INTRODUCTION
Aedes albopictus, a vector of dengue fever, Japanese encephalitis, Zika virus, and other viruses, is a great threat to human health.1,2 At present, decreasing vector populations through chemical control is the most powerful method of controlling mosquito-borne infections. According to reports, since 2000, malaria incidence has decreased to 50%, saving 660 million lives, half of which was achieved with the use of insecticides.3 However, the continuous use of pesticides has significantly increased the problem of chemical resistance in mosquitoes. Studies show that compared with past success in the Mali and Uganda regions of Africa, mosquito-borne infectious disease prevention and control programs have come to a standstill; even with the use of strengthened pesticides, malaria incidence is not decreasing any further but is, in fact, showing a trend of increase.3–5 Therefore, research into the mechanism of mosquito resistance is vital to carry out an effective resistance program. Mosquito resistance emerges from the selection of adaptive insecticides involving multiple genes.6 Studies have shown that the resistance mechanism may involve insecticide degradation, target blocking, and/or epidermal, behavioral, and microbial resistance.7–11 Metabolic resistance involves the enhanced or acquired metabolic activity of related detoxifying enzymes against insecticides.12,13 Mosquitoes have been found to be metabolically resistant to several insecticides used in public health programs, such as pyrethroids, organophosphorus, carbamates, and organochlorines.14,15
In cases of metabolic resistance, mosquitoes prevent or slow down the arrival of insecticides to the target site by metabolic detoxification, isolation, or accelerated excretion involving enhanced expression and activity of relevant metabolic detoxification enzymes.15 Metabolic detoxification enzymes in insects, which can function independently or synergistically, include primarily cytochrome P450 (CYP450), carboxylesterase, glutathione S-transferase (GST), ABC transporter, and uridine diphosphate glycosyltransferase proteins.16–19 In mosquitoes, CYP450 proteins participate in a series of physiological and biochemical reactions, including the regulation of endogenous substances such as hormones and steroids and metabolic detoxifying exogenous substances such as pesticides and plant toxins.13,20 In addition, CYP450 and reductase constitute a resistance system with a wide range of substrates and diverse catalytic functions.7,10 Therefore, it is essential to thoroughly explore the molecular mechanism of resistance-related CYP450 in mosquitoes.
With advances in transcriptomic sequencing, RNA interference (RNAi), and other molecular biological technologies, several CYP450 genes related to insecticide resistance in mosquitoes have been discovered.21,22 The CYP450 proteins can be divided into CYP6, CYP9, CYP12, and other families and their subfamilies. Resistance in Aedes mosquitoes has been related to the upregulation of the CYP6 and CYP9 genes.23–26 An accurate analysis of individual enzymes involved in insecticide metabolism is a challenging task but is crucial for the understanding of insecticide resistance.21,27 In this study, we used transcriptome sequencing on deltamethrin-resistant and sensitive strains of Ae. albopictus that were reared in our laboratory. We found that the upregulated genes in resistant strains were mainly CYPs related to metabolic resistance. Subsequently, the CYP6A14 and CYP6N6 genes were selected on the basis of messenger RNA levels for functional verification. We found that the upregulation of these two genes decreased the deltamethrin sensitivity of Ae. albopictus to a certain extent. Moreover, in vitro prokaryotic protein complexes could metabolize and degrade deltamethrin to 1-oleoyl-sn-glycero-3-phosphoethanolamine and 2′,2′-dibromo-2′-deoxyguanosine via oxidation reactions. Meanwhile, RNAi silencing of the two genes restored the deltamethrin sensitivity of Ae. albopictus.
MATERIALS AND METHODS
Ethics statement.
This study was carried out following the guidelines of the Shandong Institute of Parasitic Diseases and the Shandong Experimental Animal Society without a breach of ethics.
Mosquito rearing.
This study used two strains of Ae. albopictus. The susceptible strains, provided by Shandong Provincial Center for Disease Control and Prevention, were cultured in a mosquito cage without pesticide exposure. The resistant strains were selected from the 3rd and 4th instar larvae stage using a 50% lethal deltamethrin concentration (LC50). After 3 years of breeding, 52 generations of mosquitoes were selected as resistant strains, with a resistance ratio of 109.8 to susceptible mosquitoes. Mosquito breeding conditions were as follows: temperature 26°C; relative humidity 75%; photoperiod, 12:12 hour (bright:dark). Larvae feed included a mixture of pig liver and yeast powder (1:3). Adult mosquitoes were transferred to mosquito cages and provided 10% glucose solution and defibrillated sheep blood. According to WHO guidelines, larval resistance was determined as follows: RR = resistant strain LC50/sensitive strain LC50; the corresponding LC50 of deltamethrin for the respective strains is shown in Supplemental Table 1.
RNA extraction, library construction, and sequencing.
Total RNA was extracted separately from seven groups of samples (1st to 4th instar larvae, pupae, post-eclosion 1st adults, and post-eclosion 3rd adults) using TRIzol Reagent (Invitrogen, Carlsbad, CA) following the manufacturer’s protocol. (Each group’s sample size was 20 early larvae (1st and 2nd); 15 late larvae (3rd and 4th); 15 pupae, and 10 adult mosquitoes, and three replicons per stage were prepared). RNA concentration, purity, and integrity were measured using the NanoDrop 2000 (Thermo Fisher Scientific, Wilmington, DE) and the Agilent Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA), respectively. Qualified RNA samples were used to construct the complementary DNA (cDNA) library. Sequencing libraries for Illumina were made with 1 μg of RNA using the NEBNext Ultra Directional RNA Library Prep Kit (NEB, Beijing, China). Library quality was assessed by the Agilent Bioanalyzer 2100 system. Sequencing was performed by Biomarker (Beijing, China) on an Illumina NovaSeq 6000 platform (San Diego, CA), and 240-bp paired-end (PE150) reads were obtained.
Screening of differential genes in resistant and sensitive strains.
Twenty larvae were collected from resistant and sensitive strains of Ae. albopictus at the end of the 3rd instar stage and the beginning of the 4th instar stage, respectively, and three biological replicates were prepared. The prepared samples were used for transcriptome sequencing. Transcript expression levels were quantified on the basis of fragments per kilobase of transcript sequence per million base pairs sequenced values using StringTie. The DESeq2 (Bioconductor, available at: http://www.bioconductor.org. Accessed January 12, 2023) program was used for differential gene expression analysis of the two groups of samples: deltamethrin-resistant and susceptible mosquitoes. DESeq2 provides statistical routines for determining differential expression in digital gene expression data based on the negative binomial distribution model. The resulting P values were adjusted using Benjamini and Hochberg’s approach to control the false discovery rate. Genes with criteria of adjusted P value < 0.01 and fold change ≥ 2 were assigned as differentially expressed genes (DEGs). Gene Ontology Consortium, cluster of orthologous groups of proteins, and Kyoto Encyclopedia of Genes and Genomes functional annotation and enrichment analyses of differential genes were performed. Accordingly, 10 genes related to insecticide metabolism detoxification and highly expressed in Ae. albopictus resistant strains were identified.
Quantitative real-time polymerase chain reaction.
Quantitative real-time polymerase chain reaction (qRT-PCR) was used to validate the sequencing data and CYP6A14 and CYP6N6 expression profiles using LightCycler® 480 SYBR Green IMaster (Roche, Switzerland) with a LightCycler 96 System. The qRT-PCR reactions contained 10 μL of SYBR Premix Ex Taq II, 20 ng of cDNA template, 0.2 μM of each forward and reverse primer (sequences in Supplemental Table 2), and nuclease-free water. The qRT-PCR conditions were as follows: 50°C for 2 minutes, 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. The β-actin gene (GenBank accession no. DQ657949.1) was used as a reference gene. Raw threshold cycle (Ct) values were normalized against the β-actin standard to obtain normalized Ct values, which were then used to calculate relative expression levels of target genes by the 2−ΔΔCt method.28 All qRT-PCR measurements had three biological replicates.
Deltamethrin-induced expression analysis of CYP6A14 and CYP6N6.
Deltamethrin was dissolved in appropriate amounts of acetone and serially diluted to the semi-lethal concentration of sensitive Ae. albopictus strains (19.02 μg/L). About 600 3rd instar–sensitive Ae. albopictus larvae were collected and randomly divided into two groups. The larvae in the experimental group were exposed by immersion in deltamethrin solution. The control group larvae were placed in clean water. After the respective treatment, the surviving larvae were collected at 3, 6, 12, 24, and 48 hours (15 larvae as one group for each time point and three biological replicates). Total RNA was extracted, and then 500 ng of the total RNA was converted to cDNA using a PrimeScript RT Reagent Kit with Genomic DNA Eraser (TaKaRa, Shiga, Japan). Transcript levels were measured by qRT-PCR. Each experiment had three replicates. First, the transcript levels of 10 target genes were screened by transcriptome sequencing after 24 hours of deltamethrin exposure to mosquitoes. Next, genes with significantly upregulated expressions were analyzed for induced expression patterns at different time points. Those with high expression in resistant strains and upregulation after deltamethrin induction were considered metabolic detoxification enzyme genes related to deltamethrin resistance in Ae. albopictus and subjected to further functional verification.
Full-length cloning and verification.
To clone the full-length CYP6A14 and CYP6N6 genes, we collected 20 larvae of Ae. albopictus resistant strains at the end of the 3rd instar stage. First, the corresponding primers for validating known sequences were designed in Premier 5.0 software based on transcriptome data. Total RNA from larvae was extracted using the universal ultra-pure RNA Extraction Kit (GR2011), and the first-strand cDNA was synthesized by reverse transcription. Nested PCR was used for two rounds of PCR amplification to obtain the specific DNA sequence. The final PCR products were purified and used for TA cloning into the pMD-19T vector. The plasmids were transformed into the receptive DH5α competent cells, and the colonies were grown on an LB-ampicillin plate. The white colonies were selected and cultured in LB liquid medium to conduct PCR validation of cloned genes. Three positive clones were sent for sequencing (Sangon Biotech, Shanghai, China). The same method was used to synthesize 3-RACE and 5-RACE. Finally, full-length cDNA was obtained from two 3′/5′ RACE products with overlapping sequences and verified for predicted open-reading frame (ORF) regions. The computer program ClustalW2 was used to compare the nucleotide sequences of CYP6A14 and CYP6N6, and the P450 functional domain was labeled according to the PRINTS database.
Construction of a prokaryotic expression vector.
The lead sequence of bacterial ompA was synthesized by fusion PCR and constructed into the expression vector pET22b to obtain pET22b-HIS-ompA.24 The biological linkage between the target gene and vector was performed by seamless cloning involving 15-bp homologous sequences at the end of both the vector and target gene. Primers for CYP6A14 were designed accordingly. The cDNA of the Ae. albopictus resistant strain at the end of the 3rd instar stage was used as a template for PCR. The PCR product was gel purified and then cloned into the pET22b-HIS-ompA vector to obtain pET22b-His-Ompa-CYP6A14. Likewise, we constructed pET22b-His-Ompa-CYP6N6. All clones were PCR verified and validated by sequencing. The plasmids were successfully transformed into BL21 cells and selected on LB- ampicillin resistance plates. Also, individual positive bacterial colonies were screened by PCR and validated by sequencing.
Expression and activity analysis of recombinant proteins.
The successfully transformed strain was grown in LB liquid medium with 100 µg/mL ampicillin. The starter culture was diluted 100 times in a 500-mL culture medium and incubated at 37°C and 200 rpm until the optical density (OD) value reached 0.6. Then, the inducer isopropyl-beta-D-thiogalactopyranoside (IPTG) (0.5 mM) was added, and the culture was continued at 17°C and 250 rpm for 18–25 hours. The bacterial cells were harvested by centrifugation at low temperatures and broken by ultrasound. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze the expression of the target proteins. Proteins were purified by affinity chromatography using His-column. The supernatant of the bacteria lysate was passed through the His-column containing 50 and 500 mmol/L imidazole, which was used for washing and elution, respectively. Imidazole was removed from the eluted protein by overnight dialysis in 10 mmol/L phosphate-buffered saline (PBS), and then the protein was concentrated by ultrafiltration. Finally, purified and concentrated protein samples were checked by SDS-PAGE and estimated for protein concentration by the bicinchoninic acid assay method. Protein aliquots were snap-frozen in liquid nitrogen and then stored at −80°C. Purified recombinant proteins were transferred to the nitrocellulose membrane from SDS-PAGE. The membrane was blocked with 5% skim milk for 1 hour and then incubated with rabbit primary His antibodies for 2 hours at room temperature (RT), followed by washing in TBST thrice for 10 minutes each time. Next, the membrane was incubated with goat anti-rabbit IgG-horseradish peroxidase (HRP) (secondary antibody; BioVision, San Fransisco, CA) for 1 hour at RT and washed as before. The protein bands were illuminated by enhanced chemiluminescence chromogen, and the image was captured. The enzyme activity of CYP450 was detected according to the instructions of the insect CYP450 ELISA Kit (Jiang Lai Biological, Shanghai, China), which uses a double-antibody one-step sandwich method. Standard substances, test samples, and HRP-labeled detection antibodies were successively added to the precoated micropores coated with insect CYP450 captured antibodies. After incubation and washing, 3,3′,5,5′-tetramethylbenzidine was used for color display. Sample absorbance was measured at 450 nm using a microplate reader. The concentration of test samples was measured using the standard curve.
In vitro metabolic degradation of deltamethrin by recombinant proteins.
The recombinant CYP6A14 and CYP6N6 proteins were tested in an in vitro nicotinamide adenine dinucleotide phosphate regeneration system to determine their deltamethrin degradation ability. The reaction system in 2 mL PBS (pH, 7.4) consisted of 1 mM glucose-6-phosphate dehydrogenase, 1 mM glucose 6-phosphate, 0.25 mM MgCl2, 0.1 mM NADP+, 1 mg/L deltamethrin, and 50 mg/L recombinant protein CYP6A14/CYP6N6. The reaction was performed at 30°C and 200 rpm for 5, 10, 30, and 60 minutes. Finally, the reaction was terminated by incubating with 100 μL methanol (chromatographic grade) for 5 minutes. The reaction without the recombinant protein was used as a blank control. The samples were centrifuged at 12,000 rpm and 4°C for 15 minutes. The obtained supernatants were used to measure the concentration and degradation products of deltamethrin by gas chromatography–tandem mass spectrometry (GC-MS/MS) using a C18 column. The conditions were as follows: sample size, 1 μL; column temperature, 30°C; flow rate, 0.3 mL/minute; mobile phase composition, distilled water, 0.1% formic acid, and chromatographic pure acetonitrile; injection volume, 2 μL; and automatic injection temperature, 4°C.
Functional verification of CYP6A14 and CYP6N6 genes.
Two groups of dsRNAs (CYP6A14-dsRNA1, CYP6A14-dsRNA2, CYP6A14-dsRNA3; CYP6N6-dsRNA1, CYP6N6-dsRNA2, CYP6N6-dsRNA3; Supplemental Table 2) were designed on the basis of full-length sequences of CYP6A14 and CYP6N6, respectively, and constructed into the pgpu6 GFP Neo plasmid to produce Escherichia coli BL21 strains that consistently and stably expressed the corresponding double-stranded RNA. The bacterial strains were cultured in 100 mL LB broth at 37°C and 200 rpm to OD600 of 0.4 and then induced with 0.4 mm IPTG for 4 hours to begin dsRNA expression. Afterward, 10 mL of IPTG-induced E. coli bacterial culture was centrifuged at 10,000 g for 2 minutes. The bacterial pellet was resuspended in 2 mL sterilized distillation-distillation H2O and then used for Ae. albopictus feeding bioassay. An unrelated gene (GFP) was used as a negative control. The recombinant plasmid for GFP expression was prepared as for dsRNA. The corresponding dsRNA and GFP bacteria suspension were used as dsRNA and GFP feed. Samples of Ae. albopictus resistant strains were collected at seven different developmental stages (1st to 4th instar larvae, pupae, post-eclosion 1st adults, and post-eclosion 3rd adults). Quantitative real-time polymerase chain reaction was used to detect the expressions of CYP6A14 and CYP6N6 genes; the developmental stage with the lowest expression level was selected for RNAi. Feeding experiments were performed using a randomized block design; Ae. albopictus 1st instar larvae were randomly divided into four groups: CYP6A14 dsRNA group and CYP6N6 dsRNA group, blank control group (fed larval feed), and GFP negative control group. The different groups were provided with corresponding feed once every morning, and the water was changed before feeding. Four days later, the larvae reached the end of the 3rd instar stage. Three larvae from each group were collected as a sample pool to detect the expression levels of CYP6A14 and CYP6N6. Based on the gene expression results, the dsRNA group with the highest RNAi efficiency was selected. Finally, five groups were set, including the dsCYP6A14 group, dsCYP6N6 group, dsCYP6A14 + dsCYP6N6 group, negative control group, and blank control group. Parameters such as survival time, spawning quantity, and hatching rate were recorded to evaluate the RNAi effects on everyday life activities of mosquitoes. The larval immersion method was used to detect deltamethrin resistance in different treatment groups.
Statistical analysis.
Larval survival was evaluated by log-rank (Mental-Cox) test.25 The enzyme activity of the recombinant proteins was calculated by the linear regression method. The effect of RNAi on larval survival was analyzed by Fisher’s accurate test. A t-test was used for other statistical analyses. All analyses were performed using GraphPad Prism (San Diego, CA) or SPSS 25 (IBM Corp., Armonk, NY) statistical software, and P ≤ 0.05 was considered statistically significant.
RESULTS
Screening of DEGs in resistant and sensitive strains of Ae. Albopictus.
Transcriptome sequencing results were compared to identify changes in gene expression levels between resistant and sensitive strains of Ae. albopictus. In total, 551 DEGs were found, including 278 upregulated and 273 downregulated genes (Figure 1A) (the corresponding data can be availed from the National Center for Biotechnology Information Sequence Read Archive database, accession no. PRJNA756868). The upregulated genes mainly included detoxification enzymes, such as P450s and GSTs, which have been associated with metabolic resistance in mosquitoes, and the epidermal protein and ribosomal genes, which have also been related to mosquito resistance.26 The relative expressions of genes detected by qRT–PCR were matched with RNA sequencing (RNA-seq) data. The qRT-PCR results validated the expression pattern of the 10 highly expressed genes from the RNA-seq data (Figure 1B, Supplemental Figure 1), indicating the reliability of RNA-seq. Subsequently, qRT-PCR was performed to detect induced expression of these 10 genes in sensitive strains for 24 hours; CYP6A14 and CYP6N6 were highly upregulated (Figure 1C). Also, gene expression profiles were determined at different times, displaying different upregulation patterns (Figure 1D and E). Compared with the control group, CYP6A14 expression was the highest at 12 hours, showing an increase of 2.96 ± 0.21 times; CYP6N6 expression was the highest at 24 hours, with an increase of 3.35 ± 0.22 times. After reaching the peak, the expression levels of both genes showed a downward trend but was still sustained at about 2 times higher levels than the control group.
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.
Citation: The American Journal of Tropical Medicine and Hygiene 108, 3; 10.4269/ajtmh.22-0524
Full-length cloning and sequence comparison of CYP6A14 and CYP6N6.
Full-length cDNA of CYP6A14 and CYP6N6 was amplified from Ae. albopictus by 3′- and 5′-RACE PCR (Supplemental Figure 2). The full-length CYP6A14 is 1,709 bp including a 1,497-bp ORF encoding 499 amino acids (GenBank accession no. ON995105). The start codon is ATG (position 53–55), and the stop codon is TAG (position 1547–1549). Likewise, the full-length CYP6N6 is 1,702 bp including a 1,494-bp ORF encoding 498 amino acids (GenBank accession no. ON995106). The start codon ATG and stop codon TAG are positioned at 49–51 and 1539–1542, respectively (Figure 2A). ClustalW2 software was used to compare the amino acid sequences of CYP6A14 and CYP6N6, which share a high nucleotide sequence similarity (∼83.94%). PRINTS (http://www.bioinf.manchester.ac.uk/dbbrowser/PRINTS/) protein family fingerprint analysis indicated that CYP6A14 and CYP6N6 have a function similar to that of the P450 structure domain (Figure 2B).
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.
Citation: The American Journal of Tropical Medicine and Hygiene 108, 3; 10.4269/ajtmh.22-0524
Expression of CYP6A14 and CYP6N6 recombinant proteins and in vitro degradation of deltamethrin.
The empty vector and vectors expressing recombinant proteins were analyzed by SDS-PAGE (Figure 3A). After the 0.5 mM IPTG induction of the corresponding bacterial cells at 17°C and 250 rpm for 20 hours, the target proteins (∼60 kDa) were released in the bacterial cell lysate in soluble form. The proteins were purified by an affinity column in high purity (Figure 3B). Western blotting was performed to identify the recombinant proteins using the corresponding antibodies, showing a reaction band at 60 kDa (Figure 3C, Supplemental Figure 3). The concentrations of CYP6A14 and CYP6N6 proteins were 0.44 and 0.58 mg/mL, and the enzyme activities were 5.84 and 6.3 U/L (18.25 and 19.69 times higher than that of the blank group), respectively Supplemental Table 3). The results demonstrate that both CYP6A14 and CYP6N6 have strong P450 activity. High Performance Liquid Chromatography (HPLC) analysis showed that most of the deltamethrin degradation by the recombinant proteins was done in the first 10 minutes and was fully completed after 30 minutes (Table 1). The deltamethrin degradation efficiencies of CYP6A14 and CYP6N6 were 70% and 75%, respectively, indicating saturation at this level. In addition, GC-MS/MS results showed that the main products of deltamethrin degradation were 1-oleoyl-sn-glycero-3-phosphoethanolamine and 2′, 2′-dibromo-2′-deoxyguanosine (Figure 3D).
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.
Citation: The American Journal of Tropical Medicine and Hygiene 108, 3; 10.4269/ajtmh.22-0524
Average residual concentration of deltamethrin at different reaction times
| Concentration Sample | CYP6A14 | CYP6N6 | Control |
|---|---|---|---|
| Initial concentration | 1 mg/mL | 1 mg/mL | 1 mg/mL |
| 5 minutes | 0.63 mg/mL | 0.58 mg/mL | 1 mg/mL |
| 10 minutes | 0.31 mg/mL | 0.27 mg/mL | 1 mg/mL |
| 30 minutes | 0.30 mg/mL | 0.25 mg/mL | 0.98 mg/mL |
| 60 minutes | 0.30 mg/mL | 0.25 mg/mL | 0.98 mg/mL |
In vivo functional verification of CYP6A14 and CYP6N6.
To further verify the function of CYP6A14 and CYP6N6 in vivo, we used RNAi to knock out these genes and then detected the change in deltamethrin resistance in Ae. albopictus larvae. Previously, we analyzed the expression profiles of these two genes in Ae. albopictus resistance strains at different developmental stages and found that the levels were the highest in the 3rd instar larvae (CYP6A14: 167.14 ± 14.45 times; CYP6N6: 309.54 ± 36.97 times). The expression levels gradually decreased, were lowest at the pupal stage, and then gradually increased after eclosion (Figure 4A and B). Therefore, larvae at the end of the 1st instar were selected for RNAi experiments. First, two groups of dsRNAs with the highest efficiency for CYP6A14 and CYP6N6 RNAi were screened by preliminary screening. The results showed that after 4 days of RNAi treatments, the gene expression of CYP6A14 and CYP6N6 decreased by 53 ± 0.02 and 48 ± 0.03%, respectively, showing no statistical difference from the positive control. These results indicate that dsCYP6A14(3) and dsCYP6N6(2) have good specificity and interference efficiency (Supplemental Figure 4). After treatment with CYP6A14 alone, CYP6N6 alone, or both genes combined, the interference efficiency of the two genes was ∼50%, as in the preliminary screening (Figure 4C). The larval resistance assay showed that both treatments in isolation were not statistically different from the control groups, whereas the combined treatment significantly increased mortality. It indicated that the simultaneous knockout of CYP6A14 and CYP6N6 increased Ae. albopictus sensitivity to deltamethrin (Figure 4D). In addition, we checked for any adverse changes in the life history traits of mosquitoes after the disruption of CYP6A14 and/or CYP6N6 expression by determining parameters such as survival, egg production, and hatching rate; the results were the same as with the control group (Figure 4E–G).
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.
Citation: The American Journal of Tropical Medicine and Hygiene 108, 3; 10.4269/ajtmh.22-0524
DISCUSSION
Deltamethrin, a type II pyrethroid, has been an intensively used insecticide for several decades,29 and its prolonged use has induced resistance in many insect species.30 In recent years, transcriptome studies about mosquito metabolic resistance have focused mainly on large-scale screening of related CYP450 genes in different mosquito species from other regions.31–33 However, further functional verification and exploration of the mechanism of screened CYP450 remain relatively lacking.34,35 In this study, two proteins, CYP6A14 and CYP6N6, were expressed and purified in a prokaryotic expression system and were found to directly metabolize deltamethrin to 1-oleoyl-sn-glycero-3-phosphoethanolamine and 2, 2′-dibromo-2-deoxyguanosine, which have a nontoxic effect. This phenomenon is similar to the CYP450s proteins, CYP6A14, and CYP6N6, which are involved in mosquito resistance by directly metabolizing deltamethrin.4,36
In this study, we initially screened deltamethrin resistance-related genes using transcriptome sequencing and then validated them by deltamethrin-induced expression experiments. Finally, CYP6A14 and CYP6N6 were found to be upregulated in resistant lines; moreover, their expressions were increased about 3-fold after deltamethrin stress treatment of 12 and 24 hours, respectively. These results indicate that CYP6A14 and CYP6N6 are highly correlated with deltamethrin resistance. Meanwhile, the significantly upregulated expressions of the two CYP450 genes were observed only after a delay of 12 hours, which may be due to the time required for deltamethrin penetration through the mosquito cuticle. Also, the different peak time durations (12 and 24 hours) of the deltamethrin-induced expression of the two genes indicate that the resistance could be a result of their mutual support and interaction. In other words, different CYPs could take different exposure times to induce insecticide resistance in a tissue-specific physiological manner.36–38 Altogether, our data show that CYP6A14 and CYP6N6 are likely related to deltamethrin resistance. The protein sequences of CYP6A14 and CYP6N6, having similar CYP450 functional domains, are 83.94% similar; therefore, the two proteins should have identical functions.
To explore whether these two CYP450s can directly metabolize deltamethrin, we conducted an in vitro functional assay. CYP6A14 and CYP6N6 were expressed using the E. coli expression system,39–41 and the corresponding recombinant proteins of ∼60 kDa were obtained in high purity, both of which showed CYP450 enzymatic activity. Subsequently, HPLC was used to detect their deltamethrin degradation activity in vitro, which is similar to some other CYP450s that degrade deltamethrin through a hydroxylation reaction.42,43 Both CYP6A14 and CYP6N6 can directly catalyze the degradation of deltamethrin to 1-oleoyl-sn-glycero-3-phosphoethanolamine and 2′, 2-dibromo-2′-deoxyguanosine through oxidation and hydroxylation.44,45 Next, more robust validation was provided by in vivo RNAi experiments. First, we estimated the right developmental stage of larvae to induce RNAi. The expressions of CYP6A14 and CYP6N6 gradually increased from the egg stage to maximum in the 3rd instar stage, 167.14 ± 14.45 and 309.54 ± 36.97 times, respectively; the lowest expression levels were in the pupal stage. This was done in resistant strains reared in our laboratory. Finally, the late 3rd instar and early 4th instar larvae were selected for RNAi. Also, under long-term deltamethrin stress, the expressions of CYP6A14 and CYP6N6 reached the highest level in the late 3rd instar larvae. Therefore, we started RNAi treatment at instar 1 when CYP6A14 and CYP6N6 expression levels were the lowest. Because the larvae in this stage were too small, the microinjection method was not feasible, causing larvae death. Therefore, larvae ingestion of bacteria expressing the corresponding dsRNA was selected as the RNAi method.46–48 RNAi downregulated the expressions of both genes by ∼50% compared with the control groups. Moreover, the larval impregnation method indicated no statistical difference in deltamethrin sensitivity of the single CYP450 silencing groups. Also, there were statistically significant differences between the two CYP450 gene combination treatment groups. And its mortality increased significantly after 12 hours, indicating that mosquitoes take some time to upregulate CYP450 and may respond by other resistance mechanisms in the beginning.49,50 Meanwhile, we also assessed life activities such as survival time, egg production, and hatching rate of mosquitoes, and no significant changes between the control and RNAi groups were observed. This indicates that the increase in mosquito mortality was due to a direct effect of CYP6A14 and CYP6N6 on the insecticide and not because of the changed overall fitness of the mosquitoes. Importantly, although the recombinant proteins showed individual deltamethrin degradation ability in vitro, the sensitivity of Ae. albopictus to deltamethrin was significantly decreased only when the two CYP450 genes were silenced simultaneously in vivo.
The individual contributions of CYP450 genes to insecticide resistance in mosquitoes have not been discussed in detail, which may be attributable to the occurrence and development of resistance, mostly involving multiple detoxification enzymes.35,51 It is possible that the silencing of a specific gene upregulates a compensatory gene. However, such a hypothesis needs to be tested. Also, the interactions between CYP450, the effect of other factors such as cuticle resistance, and synergy between other resistance mechanisms need to be explored to fully understand the insecticide resistance mechanisms in mosquitoes.52
CONCLUSION
In this study, we investigated the functions of two highly expressed metabolic detoxification enzymes of CYP450, CYP6A14 and CYP6N6, in resistant strains of Ae. albopictus. Both genes were expressed in all developmental stages of Ae. albopictus, especially at the end of the 3rd instar stage, which was ∼200 times higher than at other developmental stages. In addition, in vitro and in vivo RNAi metabolic and function analyses confirmed the deltamethrin degradation ability of the two proteins. These results highlight the deltamethrin resistance mechanisms of CYP6A14 and CYP6N6 genes in Ae. albopictus, which can be exploited for vector resistance management.
Supplemental Materials
ACKNOWLEDGMENTS
We thank all the reviewers who participated in the review and MJEditor (www.mjeditor.com) for their linguistic assistance during the preparation of this manuscript.
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