A novel miRNA, miR-13664, targets CpCYP314A1 to regulate deltamethrin resistance in Culex pipiens pallens

Abstract

Extensive insecticide use has led to the resistance of mosquitoes to these insecticides, posing a major barrier to mosquito control. Previous Solexa high-throughput sequencing of Culex pipiens pallens in the laboratory has revealed that the abundance of a novel microRNA (miRNA), miR-13664, was higher in a deltamethrin-sensitive (DS) strain than a deltamethrin-resistant (DR) strain. Real-time quantitative PCR revealed that the miR-13664 transcript level was lower in the DR strain than in the DS strain. MiR-13664 oversupply in the DR strain increased the susceptibility of these mosquitoes to deltamethrin, whereas inhibition of miR-13664 made the DS strain more resistant to deltamethrin. Results of bioinformatic analysis, quantitative reverse-transcriptase polymerase chain reaction, luciferase assay and miR mimic/inhibitor microinjection revealed CpCYP314A1 to be a target of miR-13664. In addition, downregulation of CpCYP314A1 expression in the DR strain reduced the resistance of mosquitoes to deltamethrin. Taken together, our results indicate that miR-13664 could regulate deltamethrin resistance by interacting with CpCYP314A1, providing new insights into mosquito resistance mechanisms.

Keywords: CpCYP314A1; Culex pipiens pallens; deltamethrin resistance; miR-13664.

Fig.1

miR-13664 abundance is influenced by Ago1. (A) Ago1 transcript levels after dsCpAgo1 injection. (B) miR-13664 abundance level after dsCpAgo1 injection. (C) Ago3 transcript level after dsCpAgo3 injection. (D) miR-13664 abundance levels after dsCpAgo3 injection. Data are presented as means ± standard error (SE) of three independent experiments. **P < 0.01 compared with the negative control (NC).

Fig.2

MiR-13664 abundance levels in the DS and DR strains of C. pipiens pallens. (A) Laboratory mosquito populations. (B) Field-collected mosquito populations. Data are presented as means ± SE of three independent experiments. ***P < 0.001 compared with the DS strain.

Fig.3

MiR-13664 is involved in mediating mosquito resistance to deltamethrin. (A) MiR-13664 abundance levels after miR-13664 mimic injection. (B) Mortality of mosquitoes exposed to deltamethrin in CDC bottles after 72 h by injection of miR-13664 mimic. (C) MiR-13664 abundance levels after miR-13664 inhibitor injection. (D) Mortality of mosquitoes exposed to deltamethrin in CDC bottles after 72 h by injection of miR-13664 inhibitor. Data are presented as mean ± SE of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig.4

CpCYP314A1 is a direct target of miR-13664. (A) The miR-13664 binding in the 3ʹ-UTR of CpCYP314A1 mRNA and the constructed mutated vector sequence. (B) Luciferase activity in HEK293T cells cotransfected with CpCYP314A1-3ʹ-UTR (WT) or mutant CpCYP314A1-3ʹ-MUT (MUT) construct together with miR-13664. (C) CpCYP314A1 transcript levels in the DS and DR strains of C. pipiens pallens. (D) CpCYP314A1 transcript levels after injection with miR-13664 mimic. (E) CpCYP314A1 transcript levels after miR-13664 inhibitor injection. Luciferase activity was assayed in HEK293T cells cotransfected with CpCYP314A1-3ʹ-UTR (WT) or the mutant CpCYP314A1-3ʹ-MUT (MUT) construct with miR-13664. Data are presented as means ± SE of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig.5

CpCYP314A1 affects deltamethrin resistance in DR mosquitoes. (A) Expression levels of CpCYP314A1 after microinjection with CpCYP314A1 dsRNA. (B) Microinjected mosquito mortality observed after exposure to deltamethrin in CDC bottles for 2 h. Data are presented as the means ± SE of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.