TRE1 and CHS1 contribute to deltamethrin resistance in Culex pipiens pallens.

Cuticular resistance, characterized by decreased epidermal penetration, has been reported on highly pyrethroid-resistant mosquitoes. In this study, we examined the role of genes in the chitin biosynthetic pathway in the context of deltamethrin-resistant (DR) Culex pipiens pallens. We found that expression of the trehalase (TRE1) gene and chitin synthase (CHS1) gene was upregulated 1.65- and 1.75-fold with quantitative reverse transcription polymerase chain reaction, respectively, in the DR strain as compared with the deltamethrin-susceptible (DS) strain. Examination of chitin content in DR and DS pupae showed an increased amount of chitin in DR pupae. To further establish the role of TRE1 and CHS1 in deltamethrin resistance, we injected mosquitoes with small interfering RNA (siRNA) for knockdown of TRE1 or CHS1 expression. The mortality rates of DR mosquitoes exposed to insecticides increased 17% and 26% after siTRE1 and siCHS1 injection, respectively. The siRNA treatment against TRE1 resulted in decreased expression of the downstream gene CHS1. Together, our findings support a role of TRE1 and CHS1 in the regulation of pyrethroid resistance.

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

Piwi-interacting RNAs (piRNAs) are a novel class of noncoding single-strand RNAs. They play an important role in the germ cell maintenance, brain development, epigenetic regulation of cancer, and antiviral function. However, little is known about the relationship between the piRNAs and insecticide resistance in mosquitoes. In this study, we reported that piRNA-3878 was related with pyrethroid resistance in Culex pipiens pallens. The expression level of piRNA-3878 was lower in both laboratory and field-collected deltamethrin-resistant (DR) strains. Overexpression of piRNA-3878 increased the susceptibility of the DR strain, while inhibiting the expression of piRNA-3878 in DS strain made the mosquitoes more resistant to deltamethrin. Furthermore, we identified that CpCYP307B1 was the target of piRNA-3878. The mosquito mortality rate was increased after downregulating the expression of CpCYP307B1. These findings revealed that piRNA-3878 could target CpCYP307B1 to regulate pyrethroid resistance.

Cuticle genes CpCPR63 and CpCPR47 may confer resistance to deltamethrin in Culex pipiens pallens.

Cuticular proteins (CPs) are implicated in insecticide resistance in mosquito populations. Here, we investigated the role of cuticular genes in regulation of insecticide resistance in Culex pipiens pallens. We identified two CpCPRs (CpCPR63 and CpCPR47) that exhibited higher transcript levels in pyrethroid-resistant strains than in susceptible strains. Mosquito mortality was increased after knockdown of CpCPR genes by dsRNA injection. The RNA interference experiment suggested an interaction between CpCPR63 and CpCPR47, as silencing of one gene resulted in decreased expression of the other. These findings revealed that CpCPRs may regulate pyrethroid resistance and could be used as a potential genetic marker to monitor pyrethroid 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.

The role of miR-2∼13∼71 cluster in resistance to deltamethrin in Culex pipiens pallens.

Excessive and continuous application of deltamethrin has resulted in the development of deltamethrin resistance among mosquitoes, which becomes a major obstacle for mosquito control. In a previous study, differentially expressed miRNAs between deltamethrin-susceptible (DS) strain and deltamethrin-resistant (DR) strain using illumina sequencing in Culex pipiens pallens were identified. In this study, we applied RNAi and the Centers for Disease Control and Prevention (CDC) bottle bioassay to investigate the relationship between miR-2∼13∼71 cluster (miR-2, miR-13 and miR-71) and deltamethrin resistance. We used quantitative real-time PCR (qRT-PCR) to measure expression levels of miR-2∼13∼71 clusters. MiR-2∼13∼71 cluster was down regulated in adult female mosquitoes from the DR strain and played important roles in deltamethrin resistance through regulating target genes, CYP9J35 and CYP325BG3. Knocking down CYP9J35 and CYP325BG3 resulted in decreased mortality of DR mosquitoes. This study provides the first evidence that miRNA clusters are associated with deltamethrin resistance in mosquitoes. Moreover, we investigated the regulatory networks formed between miR-2∼13∼71 cluster and its target genes, which provide a better understanding of the mechanism involved in deltamethrin resistance.

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

MicroRNAs play critical roles in post-transcriptional regulation of gene expression, which participate in the modulation of almost all of the cellular processes. Although emerging evidence indicates that microRNAs are related with antineoplastic drugs resistance, whether microRNAs are responsible for insecticide resistance in mosquitos is poorly understood. In this paper, we found that miR-285 was significantly upregulated in the deltamethrin-resistant strain of Culex pipiens pallens, and overexpression miR-285 through microinjection increased mosquito survival rate against deltamethrin treatement. Using bioinformatic software, quantitative reverse transcription PCR, luciferase reporter assay and microinjection approaches, we conformed that CYP6N23 was the target of miR-285. Lower expression of CYP6N23 was observed in the deltamethrin-resistant strain. While, mosquito mortality rate was decreased after downregulating expression of CYP6N23 by dsRNA against CYP6N23 or miR-285 mimic microinjection. These findings revealed that miR-285 could target CYP6N23 to regulate pyrethroid resistance, providing new insights into mosquito insecticide resistance surveillance and control.