage of insecticides had induced mosquitoes developed mutiple resistance,which threatens insecticide continued effectiveness.Resistance has become a big obstacle for the mosquito-borne disease prevention.,Mosquito cuticle,consisting of chitin and cuticle proteins,is the first barrier to prevent pesticides from entering the body.The change of cuticle thickness and structure,leads to a decrease in the penetration efficiency of the insecticide.The molecular mechanism of cuticular involvement in resistance has not been reported in depth.Exploring the detailed molecular basis of such resistance is critical to implementing suitable resistance management strategies.Methods:In our previous research,transcriptome and proteome analysis showed that the expression of some cuticular proteins were different between pyrethroid-resistance-strain(R-strain)and pyrethroid-susceptible(S-strain).We performed qPCR to analyze the relative expression levels of CPLCG5 in R-strain and S-strain.Cuticle proteins extracts from mosquito legs of 3d-old female adults(n=40)were analyzed by gradient gel electrophoresis(4-20%).A western blot analysis of the protein level of CPLCG5 between the S-strain and a laboratory produced R-strain of Culex pipiens pallens.Immunofluorescence assays were performed with polyclonal antibody to locate cuticular CPLCG5 in mosquitoes.EM and immunohistochemical EM of femur segment was analyzed to compare the cuticle between the control group and the siRNA-CPLCG5 group.Results:Compared with S-strain,the transcription level of CPLCG5 gene in R-strain was increased 8 fold,indicating that CPLCG5 gene may be related to mosquito resistance.CPLCG5 expression increased sharply after pupation(white pupa)and the first day after emergence,and then gradually decreased,and the expression of CPLCG5 became stable after 3 days,which suggest that the cuticle may be synthesised and assembled in white pupa and the first day after emergence.CPLCG5 gene encodes the cuticle protein CPLCG5,the speculated molecular weight is about 12 kD.Immunofluorescence assays performed with polyclonal antibody showed that the cuticle protein CPLCG5 was expressed in five parts of the mosquito,including head,thorax,abdomen,wings and legs,mainly located in legs and wings,suggesting that the cuticle protein CPLCG5 was tissue-specific expressed.Western blot showed that the expression of cuticle protein CPLCG5 in R-strain was 2.2 times increased than that of S-strain.We use siRNA microinjection based study to investigate the function of CPLCG5.The expression of CPLCG5 in si-CPLCG5 injection group was only about 22.7%then that of Si-NC group,which indicated that the expression of CPLCG5 gene was significantly decreased after interference.Subsequently,CDC bottle experiments showed that CPLCG5 knockdown significantly sensitized mosquito to deltamethrin treatment,suggesting that CPLCG5 plays an important role in pyrethroid resistance.Transmission Electron Microscopy(TEM)analysis of femur segment suggested that the chitin laminae become unorganized after siCPLCG5 injection.EM immunohistochemical analysis of femur segment suggested that the total cuticle thickness of femur was was significantly thinner in siCPLCG5(1.385±0.121?m)than siNC(1.721 ±0.221 ?m),and the exocuticle thickness of femur was significantly thinner in siCPLCG5(0.717±0.110?m)than siNC(0.946±0.126?m)(P<0.001).These results indicated that the depletion of CPLCG5 by RNA interference(RNAi) caused unorganized laminae and made cuticle thinner,especially the outcuticle,suggesting that the presence of cuticle protein CPLCG5 is very important for maintaining the structure of insect epidermis.The increase of CPLCG5 expression is beneficial to the formation of thicker and stronger epidermis,which leads to insect resistance.Conclusion:This is the first study to explore the basis molecular of cuticle resistance.These results supported the hypothesis that CPLCG5 may participate in pyrethroid resistance through forming a rigid matrix and increasing cuticle thickness.