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

Abstract Background Culex 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. Results First, 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. Conclusions The 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.

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

Parasites & Vectors ◽

10.1186/s13071-021-04665-x ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Xixi Li ◽

Shengli Hu ◽

Haitao Yin ◽

Hongbo Zhang ◽

Dan Zhou ◽

...

Keyword(s):

Culex Pipiens ◽

Oral Feeding ◽

Cell Counting ◽

Luciferase Reporter ◽

Culex Pipiens Pallens ◽

Deltamethrin Resistance ◽

Regulatory Functions ◽

Pipiens Pallens

Abstract Background Culex 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. Methods We 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. Results MiR-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. Conclusions In mosquitoes, miR-4448 participates in deltamethrin resistance by targeting CYP4H31. The results of the present study increase our understanding of deltamethrin resistance mechanisms.

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CPR63 Promotes Pyrethroid Resistance by Increasing Cuticle Thickness in Culex Pipiens Pallens

10.21203/rs.3.rs-770891/v1 ◽

2021 ◽

Author(s):

Yang Xu ◽

Yang Zhou ◽

Xixi Li ◽

Lei Ma ◽

Dan Zhou ◽

...

Keyword(s):

Electron Microscopy ◽

Culex Pipiens ◽

Pyrethroid Resistance ◽

Resistance Mechanism ◽

Negative Control Group ◽

Negative Control ◽

Control Group ◽

Culex Pipiens Pallens ◽

Cuticle Thickness ◽

Pipiens Pallens

Abstract The cuticle protein (CP) encoded by CPR63 plays a role in deltamethrin resistance in Culex pipiens pallens. Herein, we investigated the distribution of CPR63 transcripts in this organism, and revealed high expression levels in legs and wings. Furthermore, expression of CPR63 in the legs of deltamethrin-resistant (DR) strains were 1.79-fold higher than in deltamethrin-susceptible (DS) strains. Cuticle analysis of small interfering RNA (siRNA) groups by scanning electron microscopy (SEM) revealed a significantly thinner procuticle of the tarsi in the siCPR63 group than the siNC (negative control (group). Transmission electron microscopy (TEM) revealed that the procuticle, exocuticle and endocuticle thickness of the tarsi were significantly thinner in the siCPR63 group than the siNC group. Our results illuminate the resistance mechanism of CPRs and demonstrate that CPR63 contributes to the resistance phenotype by thickening the cuticle and substantially reducing uptake of insecticides.

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MiR-279-3p regulates deltamethrin resistance through CYP325BB1 in Culex pipiens pallens

Parasites & Vectors ◽

10.1186/s13071-021-05033-5 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Xixi Li ◽

Shengli Hu ◽

Hongbo Zhang ◽

Haitao Yin ◽

Huan Wang ◽

...

Keyword(s):

Insecticide Resistance ◽

Culex Pipiens ◽

Oral Feeding ◽

Luciferase Reporter ◽

Culex Pipiens Pallens ◽

Mosquito Mortality ◽

Deltamethrin Resistance ◽

Pipiens Pallens

Abstract Background The overuse of insecticides to control insect vectors has promoted extensive insecticide resistance in mosquitoes. In this study, the functions of microRNA (miR)-279-3p and its target CYP325BB1 in the regulation of deltamethrin resistance in Culex pipiens pallens was investigated. Methods Quantitative real-time reverse transcription PCR was used to detect the expression levels of miR-279-3p and CYP325BB1. Then, the dual-luciferase reporter assay system, RNA interference, CDC bottle bioassay and Cell Counting Kit-8 (CCK-8) assay were used to explore the roles of these molecules in deltamethrin resistance both in vivo and in vitro. Results The expression patterns of miR-279-3p and CYP325BB1 were compared between deltamethrin-sensitive (DS-strain) and deltamethrin-resistant (DR-strain) mosquitoes. Luciferase activity was downregulated by miR-279-3p, the effect of which was ablated by a mutation of the putative binding site for CYP325BB1. In DR-strain mosquitoes, the expression of miR-279-3p was increased by microinjection and oral feeding of miR-279-3p agomir (mimic). CYP325BB1 mRNA levels were downregulated, which resulted in a higher mortality of the mosquitoes in miR-279-3p mimic-treated groups. In the DS-strain mosquitoes, microinjection of a miR-279-3p inhibitor decreased miR-279-3p expression, whereas the expression of CYP325BB1 was increased; the mortality of these mosquitoes decreased significantly. In addition, overexpression of pIB/V5-His-CYP325BB1 changed the sensitivity of C6/36 cells to deltamethrin in vitro. Also in DR-strain mosquitoes, downregulation of CYP325BB1 expression by microinjection of si-CYP325BB1 increased mosquito mortality in vivo. Conclusions These findings provide empirical evidence of the involvement of miRNAs in the regulation of insecticide resistance and indicate that miR-279-3p suppresses the expression of CYP325BB1, which in turn decreases deltamethrin resistance, resulting in increased mosquito mortality. Taken together, the results provide important information for use in the development of future mosquito control strategies. Graphical abstract

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

Parasites & Vectors ◽

10.1186/s13071-017-2567-9 ◽

2018 ◽

Vol 11 (1) ◽

Cited By ~ 16

Author(s):

Yun Huang ◽

Qin Guo ◽

Xiaohong Sun ◽

Cheng Zhang ◽

Na Xu ◽

...

Keyword(s):

Culex Pipiens ◽

Pyrethroid Resistance ◽

Cuticular Protein ◽

Culex Pipiens Pallens ◽

Rigid Matrix ◽

Pipiens Pallens

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

Journal of Medical Entomology ◽

10.1093/jme/tjx043 ◽

2017 ◽

Vol 54 (4) ◽

pp. 1013-1018 ◽

Cited By ~ 3

Author(s):

Juxin Guo ◽

Wenyun Ye ◽

Xianmiao Liu ◽

Xueli Sun ◽

Qin Guo ◽

...

Keyword(s):

Culex Pipiens ◽

Pyrethroid Resistance ◽

Culex Pipiens Pallens ◽

Putative Role ◽

Pipiens Pallens

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MiR-285 targets P450 (CYP6N23) to regulate pyrethroid resistance in Culex pipiens pallens

Parasitology Research ◽

10.1007/s00436-016-5238-4 ◽

2016 ◽

Vol 115 (12) ◽

pp. 4511-4517 ◽

Cited By ~ 15

Author(s):

Mengmeng Tian ◽

Bingqian Liu ◽

Hongxia Hu ◽

Xixi Li ◽

Qin Guo ◽

...

Keyword(s):

Culex Pipiens ◽

Pyrethroid Resistance ◽

Culex Pipiens Pallens ◽

Pipiens Pallens

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RNA-seq screening of the CPGs in Culex pipiens pallens among cypermethrin-resistant populations

10.21203/rs.2.19260/v1 ◽

2019 ◽

Author(s):

Qiqi Shi ◽

Peng Cheng ◽

Chongxing Zhang ◽

Lijuan Liu ◽

Xiao Song ◽

...

Keyword(s):

Culex Pipiens ◽

Noncoding Rna ◽

Pyrethroid Resistance ◽

Fold Change ◽

Functional Enrichment ◽

Rna Seq ◽

Culex Pipiens Pallens ◽

Metabolic Resistance ◽

Illumina Hiseq ◽

Pipiens Pallens

Abstract Background Long-lasting overdependence on insecticides has led to the rapid spread of pyrethroid resistance in mosquito vectors, which is of great concern to the general public. There are many studies on metabolic resistance and target resistance, but fewer studies have been conducted on cuticle resistance and behaviour resistance. The cuticle of mosquitoes has been hypothesized to play a role in insecticide resistance by reducing penetration or sequestering insecticides. Methods We used RNA sequencing (RNA-seq) to analyse the transcriptome of cypermethrin-resistant and cypermethrin-susceptible strains of Culex pipiens pallens . Sequenced 6 samples using an Illumina HiSeq platform, and generated approximately 6.66 Gb bases from each sample on average. Mapping the sequenced reads to a reference genome and reconstructing the transcripts, through gene expression analysis, we detected differentially expressed genes (DEGs) among the samples. Followed Gene Ontology (GO) classification and functional enrichment. Finally, we screened the genes of cuticle proteins associated with drug resistance throughout the genome, selected the significant DEGs with a log2 fold change>3.0 and Padj<0.05, and applied real-time fluorescence quantitative PCR to verify the DEGs. Results We obtained 13,517 novel transcripts, of which 8,653 were previously unknown splicing events for known genes, 665 were novel coding transcripts without any known features, and 4,199 were long noncoding RNA. A total of 1035, 944, and 657 genes were upregulated in comparisons between samples, and 2680, 1215, and 975 genes were downregulated in comparisons between samples. Finally, among all samples, 167 genes upregulated, and 145 genes downregulated. The GO classification and functional enrichment of DEGs as follows: molecular function, 224 genes; cellular component, 149 genes; and biological process, 272 genes. The expression of XM_001863852 and XM_001845881 in resistant strains of Culex pipiens pallens was lower than that in the laboratory sensitive strain, with fold changes in expression of 0.177 and 0.548, respectively; the expression of the XM_001845883.1 in the resistant strain was higher than that in the susceptible strain, and a 2.281-fold change in expression. Conclusions The results provide a reference for resistance mechanisms through the mosquito cuticle, furthermore, could provide a new perspective for disease vector control.

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piRNA-3878 targets P450 (CpCYP307B1) to regulate pyrethroid resistance in Culex pipiens pallens

Parasitology Research ◽

10.1007/s00436-017-5554-3 ◽

2017 ◽

Vol 116 (9) ◽

pp. 2489-2497 ◽

Cited By ~ 3

Author(s):

Wenyun Ye ◽

Xianmiao Liu ◽

Juxin Guo ◽

Xueli Sun ◽

Yan Sun ◽

...

Keyword(s):

Culex Pipiens ◽

Pyrethroid Resistance ◽

Culex Pipiens Pallens ◽

Pipiens Pallens

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Diverse sensitivities of TRPA1 from different mosquito species to thermal and chemical stimuli

Scientific Reports ◽

10.1038/s41598-019-56639-w ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Tianbang Li ◽

Claire T. Saito ◽

Tomoyuki Hikitsuchi ◽

Yoshihiro Inoguchi ◽

Honami Mitsuishi ◽

...

Keyword(s):

Culex Pipiens ◽

Transient Receptor Potential ◽

Mosquito Control ◽

Splice Variants ◽

Mosquito Species ◽

Temperature Threshold ◽

Culex Pipiens Pallens ◽

Chemical Stimuli ◽

Pipiens Pallens

AbstractTemperature and odors profoundly affect the behavior of animals. Transient receptor potential channel, subfamily A, member 1 (TRPA1) functions as a polymodal nociceptor for sensing both vital environmental cues in insects. Mosquitoes are recognized as disease vectors, and many efforts have been devoted to investigations of their host-seeking behaviors and repellents. However, the physiological characteristics of mosquito TRPA1 have not been systematically studied. We identified multiple alternative splice variants of the TrpA1 gene from Anopheles gambiae, Anopheles stephensi, Aedes aegypti and Culex pipiens pallens mosquitoes. And we performed comparative analyses of the responses of mosquito TRPA1s to heat or chemical stimuli with calcium-imaging and whole-cell patch-clamp methods. Comparison of TRPA1 among four mosquito species from different thermal niches revealed that TRPA1 of Culex pipiens pallens inhabiting the temperate zone had a lower temperature threshold for heat-evoked activation, which was supported by the in vivo heat-avoidance test. Notably, the chemosensitivity of mosquito TRPA1 channels revealed differences not only between variants but also among species. Moreover, we discovered three novel mosquito TRPA1 agonists. Thermal niches selection and evolutionary trajectories significantly affect the functional properties of mosquito TRPA1, which represents a hallmark of the behaviors that may permit the design of improved mosquito control methods.

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