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Dan Zhou,
Shunhua Hao,
Yan Sun,
Lin Chen, Chunrong Xiong,
Lei Ma,
Donghui Zhang,
Shanchao Hong,
Linna Shi,
Maoqing Gong,
Huayun Zhou,
Xinyou Yu,
Bo Shen,
Changliang Zhu
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ABSTRACT: The prophenoloxidase subunit A3 (proPOA3) gene was cloned from Culex pipiens pallens, which had an open reading frame of 2061 bp encoding a putative 686 amino acid protein. The deduced amino acid sequence shares 98% with proPOA3 from Culex quinquefasciatus. ProPOA3 is expressed at all developmental stages of C. pipiens pallens. Significant negative correlation was observed between proPOA3 expression and deltamethrin resistance in resistant C. pipiens pallens. Furthermore, proPOA3 expression levels were significantly lower in deltamethrin-resistant mosquitoes than in susceptible mosquitoes collected at four locations in Eastern China. However, we did not find any substantial change in proPOA3 expression in field-collected resistant Anopheles mosquitoes. Moreover, overexpressing proPOA3 in C6/36 cells led to more sensitivity to deltamethrin treatment. In laboratory and field-collected resistant C. pipiens pallens, a valine to isoleucine mutation (769G>A) and two synonymous mutations (1116G>C and 1116G>A) were identified in proPOA3. In addition, the mutation frequency of 769G>A and 1116G>C increased gradually, which corresponded with raised deltamethrin resistance levels. Taken together, our study provides the first evidence that proPOA3 may play a role in the regulation of deltamethrin-resistance in C. pipiens pallens.
Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology 04/2012; 162(4):57-65. · 1.61 Impact Factor
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Lin Chen,
Daibin Zhong,
Donghui Zhang,
Linna Shi,
Guofa Zhou,
Maoqing Gong,
Huayun Zhou,
Yan Sun,
Lei Ma,
Ji He,
Shanchao Hong,
Dan Zhou, Chunrong Xiong,
Chen Chen,
Ping Zou,
Changliang Zhu,
Guiyun Yan
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ABSTRACT: Pyrethroid insecticides have been extensively used in China and worldwide for public health pest control. Accurate resistance monitoring is essential to guide the rational use of insecticides and resistance management. Here we examined the nucleotide diversity of the para-sodium channel gene, which confers knockdown resistance (kdr) in Culex pipiens pallens mosquitoes in China. The sequence analysis of the para-sodium channel gene identified L1014F and L1014S mutations. We developed and validated allele-specific PCR and the real-time TaqMan methods for resistance diagnosis. The real-time TaqMan method is more superior to the allele-specific PCR method as evidenced by higher amplification rate and better sensitivity and specificity. Significant positive correlation between kdr allele frequency and bioassay-based resistance phenotype demonstrates that the frequency of L1014F and L1014S mutations in the kdr gene can be used as a molecular marker for deltamethrin resistance monitoring in natural Cx. pipiens pallens populations in the East China region. The laboratory selection experiment found that L1014F mutation frequency, but not L1014S mutation, responded to deltamethrin selection, suggesting that the L1014F mutation is the key mutation conferring resistance to deltamethrin. High L1014F mutation frequency detected in six populations of Cx. pipens pallens suggests high prevalence of pyrethroid resistance in Eastern China, calling for further surveys to map the resistance in China and for investigating alternative mosquito control strategies.
PLoS ONE 01/2010; 5(7):e11681. · 4.09 Impact Factor
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ABSTRACT: Two deltamethrin resistance-associated serine protease genes (NYD-tr and NYD-ch) were isolated from Culex pipiens pallens in our previous study. To study the function of NYD-Tr and NYD-Ch in the metabolism of deltamethrin, we constructed the recombinant plasmid pET32a(+)/NYD-tr and pET32a(+)/NYD-ch with a 6x histidine tag. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analyses of the recombinant proteins revealed that the molecular weights of NYD-Tr and NYD-Ch are 42 and 50 kDa. Enzyme activity assay indicated that the recombinant NYD-Tr and NYD-Ch had the corresponding features of trypsin and chymotrypsin. Using BApNA as the substrate, NYD-Tr gave optimal activity between pH 9.0 and 10.5, while NYD-Ch was optimally active over the range of pH 8.0-11.0 using the S(Ala)(2)ProPhe-pNA as the substrate. Then, we investigated the metabolism of deltamethrin by NYD-Tr and NYD-Ch. Our results showed that NYD-Tr and NYD-Ch could hydrolyze deltamethrin. The acute oral toxicity of the metabolite to Wistar rats was much lower than deltamethrin.
Parasitology Research 09/2008; 103(3):507-16. · 2.15 Impact Factor
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ABSTRACT: The 60S ribosomal protein L22 (GenBank accession no. EF990190) was cloned from Culex pipiens pallens. An open reading frame (ORF) of 447 bps was found to encode a putative 148 amino acids protein which shares 90% and 80% identity with RPL22 genes from Aedes aegypti and Anopheles gambiae respectively. Real-time quantitative PCR analysis demonstrated that the transcription level of RPL22 in deltamethrin-resistant strain was 2.57 folds higher than in deltamethrin-susceptible strain of Cx. pipiens pallens. Overexpression of RPL22 in C6/36 cells showed that the deltamethrin-resistance was decreased in C6/36-RPL22 cell compared to the control. The mRNA level of cytochrome P450 6A1 (CYP6A1, GenBank accession no. FJ423553) showed that CYP6A1 was down-regulated in the C6/36 transfected with RPL22 (C6/36-RPL22) cells, suggesting that CYP6A1 was repressed by RPL22. Our study provides the first evidence that RPL22 may play some role in the regulation of deltamethrin-resistance in Cx. pipiens pallens.
Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology.
