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The widespread and improper use of pyrethroid insecticides, such as deltamethrin, has resulted in the evolution of resistance in many mosquito species, including Culex pipiens pallens. With the development of high-throughput sequencing, it is possible to massively screen pyrethroid resistance-associated gene. In this study, we used Illumina-Solexa transcriptome sequencing to identify genes that are expressed differently in deltamethrin-susceptible and -resistant strains of Culex pipiens pallens as a critical knowledge base for further studies. A total of 4,961,197,620 base pairs and 55,124,418 reads were sequenced, mapped to the Culex quinquefasciatus genome and assembled into 17,679 known genes. We recorded 1826 significantly differentially expressed genes (DEGs). Among them, 1078 genes were up-regulated and 748 genes were down-regulated in the deltamethrin-resistant strain compared to -susceptible strain. These DEGs contained cytochrome P450 s, cuticle proteins, UDP-glucuronosyltransferases, lipases, serine proteases, heat shock proteins, esterases and others. Among the 1826 DEGs, we found that the transcriptional levels of CYP6AA9 in the laboratory populations was elevated as the levels of deltamethrin resistance increased. Moreover, the expression levels of the CYP6AA9 were significantly higher in the resistant strains than the susceptible strains in three different field populations. We further confirmed the association between the CYP6AA9 gene and deltamethrin resistance in mosquitoes by RNA interfering (RNAi). Altogether, we explored massive potential pyrethroid resistance-associated genes and demonstrated that CYP6AA9 participated in the pyrethroid resistance in mosquitoes.

Insecticide resistance has been a major public health challenge. It is impendent to study the mechanism on insecticide resistance. In our previous study, 14 differentially accumulated insect cuticle proteins (ICPs) based on insecticide resistance proteomes and transcriptomes were found in the deltamethrin-resistant (DR) and -susceptible (DS) strains of Culex pipiens pallens. To investigate if these ICPs are associated with deltamethrin resistance, different transcriptional levels of the 14 ICPs were detected in the DS and DR strains from laboratory and field populations by using quantitative real-time polymerase chain reaction (qRT-PCR). The expression levels of the 14 ICPs were also measured after short-term exposure of the DS strain to deltamethrin. The full-length complementary DNA (cDNA) of CpCPLCG5 gene, which encodes one of the 14 ICPs, was cloned from Cx. pipiens pallens. Homology analysis and phylogenetic analysis were carried out with some other insects. Furthermore, small interfering RNA (siRNA) was used to knockdown the expression level of CpCPLCG5 gene for characterizing its contribution to deltamethrin resistance. The results showed that the expression level of CpCPLCG5 gene was higher in DR strain than in DS strain both in laboratory and field populations while the other 13 ICPs were downregulated. The full-length cDNA of CpCPLCG5 gene was 732 bp, with the ORF of 390 bp and deduced 129 amino acids (GenBank/KF723314,2013). Knockdown of CpCPLCG5 gene increased the susceptibility of the DR strain while the expression level of the other 13 ICPs elevated. Our findings indicate that the cuticle proteins are associated with deltamethrin resistance in Cx. pipiens pallens.

Culex pipiens pallens is the most abundant Culex mosquito species in northern China and is an important vector of bancroftian filariasis and West Nile virus. Deltamethrin is an insecticide that is widely used for mosquito control, however resistance to this and other insecticides has become a major challenge in the control of vector-borne diseases that appear to be inherited quantitatively. Furthermore, the genetic basis of insecticide resistance remains poorly understood. In this study, quantitative trait loci (QTL) mapping of resistance to deltamethrin was conducted in F2 intercross segregation populations using bulked segregation analysis (BSA) and amplified fragment length polymorphism markers (AFLP) in Culex pipiens pallens. A genetic linkage map covering 381 cM was constructed and a total of seven QTL responsible for resistance to deltamethrin were detected by composite interval mapping (CIM), which explained 95% of the phenotypic variance. The major QTL in linkage group 2 accounted for 62% of the variance and is worthy of further study. 12 AFLP markers in the map were cloned and the genomic locations of these marker sequences were determined by applying the Basic Local Alignment Search Tool (BLAST) tool to the genome sequence of the closely related Culex quinquefasciatus. Our results suggest that resistance to deltamethrin is a quantitative trait under the control of a major QTL in Culex pipiens pallens. Cloning of related AFLP markers confirm the potential utility for anchoring the genetic map to the physical map. The results provide insight into the genetic architecture of the trait.

Current vector control programs are largely dependent on pyrethroids, which are the most commonly used and only insecticides recommended by the World Health Organization for insecticide-treated nets (ITNs). However, the rapid spread of pyrethroid resistance worldwide compromises the effectiveness of control programs and threatens public health. Since few new insecticide classes for vector control are anticipated, limiting the development of resistance is crucial for prolonging efficacy of pyrethroids. In this study, we exposed a field-collected population of Culex pipiens pallens to different insecticide selection intensities to dynamically monitor the development of resistance. Moreover, we detected kdr mutations and three detoxification enzyme activities in order to explore the evolutionary mechanism of pyrethroid resistance. Our results revealed that the level of pyrethroid resistance was proportional to the insecticide selection pressure. The kdr and metabolic resistance both contributed to pyrethroid resistance in the Cx. pipiens pallens populations, but they had different roles under different selection pressures. We have provided important evidence for better understanding of the development and mechanisms of pyrethroid resistance which may guide future insecticide use and vector management in order to avoid or delay resistance.

The mosquito, Culex pipiens pallens (L.), is an important vector of encephalitis and filariasis in northern China. The control of these mosquitoes occurs primarily via the use of pyrethroid insecticides, such as deltamethrin. The widespread and improper application of pyrethroid has resulted in the evolution of pyrethroid resistance amongst many mosquito populations, including Cx. pipiens pallens. Previous studies using high-throughput transcriptome sequencing have identified that the venom allergen 5 gene is differentially expressed between deltamethrin-susceptible and deltamethrin-resistant Cx. pipiens pallens. In this study, quantitative real-time polymerase chain reaction analyses revealed that venom allergen 5 was significantly overexpressed in adult females of both deltamethrin-resistant laboratory populations and two field populations. The transcriptional level of venom allergen 5 in the laboratory populations was elevated as the levels of deltamethrin resistance increased. Full-length cDNAs of the venom allergen 5 gene were cloned from Cx. pipiens pallens, and contained an open reading frame of 765 bp, encoding a protein with 254 amino acids. The deduced amino acid sequence shared 100% identity with the ortholog in Culex quinquefasciatus Say. The overexpression of venom allergen 5 decreased the susceptibility of mosquito cells to deltamethrin, while knockdown of this gene by RNAi increased the susceptibility of mosquitoes to deltamethrin. This study provides the first evidence of the association between the venom allergen 5 gene and deltamethrin resistance in mosquitoes., (© The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: Mosquito control based on chemical insecticides is considered as an important element in the current global strategies for the control of mosquito-borne diseases. Unfortunately, the development of pyrethroid resistance in important vector mosquito species jeopardizes the effectiveness of insecticide-based mosquito control. To date, the mechanisms of pyrethroid resistance are still unclear. Recent advances in proteomic techniques can facilitate to identify pyrethroid resistance-associated proteins at a large-scale for improving our understanding of resistance mechanisms, and more importantly, for seeking some genetic markers used for monitoring and predicting the development of resistance., Methods: We performed a quantitative proteomic analysis between a deltamethrin-susceptible strain and a deltamethrin-resistant strain of laboratory population of Culex pipiens pallens using isobaric tags for relative and absolute quantitation (iTRAQ) analysis. Gene Ontology (GO) analysis was used to find the relative processes that these differentially expressed proteins were involved in. One differentially expressed protein was chosen to confirm by Western blot in the laboratory and field populations of Cx. pipiens pallens., Results: We identified 30 differentially expressed proteins assigned into 10 different categories, including oxidoreductase activity, transporter activity, catalytic activity, structural constituent of cuticle and hypothetical proteins. GO analysis revealed that 25 proteins were sub-categorized into 35 hierarchically-structured GO classifications. Western blot results showed that CYP6AA9 as one of the up-regulated proteins was confirmed to be overexpressed in the deltamethrin-resistant strains compared with the deltamethrin-susceptible strains both in the laboratory and field populations., Conclusions: This is the first study to use modern proteomic tools for identifying pyrethroid resistance-related proteins in Cx. pipiens. The present study brought to light many proteins that were not previously thought to be associated with pyrethroid resistance, which further expands our understanding of pyrethroid resistance mechanisms. CYP6AA9 was overexpressed in the deltamethrin-resistant strains, indicating that CYP6AA9 may be involved in pyrethroid resistance and may be used as a potential genetic marker to monitor and predict the pyrethroid resistance level of field populations.

MicroRNAs (miRNAs) regulate gene expression and biological processes including embryonic development, innate immunity, and infection in many species. Emerging evidence indicates that miRNAs are involved in drug resistance. However, little is known about the relationship between the miRNAs and insecticide resistance in mosquitos. Here, we reported that conserved miR-278-3p and its target gene are critical for pyrethroid resistance in Culex pipiens pallens. We found that CYP6AG11 is the target of miR-278-3p, through bioinformatic analysis and experimental verification. The expression level of miR-278-3p was lower, whereas the level of CYP6AG11 was higher in deltamethrin-resistant strain, which were detected using quantitative reverse transcription PCR (qRT-PCR). We also found that CYP6AG11 was regulated by miR-278-3p via a specific target site with the 3' untranslated region (UTR) by luciferase reporter assay. In addition, overexpression of CYP6AG11 in the mosquito C6/36 cells showed better proliferation than the cells with empty vector when treated by deltamethrin at different concentrations. Moreover, the overexpression of miR-278-3p through microinjection led to a significant reduction in the survival rate, and the level of CYP6AG11 was simultaneously reduced. These results indicated that miR-278-3p could regulate the pyrethroid resistance through CYP6AG11.

Pyrethroids are the major class of insecticides used for mosquito control. Excessive and improper use of insecticides, however, has resulted in pyrethroid resistance, which has become a major obstacle for mosquito control. The development of pyrethroid resistance is a complex process involving many genes, and information on post-transcription regulation of pyrethroid resistance is lacking. In this study, we extracted RNA from mosquitoes in various life stages (fourth-instar larvae, pupae, male and female adult mosquitoes) from deltamethrin-sensitive (DS) and resistant (DR) strains. Using illumina sequencing, we obtained 13760296 and 12355472 reads for DS-strains and DR-strains, respectively. We identified 100 conserved miRNAs and 42 novel miRNAs derived from 21 miRNA precursors in Culex pipiens. After normalization, we identified 28 differentially expressed miRNAs between the two strains. Additionally, we found that cpp-miR-71 was significant down regulated in female adults from the DR-strain. Based on microinjection and CDC Bottle Bioassay data, we found that cpp-miR-71 may play a contributing role in deltamethrin resistance. The present study provides the firstly large-scale characterization of miRNAs in Cu. pipiens and provides evidence of post-transcription regulation. The differentially expressed miRNAs between the two strains are expected to contribute to the development of pyrethroid resistance., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

To explore if trypsin could catalyze the degradation of non-protein molecule deltamethrin, we compared in vitro hydrolytic reactions of deltamethrin in the presence and absence of trypsin with ultraviolet-visible (UV/Vis) spectrophotometry and gas chromatography-mass spectrometry (GC/MS). In addition, acute oral toxicity of the degradation products was determined in Wistar rats. The results show that the absorption peak of deltamethrin is around 264 nm, while the absorption peaks of deltamethrin degradation products are around 250 nm and 296 nm. In our GC setting, the retention time of undegraded deltamethrin was 37.968 min, while those of deltamethrin degradation products were 15.289 min and 18.730 min. The LD50 of deltamethrin in Wistar rats is 55 mg/kg, while that of deltamethrin degradation products is 3358 mg/kg in female rats and 1045 mg/kg in male rates (61-fold and 19-fold reductions in toxicity), suggesting that trypsin could directly degrade deltamethrin, which significantly reduces the toxicity of deltamethrin. These results expand people's understanding of the functions of proteases and point to potential applications of trypsin as an attractive agent to control residual pesticides in the environment and on agricultural products.

Deltamethrin (DM) insecticides are currently being promoted worldwide for mosquito control, because of the high efficacy, low mammalian toxicity and less environmental impact. Widespread and improper use of insecticides induced resistance, which has become a major obstacle for the insect-borne disease management. Resistance development is a complex and dynamic process involving many genes. To better understand the possible molecular mechanisms involved in DM resistance, a proteomic approach was employed for screening of differentially expressed proteins in DM-susceptible and -resistant mosquito cells. Twenty-seven differentially expressed proteins were identified by two-dimensional electrophoresis (2-DE) and mass spectrometry (MS). Four members of the ubiquitin-proteasome system were significantly elevated in DM-resistant cells, suggesting that the ubiquitin-proteasome pathway may play an important role in DM resistance. Proteasome subunit beta type 6 (PSMB6) is a member of 20S proteasomal subunit family, which forms the proteolytic core of 26S proteasome. We used pharmaceutical inhibitor and molecular approaches to study the contributions of PSMB6 in DM resistance: the proteasome inhibitor MG-132 and bortezomib were used to suppress the proteasomal activity and siRNA was designed to block the function of PSMB6. The results revealed that both MG-132 and bortezomib increased the susceptibility in DM-resistant cells and resistance larvae. Moreover, PSMB6 knockdown decreased cellular viability under DM treatment. Taken together, our study indicated that PSMB6 is associated with DM resistance in mosquitoes and that proteasome inhibitors such as MG-132 or bortezomib are suitable for use as a DM synergist for vector control.

Ribose-phosphate pyrophosphokinase 1 (PRPS1) was identified and isolated as a differentially expressed gene between deltamethrin-susceptible (DS) and deltamethrin-resistant (DR) Culex pipiens pallens and Aedes albopictus C6/36 cell line through microarray and 2D-Gel. An open reading frame of PRPS1 cloned from C. pipiens pallens has 1,011 bp and encodes for a 336 amino acids protein which shares high homology with Culex quinquefasciatus. Real-time polymerase chain reaction was used to determine the transcript expression level of PRPS1 in DS and DR strains. The expression levels of PRPS1 were higher in DR laboratory strains and natural population JXZ-DR, JXZ-LDR. PRPS1 was also detected and expressed at all developmental stages of C. pipiens pallens and increased expression level in DR3 strain than DS strain in the third and fourth instar larvae, female and male stages. In addition, to further investigate the role of PRPS1 in deltamethrin resistance, PRPS1 was transiently expressed in A. albopictus C6/36 cells and detected by western blotting. Cells transfected with PRPS1 had an increased resistance to deltamethrin compared with control cells. These results suggested that the increased expression level of PRPS1 may play roles in the regulation of deltamethrin resistance.

The 40S ribosomal protein S29 (RPS29) was shown differentially expressed in deltamethrin (DM)-susceptible and resistant Culex pipiens pallens previously. Here, we cloned RPS29 from this mosquito. An open reading frame (ORF) of 171 bp was found to encode a putative protein with 56 amino acid residues, which shares more than 90% identities with other mosquito RPS29 genes. Moreover, quantitative real-time PCR analysis demonstrated that the transcriptional level of RPS29 was significantly up-regulated both in DM-resistant Cx. p. pallens and C6/36 cells compared with the susceptible ones. Over-expression of RPS29 showed a slight decrease of cell viability in DM-susceptible C6/36 cells under DM treatment and knockdown of RPS29 by siRNA comparably increased cell viability in DM-resistant cells. Our study provides evidence that RPS29 might be associated with fitness cost of DM resistance in mosquitoes for the first time.

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.

Deltamethrin degradation was assessed by measuring deltamethrin reduction with GC-MS and UV-Vis spectrophotometry following incubation of various concentrations (4.96-24.80 microM) of deltamethrin with alpha-chymotrypsin from bovine pancreas. The retention times of crude products of deltamethrin metabolic reactions were 37.968 min, 37.415 min, 36.490 min, and 35.895 min. The UV-Vis spectrophotometric peak absorbance of deltamethrin was at 264 nm, and the peak absorbance of deltamethrin metabolic products were at 250 nm and 296 nm, respectively. Michaelis-Menten metabolic rate constants (V(max) and K(m)) were calculated by nonlinear regression for chymotrypsin using GraphPad Prism 4.0, the V(max) was 97.97+/-26.57 nmol/L/min and K(m) was 7.84+/-3.83 microM. The larvicidal bioassay tests indicated that the mixture from the degradation reaction showed LC(50) increased significantly (P<0.05). This is the first report demonstrating deltamethrin metabolism by chymotrypsin.

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.

The glycogen branching enzyme (GBE) gene has been cloned from Culex pipiens pallens. An open reading frame of 2,070 bp was found to encode a putative 689 amino acid protein. The deduced amino acid sequence shares 82% and 60% identity with GBE genes from Aedes aegypti and Homo sapiens. Transcript expression of GBE was determined by real-time polymerase chain reaction in deltamethrin-susceptible and deltamethrin-resistant strains of the Culex mosquito. The results demonstrated that this gene has 19.7-fold higher expression in deltamethrin-resistant strain. Unexpectedly, the following study showed that cell proliferation decreased obviously in the GBE overexpression group compared to the empty vector control or the unrelated gene when treated by deltamethrin in the mosquito C6/36 cells whose mechanism is still unexplained. Our study provides the first direct evidence that GBE gene may play some role in the development of deltamethrin resistance in C. pipiens pallens.

The complete sequence of ribosomal protein L39 has been cloned from deltamethrin-resistant strain of Culex pipiens pallens (DR1 strain). Quantitative RT-PCR analysis indicated that the RPL39 transcription level was 23.4 times higher in DR1 strain than in susceptible strain at 4th instar larvae. The RPL39 expression was also found to be consistently higher throughout the life cycle of DR1 strain. A protein of predicted size 17 kDa has been detected by Western blotting in RPL39-transfected mosquito C6/36 cells. These RPL39-transfected cells also showed enhanced deltamethrin resistance compared to plasmid vector-transfected cells as determined by methyl tritiated thymidine ((3)H-TdR) incorporation. These results indicate that RPL39 is expressed at higher levels in DR1 strain, and may confer some insecticide resistance in Cx. pipiens pallens.

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.