Cloning And Characterization Of ProPOA3, A Novel Deltamethrin Resistance Associated Gene From Culex Pipiens Pallens

Mosquitoes transmit many mosquito-borne diseases which threaten the public health. Among all applicable strategies, chemical control played a major role in controlling disease-spreading vectors. Unfortunately, excessive and continuous usage of insecticides induced the development and spread of resistance, which presents the major obstacle in arbo-diseases control. Because the mechanisms of insecticide resistance are polygenetic and incompletely defined, identification and characterization of insecticide resistance genes will contribute to clarifying the mechanism of insecticide resistance and managing insecticide resistance.To investigate the insecticide resistance in mosquitoes, we identified differentially expressed genes between deltamethrin-susceptible and-resistant strains of Cx. pipiens pallens in our previous experiments, and some deltamethrin resistance related genes were isolated, one of which was prophenoloxidase subunit A3(proPOA3) gene. In this study, the full length sequence of proPOA3gene was acquired using RT-PCR and RACE techniques, and then proPOA3was exogenous overexpressed in mosquito cells to investigate its role in deltamethrin resistance. Moreover, the expression profile of proPOA3in deltamethrin-susceptible and-resistant mosquitoes were assessed with quantitative PCR. In addition, sequence analysis of proPOA3was performed to identify point mutations associated with deltamethrin resistance in Cx. pipiens pallens.The full length sequence of proPOA3gene, amplified from Cx. pipiens pallens, contains2,349base pairs, while the ORF region of proPOA3has2,061bps and encodes for a protein with686amino acids (GeneBank Accession No:FJ755838.1,2009). Amino acid sequence alignment indicating that proPO are conserved among species, and phylogenetic relationships showed that proPOA3of Cx. pipiens pallens has the highest homo logy with that of Cx. quinquefasciatus (98%identity) by the neighbor-joining method.To further investigate the mechanism of proPOAS involving in deltamethrin resistance, C6/36cell line transiently transfected with pIB/V5-His-proPOA3was used. The significantly decreased viability in C6/36-proPOA3cells compared with the controls under high dosage deltamethrin treatment (101.5μg/ml and102.0μg/ml) suggested that proPOA3overexpression may sensitize C6/36cells to deltamethrin treatment.The proPOA3differential expressions between deltamethrin-susceptible and-resistant strains were also analyzed in field populations of Cx. pipiens pallens. The expression level of proPOA3was significantly higher in deltamethrin-susceptible individuals than-resistant in four different regions of mainland China (2.60-fold in Wuxi,6.33-fold in Nanjing4.33-fold in Tangkou, and5.02-fold in Yucheng, respectively, p<0.05). To investigate whether decreased proPOA3transcription was associated with deltamethrin-resistance in other mosquito species, expression levels between susceptible and resistant mosquitoes from Cx. tritaeniorhynchus, An. sinensis and An. minimus were also evaluated. The transcriptional level of proPOA3was5.03-fold higher in deltamethrin-susceptible stain compared with-resistant strain in Cx. tritaeniorhynchus (p<0.05), which was similar to the results got in Cx. pipiens pallens. But the expression of proPOA3had no significant differences between two strains in Anopheles mosquitoes (p>0.05). To further investigate the role of proPOA3expression in deltamethrin-resistance, laboratory populations of Cx pipiens pallens with various resistance levels were used for proPOA3expression analysis. The transcriptional expression of proPOA3significantly decreased during deltamethrin selection.We did sequence analysis on proPOA3genes in field-collected Cx. pipiens pallens populations. Three point mutations were found in the769th and1116th base, which were all located in the exon region of proPOA3. The non-synonymous point mutation (769G>A) resulted in a switch from valine to isoleucine, whereas two other mutations (1116G>C and1116G>A) are synonymous. Interestingly, the769G>A and1116G>C mutations could be detected simultaneously. When homozygous mutation (G/Gâ†'A/A)/heterozygous mutation (G/Gâ†'G/A) occurred in769th base, homozygous mutation (G/Gâ†'C/C)/heterozygous mutation (G/Gâ†'G/C) in1116th base could also be detected. The mutation769G>A (1116G>C) were mostly detected in the deltamethrin-resistant individuals from Qingdao (42.85%), Weishan (11.54%) and Nanjing (23.92%) populations. The1116G>A mutation was detected both in deltamethrin-resistant (13.64%) and deltamethrin-susceptible (9.52%) individuals from Wuxi. However, none mutation was detected in deltamethrin-resistant or-susceptible individuals from Huaibei populations. We also performed nucleotide diversity analysis in laboratory deltamethrin-selected Cx. pipiens pallens. There was a significant positive correlation between the769G>A (1116G>C) mutation frequency and the LC50of deltamethrin in Cx. pipiens pallens. While, mutation1116G>A was disappeared after14generation of deltamethrin-selection. These results suggested that mutations769G>A and1116G>C may be associated with deltamethrin-resistance in Cx. pipiens pallens population.In this study, we cloned the full length of proPOA3, and presented the first evidence that proPOA3confer the deltamethrin resistance in mosquito. And our results provided new evidence for clarifying the molecular mechanisms of insecticide resistance and establishing the new resistance testing methods. It has important theoretical value and potentially practical application.