Mosquito strains and cell lines
Two strains of Cx. pipiens pallens with different resistance levels to deltamethrin were used in this study. The DS-strain of Cx. pipiens pallens used in this study was obtained from Ji Nan University and subsequently reared in our laboratory at 28 °C, 70–80% relative humidity, 14:10 h light/dark cycle. The DS-strain mosquitoes had not been exposed to any insecticides. The DR-strain was selected from the DS-strain by constant exposure to deltamethrin (at lethal concentration 50 [LC50]; concentration which kills 50% of sample population) and was screened over 80 generations. There were 4000 larvae screened for each pool (three pools/generation). The LC50 of the DS- and DR-strains was 0.05 and 8.5 mg/l, respectively. The resistance ratio of LC50 (RR50) of the DR-strain was 170. Deltamethrin (technical grade, 99.0%) was obtained from Jiangsu Provincial Center for Disease Control and Prevention (Jiangsu, China). Larvae were grown in dechlorinated tap water and fed with fish food powder (Tetramin; Tetra, Pirmasens, Germany) every 2 days. Adult mosquitoes were maintained in cages with constant access to a 5% glucose solution. Female mosquitoes were fed on mouse blood to reproduce the next generation every 3–4 weeks. Procedures for blood-feeding with mice in our laboratory were approved by The National Science and Technology of China and People’s Government of Jiangsu Province Animal Care and Use Committee and Institutional Review Board (No. IACUC-1812047).
The HEK293T cell line was grown in complete high glucose Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY, USA) containing 10% (v/v) fetal bovine serum (FBS; Gibco) and 100 U/ml penicillin–streptomycin solution (Gibco) at 37 °C under 5% CO2. Cells (6 × 104/well) were seeded and incubated in 2.5 ml of complete growth medium in a 6-well plate for 24 h until they achieved > 80% confluency. The mosquito C6/36 cell line (CRL-1660; ATCC, Manassas, VA, USA) was cultured in DMEM supplemented with 10% FBS. The cells were plated in a 6-well plate and were grown in a 5% CO2-humidified incubator at 28 °C. Cells (5 × 105 /well) were plated and incubated in 2.5 ml of complete growth medium in a 6-well plate for 24 h, until they achieved the required density of 60–80% for transfection. All cells were confirmed to be negative for mycoplasma contamination regularly.
RNA and genomic DNA extraction
The RNAiso Plus reagent (Takara, Dalian, China) was used to extract total RNA from the female adult mosquitoes (n = 5) of the DS- and DR-strains at 3 days post-emergence (3 days PE). Genomic DNA (gDNA) was extracted from 3-day PE female adult mosquitoes using a MiniBEST Universal Genomic DNA Extraction Kit Ver. 5.0 (Takara) according to the manufacturer’s protocol. The quality and quantity of the RNA and gDNA were checked using a Thermo Scientific™ NanoDrop 2000 instrument (Thermo Fisher Scientific, Waltham, MA, USA).
Quantitative real-time reverse transcription PCR analyses
A PrimeScript RT Reagent Kit (Takara) and PrimeScript™ RT Master Mix (Takara) were used to synthesize complementary DNA (cDNA) from 1 μg of total RNA following the manufacturer’s instructions. Then, 4 μl of 1:10 diluted cDNA solution was used as the template for quantitative real-time PCR (qPCR), which was performed using the Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA). The PCR reaction mix (20 μl) contained forward and reverse PCR primers (10 pmol) for miR-279-3p, U6, CYP325BB1 and β-actin, designed using Primer Premier 6.0 software (Premier Biosoft, Palo Alto, CA, USA; Table 1). Stem-loop reverse transcription (RT)-PCR was used to measure the expression of miR-279-3p . The PCR cycling conditions were: 50 °C for 2 min; then 95 °C for 10 min; followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min; melting-curve analysis was then performed on an ABI Prism 7300 real-time PCR Instrument (Applied Biosystems). The miR-279-3p relative expression level was normalized to that of the internal control small nuclear RNA (U6), which is standard for miRNA expression normalization , and the expression level of CYP325BB1 was normalized to that of β-actin from the DS- and DR-strains . The DS-strain expression level was designated as 1, and the negative control (NC) was used to compare the gene expression levels. Each experiment used RNA from three biological replicates, and each cDNA sample was PCR amplified in triplicate. The 2−ΔΔCt method was used to calculate the expression levels .
Identifying the potential target of miR-279-3p
To identify the putative gene targets of miR-279-3p, we used 3′-UTR sequences from the Culex quinquefasciatus genome in the RNAhybrid target prediction program . We focused on the cytochrome P450 family of genes (CYP) that participate in the regulation of insecticidal resistance of mosquitoes, and only CYP325BB1 was identified as a potential target of miR-279-3p. To assess the conservation of the 3′-UTR, we amplified the 3′-UTR from Cx. pipiens pallens. The 3′-UTR sequence of CYP325BB1 in Cx. pipiens pallens was 100% identical with that from Cx. quinquefasciatus.
PMIR-REPORT vector construction and dual-luciferase reporter assay
To amplify the wild-type 3′-UTR (3′-UTR-WT) and the mutated 3′-UTR (3′-UTR-Δ) of CYP325BB1 of Cx. pipiens pallens, primers for CYP325BB1 3′-UTR-WT/Δ (Table 1) were designed based on Cx. quinquefasciatus transcripts, which amplified the 3′-UTR region containing the miR-279-3p complementary sequences. Mutagenesis of the 3′-UTR (CTAGTCA) comprised replacing the WT binding site with CGACTGA. The Cx. pipiens pallens CYP325BB1 3′-UTR-WT and 3′-UTR-Δ sequences containing the putative seed region of the miR-279-3p binding sites were amplified and sequenced using the T/A cloning method. PCR products were run in 2.0% agarose gels and purified using a MiniBEST Agarose Gel DNA Extraction Kit Ver. 4.0 (Takara), and then cloned into vector pMD 19-T (Takara). The resultant plasmid was transferred into Escherichia coli One Shot® TOP10 Competent Cells (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, USA). The UTR sequences were then ligated into the HindIII and XbaI sites, which are located downstream of the Renilla translational stop codon in the pMIR-REPORT miRNA Expression Reporter Vector (Promega, Madison, WI, USA), to create the luciferase constructs. The pMIR-UTR-WT (6 ng) or pMIR-UTR-Δ were treated with miR-279-3p mimic (6 µl; GenePharma, Shanghai, China) and NC (GenePharma) along with PGL4.7 (6 ng; Promega), which were cotransfected using the FuGENE HD transfection reagent (Promega) into HEK293T cells (> 80% confluency). The Dual-Luciferase® Reporter Assay System (Promega) was then used for the reporter assay at 48 h after transfection. PGL4.7 provided the constitutive Firefly luciferase expression and was cotransfected as an internal control. Renilla luciferase was normalized to Firefly luciferase expression in each sample. The luciferase activity was detected at 560 and 480 nm using an M200 microplate fluorescence reader (Tecan, Lyon, France). The transfections were performed three times, and treatment was performed in triplicate.
For the microinjection of miRNA, female adult mosquitoes were collected within 12 h PE and frozen at − 20 °C for 3–5 min. These mosquitoes were divided into three groups and prepared for injection through the thorax using a Nanoject III instrument (Drummond, Broomall, PA, USA). The miR-279-3p mimic or NC (0.5 μl; 20 nmol/l) were injected into the DR-strain mosquitoes (miR-279-3p mimic/NC group) under the same conditions. The miR-279-3p inhibitor or NC 1 (NC1, 0.5 μl; 20 nmol/l) was injected into the DS-strain mosquitoes (miR-279-3p inhibitor/NC1). For the microinjection of small interfering RNA (siRNA), 12-h PE DR-strain mosquitoes from the experimental group (si-CYP325BB1) and NC group were injected with 69 nl of si-CYP325BB1 or NC (5 μg/μl) under the same conditions. Thereafter, the mosquitoes were maintained with a constant light/dark cycle (14/10 h) at 28 °C and 70–80% humidity. After 72 h, the expression levels of miR-279-3p and CYP325BB1 were validated using qRT-PCR. Three biological replicates (each comprising 15 female mosquitoes) with three technical replicates were performed. GenePharma designed and produced all the injected RNA products (Table 2).
For the oral feeding experiments, pupae of the DR-strain were collected and placed in three cages until eclosion, and then fasted for 12 h. The mosquitoes of the control group were fed with 5% (w/v) glucose on a sponge wick (3 M, Minneapolis, MN, USA), while the NC group and the experimental group (miR-279-3p mimic) were given NC and miR-279-3p mimic (100 nmol/l) dissolved in 5% (w/v) glucose dissolved in DEPC-treated water, respectively. After 48 h of treatment, the RNAs of female adult mosquitoes were extracted to validate the expression levels of miR-279-3p and CYP325BB1. The miR-279-3p mimic and NC were obtained from GenePharma.
U.S. Centers for Disease Control and Prevention bottle bioassay
To detect the mosquitoes’ sensitivity to deltamethrin after alteration of the miR-279-3p and CYP325BB1 levels, U.S. Centers for Disease Control and Prevention (CDC) bottle bioassays were conducted according to published guidelines (https://www.cdc.gov/malaria/resources/pdf/fsp/ir_manual/ir_cdc_bioassay_en.pdf) . Each bottle (250 ml) and its cap were rolled and inverted to coat them with 1 ml deltamethrin solution (7 mg/l for DR-strain; 0.01 mg/l for DS-strain). In parallel, a control bottle was coated with 1 ml of acetone, and then all bottles were left to dry in the dark. Mosquitoes from the designated groups (20/bottle) were exposed to deltamethrin or acetone for 2 h. Following exposure, the mosquitoes were monitored at 15-min intervals for 2 h and the percentage mortality (y-axis) was plotted against time (x-axis) using a linear scale.
PIB/V5-His vector construction, transfection and detection
Standard molecular biology procedures were used for plasmid constructions. Standard overlap PCR was performed to amplify the open reading frame (ORF) of CYP325BB1 using the pIB/V5-His-CYP325BB1 primer pair (Table 1) from Cx. quinquefasciatus, which was ligated between unique restriction enzyme sites (SpeI/XhoI) of the eukaryotic expression vector pIB/V5-His. The positive recombinant plasmid was named pIB/V5-His-CYP325BB1 and was confirmed using DNA sequencing.
C6/36 cells at 60–80% confluence were used for transfection. Plasmid DNA (pIB/V5-His-CYP325BB1) was diluted in complete growth medium to 1.5 ng/100 µl and 5 µl of FuGENE HD transfection reagent was added, followed by incubaion at room temperature with shaking for 25 min. pIB/V5-His was transfected as a control. Three biological replicates with three technical replicates were performed.
To evaluate the transfection efficiency of CYP325BB1, after 48 h of transfection, the mRNA and protein levels of CYP325BB1 in transiently transfected C6/36 cells were detected. Total RNA was isolated from the transfected cells and subjected to qRT-PCR to check the expression level of CYP325BB1. Protein was extracted from transfected cells, followed by washing in phosphate buffered saline (PBS), digestion in trypsin solution and lysis using radioimmunoprecipitation assay (RIPA) buffer (Beyotime, Jiangsu, China). The protein concentration was tested using a bicinchoninic acid (BCA) Protein Assay kit (Pierce, Rockford, IL, USA). Soluble protein (50 µg) was denatured and subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins were transferred to a nitrocellulose membrane using a Trans-Blot System for 60 min at 300 mA (Bio-Rad, Hercules, CA, USA), and then washed twice in 1× Tris-buffered saline-Tween20 (TBS-T),and blocked for 60 min at 37 °C in 5% Difco™ Skim Milk (BD Biosciences, San Jose, CA, USA). The membrane was then incubated with anti-His-Tag monoclonal primary antibodies (1:1000; NovaGen, Madison, WI, USA) and anti-β-actin monoclonal primary antibodies (1:2000; ABGENT, Suzhou, China), with shaking overnight at 4 °C. Thereafter, the membranes were washed using TBS-T, and incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse secondary antibody (1:2000; Bioworld, Shenzhen, China) in blocking buffer at 37 °C for 2 h. The membranes were washed thoroughly using TBS-T, and the immunoreactive protein bands were visualized using Pierce™ ECL Western Blotting Substrate, before imaging using the Universal Hood Gel Doc System (Bio-Rad).
C6/36 cell viability assay
The viability of cells overexpressing CYP325BB1 was investigated using a CCK-8 kit assay (Dojindo, Kumamoto, Japan) . C6/36 cells (100 µl) were added to each well of a 96-well plate at 5 × 103 cells/well and incubated in a 5% CO2-humidified incubator at 28 °C for 24 h. At 24 h after transfection the cells were treated with 100 µl of deltamethrin at concentrations of 0, 100.5, 101, 101.5, 102 and 102.5 mg/l . After a further 24 h, the CCK-8 solution (10 µl) was added to each well and incubated at 28 °C for 3 h. The absorbance was then measured using a dual wavelength spectrophotometer in a microplate reader at 450 and 630 nm. Dimethyl sulfoxide (DMSO; Sigma, St. Louis, MO, USA) was used to dissolve deltamethrin at a final concentration of 0.5% (v/v). Three biological replicates with three technical replicates were performed.
Statistically significant qualitative variables were detected using GraphPad Prism 8.0 software (GraphPad Software Inc., La Jolla, CA, USA). Data from independent experiments are presented as the mean ± the standard error of mean (SEM). Student’s t-test was used to determine the statistical significance of gene expression compared with that in the NC. The Chi-squared test (χ2) was used to analyze mosquito mortality. Statistical significance was indicated by P < 0.05 [28, 29]. All experiments were performed in at least three independent cohorts.
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