In Vitro and In Vivo Validation of CYP6A14 and CYP6N6 Participation in Deltamethrin Metabolic Resistance in Aedes albopictus

Abstract

The extensive use of chemical insecticides for public health and agricultural purposes has increased the occurrence and development of insecticide resistance. This study used transcriptome sequencing to screen 10 upregulated metabolic detoxification enzyme genes from Aedes albopictus resistant strains. Of these, CYP6A14 and CYP6N6 were found to be substantially overexpressed in the deltamethrin-induced expression test, indicating their role in deltamethrin resistance in Ae. albopictus. Furthermore, the corresponding 60-kDa recombinant proteins, CYP6A14 and CYP6N6, were successfully expressed using the Escherichia coli expression system. Enzyme activity studies revealed that CYP6A14 (5.84 U/L) and CYP6N6 (6.3 U/L) have cytochrome P450 (CYP450) enzyme activity. In vitro, the metabolic analysis revealed that the recombinant proteins degraded deltamethrin into 1-oleoyl-sn-glycero-3-phosphoethanolamine and 2',2'-dibromo-2'-deoxyguanosine. Subsequently, the CYP450 genes in larvae of Ae. albopictus were silenced by RNA interference technology to study deltamethrin resistance in vivo. The silencing of CYP6A14 and CYP6N6 increased the mortality rate of mosquitoes without affecting their survival time, spawning quantity, hatching rate, and other normal life activities. Altogether, CYP6A14 and CYP6N6 belong to the CYP6 family and mutually increase deltamethrin resistance in Ae. albopictus.

Figure 1.

Screening of DEGs in resistant and sensitive strains of Aedes albopictus. (A) Transcriptome sequencing volcano plot of DEGs (green, red, and black dots represent downregulated, upregulated, and unchanged DEGs, respectively). (B) Comparison of expression profiles of the 10 genes selected by RNA-seq and qRT-PCR. The red bars are the log₂ (FPKM + 1) values of RNA-seq data, and the black bars are the relative expressions from qRT-PCR. (C) The expression levels of six upregulated genes in the transcriptome and qRT-PCR of Ae. albopictus sensitive strain after 24-hour exposure to deltamethrin (C, control group; T, processing group). (D and E) CYP6A14 and CYP6N6 expression levels in Ae. albopictus sensitive strain at different time points after exposure to deltamethrin. Data are the mean ± SD of three biological replicates. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. DEG = differentially expressed gene; FC = fold change; FDR = false discovery rate; FPKM = fragments per kilobase of transcript sequence per million base pairs sequenced; qRT-PCR = quantitative real-time polymerase chain reaction; RNA-seq = RNA sequencing.

Figure 2.

Full-length gene cloning and sequence alignment. (A) Nested PCR electrophoresis band of CYP6A14 and CYP6N6. (B) Comparison of CYP6A14 and CYP6N6 amino acid sequences; the black boxes represent the conserved functional domain of the CYP450 protein family. CYP450 = cytochrome P450; PCR = polymerase chain reaction.

Figure 3.

Expression analysis of recombinant proteins and in vitro functional verification. (A) SDS-PAGE analysis of induced expression of recombinant proteins. Marker: protein marker; 1: the empty vector whole bacteria; 2: CYP6A14 whole bacteria; 3: CYP6N6 whole bacteria; 4: CYP6A14 bacterial cell pellet; 5: CYP6A14 bacterial lysate supernatant; 6: CYP6N6 bacterial cell pellet; and 7: CYP6N6 bacterial lysate supernatant. (B) SDS-PAGE analysis of purified recombinant proteins. (C) Western blotting of purified recombinant proteins. (D) The main degradation products of deltamethrin metabolized by the recombinant proteins in vitro (a) deltamethrin degradation pathways to (b) 1-oleoyl-sn-glycero-3-phosphoethanolamine and (c) 2′, 2′-dibromo-2′-deoxyguanosine substances through oxidation and hydration. SDS-PAGE = sodium dodecyl sulfate–polyacrylamide gel electrophoresis.

Figure 4.

In vivo functional verification of CYP6A14 and CYP6N6. (A and B) CYP6A14 and CYP6N6 expression patterns during the seven critical developmental stages of Aedes albopictus. (C) RNAi efficiency after the silencing of deltamethrin resistance–related genes alone (dsCYP6A14, dsCYP6N6) and in combination (dsCYP6A14 + dsCYP6N6). Horizontal dashed line represents gene expression normalized to 1.0 to facilitate comparison. (D) Deltamethrin resistance analysis of the larval immersion method in Ae. albopictus. A through D error bars represent mean ± standard deviation of three biological replicates; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. (E–G) The effects of RNAi on life activities of Ae. albopictus, including (E) survival time, (F) spawning rate, and (G) hatching rate. Horizontal lines in F and G represent mean value of the sample data for each group. Larvae were treated in groups of 10, with one dot representing the number of eggs laid and the hatching rate of a female mosquito. ns = not significant; RNAi = RNA interference.