Dan Zhou

Molecular Ecology of Pyrethroid Knockdown Resistance in Culex pipiens pallens Mosquitoes

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.

Genome sequence of Anopheles sinensis provides insight into genetics basis of mosquito competence for malaria parasites

BackgroundAnopheles sinensis is an important mosquito vector of Plasmodium vivax, which is the most frequent and widely distributed cause of recurring malaria throughout Asia, and particularly in China, Korea, and Japan.ResultsWe performed 454 next-generation sequencing and obtained a draft sequence of A. sinensis assembled into scaffolds spanning 220.8 million base pairs. Analysis of this genome sequence, we observed expansion and contraction of several immune-related gene families in anopheline relative to culicine mosquito species. These differences suggest that species-specific immune responses to Plasmodium invasion underpin the biological differences in susceptibility to Plasmodium infection that characterize these two mosquito subfamilies.ConclusionsThe A. sinensis genome produced in this study, provides an important resource for analyzing the genetic basis of susceptibility and resistance of mosquitoes to Plasmodium parasites research which will ultimately facilitate the design of urgently needed interventions against this debilitating mosquito-borne disease.

Identification of differentially expressed microRNAs in Culex pipiens and their potential roles in pyrethroid resistance

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.

MiR-278-3p regulates pyrethroid resistance in Culex pipiens pallens

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.

Identification of proteins associated with pyrethroid resistance by iTRAQ-based quantitative proteomic analysis in Culex pipiens pallens

BackgroundMosquito 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.MethodsWe 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.ResultsWe 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.ConclusionsThis 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.

Genomic Analysis of Detoxification Supergene Families in the Mosquito Anopheles sinensis

Anopheles sinensis is an important malaria vector in China and other Southeast Asian countries, and the emergence of insecticide resistance in this mosquito poses a serious threat to the efficacy of malaria control programs. The recently published An. sinensis genome and transcriptome provide an opportunity to understand the molecular mechanisms of insecticide resistance. Analysis of the An. sinensis genome revealed 174 detoxification genes, including 93 cytochrome P450s (P450s), 31 glutathione-S-transferases (GSTs), and 50 choline/carboxylesterases (CCEs). The gene number was similar to that in An. gambiae, but represented a decrease of 29% and 42% compared with Aedes aegypti and Culex quinquefasciatus, respectively. The considerable contraction in gene number in Anopheles mosquitoes mainly occurred in two detoxification supergene families, P450s and CCEs. The available An. sinensis transcriptome was also re-analyzed to further identify key resistance-associated detoxification genes. Among 174 detoxification genes, 124 (71%) were detected. Several candidate genes overexpressed in a deltamethrin-resistant strain (DR-strain) were identified as belonging to the CYP4 or CYP6 family of P450s and the Delta GST class. These generated data provide a basis for identifying the resistance-associated genes of An. sinensis at the molecular level.

Development of Resistance to Pyrethroid in Culex pipiens pallens Population under Different Insecticide Selection Pressures

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.

MiR-932 Regulates Pyrethroid Resistance in Culex pipiens pallens (Diptera: Culicidae)

Abstract MicroRNAs (miRNAs) play notable role in regulation of gene expression at the posttranscription level, and have been involved in many biological processes, including insecticide resistance. In this study, we investigated the role of miR-932 in the molecular mechanisms of pyrethroid resistance in Culex pipiens pallens (L.). Overexpression of miR-932 in the DS-strain made the mosquitoes more resistant to deltamethrin, while inhibiting the expression of miR-932 in the DR-strain made the mosquitoes more sensitive to deltamethrin. Further, we also identified CpCPR5 as a target gene of miR-932. Sustained overexpression of miR-932 resulted in repression of CpCPR5, and that knockdown of miR-932 increased CpCPR5 expression. In addition, knockdown of CpCPR5 decreased the sensitivity of mosquitoes to deltamethrin in the DS-strain. In conclusion, our study finds a molecular link between miR-932 and CpCPR5 and provides a novel insight into the mechanism of insecticide resistance.

Functional characterization of an arrestin gene on insecticide resistance of Culex pipiens pallens.

BackgroundContinuous and excessive application of insecticides has resulted in the rapid development of insecticide resistance in several mosquito species, including Culex pipiens pallens. Previous studies in our laboratory found that arrestin gene expression was higher in the deltamethrin-resistant (DR) strain than in the deltamethrin-susceptible (DS) strain of Cx. pipiens pallens. Similarly, other studies reported that arrestin was highly expressed in permethrin-resistant Cx. quinquefasciatus and in dichlorodiphenyltrichloroethane (DDT)-resistant Drosophila melanogaster.MethodsFull-length cDNAs of an arrestin gene were cloned from Cx. pipiens pallens via polymerase chain reaction (PCR) and rapid amplification of cDNA end (RACE). The mRNA levels of the arrestin gene in the whole life cycle of DR and DS strains of Cx. pipiens pallens were investigated via quantitative real-time PCR. In addition, the relationship between arrestin and deltamethrin (DM) resistance were identified using genetic overexpression strategies and arrestin RNAi in mosquito cells. Cell viability was analyzed with cholecystokinin octapeptide after DM treatment. Moreover, the mRNA levels of cytochrome P450 6A1 (CYP6A1) and opsin in the transfected cells and controls were analyzed.ResultsComplete arrestin gene sequence was cloned and expressed throughout the life cycle of Cx. pipiens pallens. Moreover, arrestin was significantly upregulated in the DR strain, compared with that in the DS strain at the egg, pupae, and adult stages. Arrestin overexpression comparably increased the mosquito cell viability, whereas arrestin knockdown by siRNA decreased mosquito cell viability with deltamethrin (DM) treatment. Meanwhile, the mRNA levels of CYP6A1 and opsin were upregulated in mosquito cells transfected with arrestin and downregulated in mosquito cells with arrestin knockdown.ConclusionThis study presented the first evidence that arrestin might be associated with insecticide resistance in Cx. pipiens pallens.

Cloning and characterization of prophenoloxidase A3 (proPOA3) from Culex pipiens pallens

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.