Deltamethrin-susceptible Strain Research Articles

CircRNA expression profiles in deltamethrin-susceptible and -resistant Culex pipiens pallens (Diptera: Culicidae)

The excessive and improper application of insecticides has caused the evolution of resistance in many mosquito populations, including Culex pipiens pallens (L.). Deltamethrin, a representative pyrethroid insecticide, is the most widely used synthetic insecticide in mosquito-borne control field. Comprehensively identifying genes and regulators associated with deltamethrin resistance and elucidating the manner in which they regulate this process is critical for effective control of mosquitoes. CircRNAs are the upstream regulatory factors of miRNAs and mRNAs, which play a role via the competitive endogenous RNA mechanism. In this study, we used high-throughput circRNA sequencing to identify circRNAs that were expressed differently in deltamethrin-susceptible strain (DS strain) and -resistant strain (DR strain) mosquitoes [NCBI Sequence Read Archive (SRA) database accession number: PRJNA714543]. We detected a total of 12,816 significantly differentially expressed circRNAs (DE-circRNAs). Among them, 6769 circRNAs were up-regulated and 6047 circRNAs were down-regulated in the DR strain compared to DS strain. Among the DE-circRNAs, we further screened that supercont3.352:252102|253283 was significantly over-expressed in the DR strain through qPCR multiple verification (P < 0.05).We used the divergent primer to amplify the rolling circle product and obtained the full-length sequence of supercont3.352:252102|253283 (GeneBank accession number: MW729338). Through software comparison and bioinformatics analysis, we predicted that supercont3.352:252102|253283 might participate in deltamethrin resistance by sponging cpp-miR-1671 and blocking its inhibition on CYP4G15.We further found that the expression of cpp-miR-1671 was significantly lower in DR strain (P < 0.01), while the expression of CYP4G15 was significantly higher in DR strain (P < 0.05).Taken together, the present study provided the most comprehensive circRNA expression profile of mosquitoes, and suggested that supercont3.352:252102|253283 might participate in deltamethrin resistance through the supercont3.352:252102|253283/cpp-miR-1671/CYP4G15 pathway.

MiR-4448 is involved in deltamethrin resistance by targeting CYP4H31 in Culex pipiens pallens

BackgroundCulex pipiens (Cx. pipiens) complex, which acts as a vector of viruses and is widespread and abundant worldwide, including West Nile virus, Japanese encephalitis virus, and Sindbis virus, can cause serious vector-borne diseases affecting human health. Unfortunately, mosquitoes have developed deltamethrin resistance because of its long-term overuse, representing a major challenge to mosquito control. Understanding the molecular regulatory mechanisms of resistance is vital to control mosquitoes. MicroRNAs (miRNAs) are short non-coding RNAs that have been demonstrated to be important regulators of gene expression across a wide variety of organisms, which might function in mosquito deltamethrin resistance. In the present study, we aimed to investigate the regulatory functions of miR-4448 and CYP4H31 in the formation of insecticidal resistance in mosquito Culex pipiens pallens.MethodsWe used quantitative real-time reverse transcription PCR to measure miR-4448 and CYP4H31 (encoding a cytochrome P450) expression levels. The regulatory functions of miR-4448 and CYP4H31 were assessed using dual-luciferase reporter assays. Then, oral feeding, RNA interference, and the American Centers for Disease Control and Prevention bottle bioassay were used to determine miR-4448’s association with deltamethrin resistance by targeting CYP4H31in vivo. Cell Counting Kit-8 (CCK-8) was also used to detect the viability of pIB/V5-His-CYP4H31-transfected C6/36 cells after deltamethrin treatment in vitro.ResultsMiR-4448 was downregulated in the deltamethrin-resistant strain (DR strain), whereas CYP4H31 was downregulated in deltamethrin-susceptible strain. CYP4H31 expression was downregulated by miR-4448 recognizing and binding to its 3′ untranslated region. Functional verification experiments showed that miR-4448 overexpression resulted in lower expression of CYP4H31. The mortality of miR-4448 mimic-injected DR strain mosquitoes was higher than that of the controls. CCK-8 assays showed that CYP4H31 decreased cellular resistance to deltamethrin in vitro and the mortality of the DR strain increased when CYP4H31 was knocked down in vivo.ConclusionsIn mosquitoes, miR-4448 participates in deltamethrin resistance by targeting CYP4H31. The results of the present study increase our understanding of deltamethrin resistance mechanisms.Graphical

Transcription factor FTZ-F1 regulates mosquito cuticular protein CPLCG5 conferring resistance to pyrethroids in Culex pipiens pallens

BackgroundCulex pipiens pallens poses a serious threat to human health because of its widespread distribution, high carrier capacity for several arboviruses, frequent human-biting, and growth in urban environments. Pyrethroid insecticides have been mainly used to control adult Cx. pipiens pallens during outbreaks of mosquito-borne diseases. Unfortunately, mosquitoes have developed resistance, rendering the insecticides ineffective. Cuticular resistance is the primary mechanism of pyrethroid resistance. Previously, we revealed that cuticular protein of low complexity CPLCG5 is a major cuticular protein associated with deltamethrin resistance in Cx. pipiens pallens, which is enriched in the cuticle of mosquitoes’ legs and participates in pyrethroid resistance by forming a rigid matrix. However, the regulatory mechanisms of its transcription remain unknown.ResultsFirst, qRT-PCR analysis revealed that the expression of FTZ-F1 (encoding Fushi tarazu-Factor 1) was ~ 1.8-fold higher in the deltamethrin-resistant (DR) than deltamethrin-susceptible (DS) strains at 24 h post-eclosion (PE) and ~ 2.2-fold higher in the DR strain than in the DS strain at 48 h PE. CPLCG5 and FTZ-F1 were co-expressed in the legs, indicating that they might play an essential role in the legs. Dual luciferase reporter assays and EMSA (electrophoretic mobility shift experiments) revealed that FTZ-F1 regulates the transcription of CPLCG5 by binding to the FTZ-F1 response element (− 870/− 864). Lastly, knockdown of FTZ-F1 not only affected CPLCG5 expression but also altered the cuticle thickness and structure of the legs, increasing the susceptibility of the mosquitoes to deltamethrin in vivo.ConclusionsThe results revealed that FTZ-F1 regulates the expression of CPLCG5 by binding to the CPLCG5 promoter region, altering cuticle thickness and structure, and increasing the susceptibility of mosquitoes to deltamethrin in vivo. This study revealed part of the mechanism of cuticular resistance, providing a deeper understanding of insecticide resistance.

Lipase is associated with deltamethrin resistance in Culex pipiens pallens.

The wide application of pyrethroids has led to the rapid development of insecticide resistance in mosquitoes, leading to a rise in mosquito-borne diseases. We previously identified five differentially expressed lipase family genes upon evaluating the transcriptomes of deltamethrin-resistant and deltamethrin-susceptible strains of Culex pipiens pallens. Herein, the gene expression levels were verified by quantitative real-time PCR, and two lipase family genes, lipase A and pancreatic triacylglycerol lipase A, were chosen for further investigations. Using cell viability assays and Centers for Disease Control and Prevention bottle bioassays, lipase A was found to increase the resistance of mosquitoes against deltamethrin both in vitro and in vivo. Our findings indicate that lipase A is involved in conferring deltamethrin resistance in Cx. pipiens pallens.

Excretion/defecation patterns in Triatoma infestans populations that are, respectively, susceptible and resistant to deltamethrin

Pyrethroid resistance has been detected in Triatoma infestans (Klug) (Hemiptera: Reduviidae) specimens from different areas of Argentina and Bolivia. Genes conferring resistance can have a pleiotropic effect with epidemiological and evolutionary consequences. This research studied excretion/defecation patterns in deltamethrin-resistant T.infestans in order to elucidate its biological performance, adaptive consequences and role in the transmission of Chagas' disease. One deltamethrin-susceptible strain and two deltamethrin-resistant strains were used. Fifth-instar nymphs were fed ad libitum and their defecations recorded during and after the first or second feeding in the stadium. Resistant insects began to defecate later, defecated less, showed a lower proportion of defecating individuals and lower defecation indices compared with susceptible insects during the first hour after feeding. The number of bloodmeals in the stadium did not affect the main variables determining the pattern of defecation. The present study suggests that alterations in the excretion/defecation pattern in resistant insects entail an adaptive cost and, considering only this pattern, determine a lower capacity for transmission of Trypanosoma cruzi (Kinetoplastida: Trypanosomatidae) compared with susceptible insects.

Culex pipiens pallens cuticular protein CPLCG5 participates in pyrethroid resistance by forming a rigid matrix

BackgroundChemical insecticides have hugely reduced the prevalence of vector-borne diseases around the world, but resistance threatens their continued effectiveness. Despite its importance, cuticle resistance is an under-studied area, and exploring the detailed molecular basis of resistance is critical for implementing suitable resistance management strategies.MethodsWe performed western blotting of cuticular protein CPLCG5 in deltamethrin-susceptible (DS) and laboratory-produced deltamethrin-resistant (DR) strains of Culex pipiens pallens. Immunofluorescence assays using a polyclonal antibody to locate cuticular CPLCG5 in mosquitoes. EM immunohistochemical analysis of the femur segment was used to compare the cuticle in control and CPLCG5-deficient siRNA experimental groups.ResultsThe gene CPLCG5 encodes a cuticle protein that plays an important role in pyrethroid resistance. Based on a prior study, we found that expression of CPLCG5 was higher in the resistant (DR) strain than the susceptible (DS) strain. CPLCG5 transcripts were abundant in white pupae and 1-day-old adults, but expression was dramatically decreased in 3-day-old adults, then remained stable thereafter. Western blotting revealed that the CPLCG5 protein was ~2.2-fold higher in the legs of the DR strain than the DS strain. Immunofluorescence assays revealed CPLCG5 expression in the head, thorax, abdomen, wing, and leg, and expression most abundant in the leg and wing. EM immunohistochemical analysis suggested that the exocuticle thickness of the femur was significantly thinner in the CPLCG5-deficient siCPLCG5 strain (0.717 ± 0.110 μm) than the siNC strain (0.946 ± 0.126 μm). Depletion of CPLCG5 by RNA interference resulted in unorganised laminae and a thinner cuticle.ConclusionsThe results suggest CPLCG5 participates in pyrethroid resistance by forming a rigid matrix and increasing the thickness of the cuticle.

Carbonic anhydrase II confers resistance to deltamethrin in Culex pipiens pallens.

Carbonic anhydrases (CAs) are metabolic enzymes that regulate the physiological equilibrium in a variety of organisms. In this study, we investigated the function of CA II in Culex pipiens pallens using real-time quantitative polymerase chain reaction and double-stranded RNA injection. CA II transcripts were more abundant in the deltamethrin-susceptible strain than the deltamethrin-resistant strain. The activities of metabolic enzymes increased when the CA II expression was silenced. These findings suggest CA II regulates deltamethrin resistance by altering metabolic enzyme activity, and could serve as a potential genetic marker for monitoring deltamethrin resistance in mosquitoes.

PiRNA-3312: A Putative Role for Pyrethroid Resistance in Culex pipiens pallens (Diptera: Culicidae).

Piwi-interacting RNAs (piRNAs) are a newly identified class of small noncoding RNAs. They are associated with chromatin organization, messenger RNA stability, and genome structure. Although the overexpression of piRNA-3312 in deltamethrin-susceptible (DS) strain of Culex pipiens pallens (L.) was observed in our previous large-scale transcriptome data, the roles of piRNA in insecticide resistance have not been clearly defined. The aim of the present study was to investigate how piRNA-3312 is involved in insecticide resistance. The lower expression level of piRNA-3312 in deltamethrin-resistant (DR) strain of Cx. pipiens pallens was confirmed by quantitative real time polymerase chain reaction (qRT-PCR). Overexpression of piRNA-3312 in the DR strain made the mosquitoes more sensitive to deltamethrin, whereas inhibiting the expression of piRNA-3312 in the DS strain made the mosquitoes more resistant to deltamethrin. Piwi-interacting RNA-3312 was also found to bind 3' UTR (Untranslated Regions) of gut esterase 1 gene and could induce its degradation. In addition, knockdown of gut esterase 1 gene increased the sensitivity of DR strain to deltamethrin. In conclusion, we found that piRNA-3312 targeted the gut esterase 1 gene to negatively regulate the insecticide resistance. This finding facilitates the understanding of various functions of piRNAs and their association with insecticide resistance.

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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-0709-5) contains supplementary material, which is available to authorized users.

Cloning and characterization of ubiquitin ribosome fusion gene RpS27a, a deltamethrin resistance associated gene from diamondback moth (Plutella xylostella L.)

The ubiquitin ribosome fusion gene RpS27a's coding sequence was cloned from diamondback moth, Plutella xylostella (L.). An open reading frame of 468 bp was found to encode a putative 155 amino acid precursor protein, which shares over 90% similarity with other insects' RpS27a protein. Real-time quantitative PCR was carried out to determine the relative expression level in the fourth instar larvae of deltamethrin-resistant and deltamethrin-susceptible strains. The results showed that the expression of RpS27a was significantly higher in the deltamethrin-resistant strain than in the deltamethrin-susceptible strain. There are reports indicating that upregulation expression of some ribosomal proteins confers some insecticide resistance. For the first time, we have predicted that the increased expression of the RpS27a gene may have some association with pesticide resistance in Plutella xylostella.

Ribose-phosphate pyrophosphokinase 1 (PRPS1) associated with deltamethrin resistance in Culex pipiens pallens

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.

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.

Prag01, a novel deltamethrin-resistance-associated gene from Culex pipiens pallens

The prag01 gene (GenBank accession no. EU073017) was cloned from Culex pipiens pallens. An open reading frame of 270 bp was found to encode a putative 89-amino-acid protein which has the highest homology with Culex quinquefasciatus and Anopheles funestus. Real-time quantitative PCR analysis demonstrated that the transcription level of prag01 gene in deltamethrin-resistant strain was 1.65-fold higher than in deltamethrin-susceptible strain of C. pipiens pallens. Overexpression of prag01 gene in the mosquito C6/36 cells showed better prolification than the cells with empty vector when treated by deltamethrin. Our data for the first time approved that prag01 gene might play some role in the development of deltamethrin resistance in C. pipiens pallens.

Cloning and characterization of 60S ribosomal protein L22 (RPL22) from Culex 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.

Cloning and characterization of myosin regulatory light chain (MRLC) gene from Culex pipiens pallens

Myosin regulatory light chain ( MRLC) (GenBank accession no. DQ140391) was cloned from Culex pipiens pallens. An open reading frame (ORF) of 630 bps was found to encode a putative 210 amino acids protein which shows 73% similarity with myosin regulatory light chain of Gryllotalpa orientalis. Real-time quantitative PCR analysis demonstrated that the transcription level of MRLC in deltamethrin-resistant strain (DR-strain) was 4.08-fold higher than in deltamethrin-susceptible strain (DS-strain) of C. pipiens pallens. Over-expression of MRLC in Aedes albopictus C6/36 cells conferred protection against deltamethrin based on tritiated methyl tritiated thymidine ( 3H-TdR) incorporation assay. These results indicate that MRLC may be a potential cause of deltamethrin resistance in C. pipiens pallens.