Insects
DS- and DR-strains of mosquito (Cullex pipiens pallens) used in the present study were obtained from Tangkou, Shandong province, and maintained in the laboratory with a constant light/dark cycle (14 h:10 h) at 28 °C and 70–80% humidity. Adults were provided with 5% sterilized sugar on sponge wick ad libitum. The DR-strain was selected from the DS-strain and was maintained via treatment with deltamethrin at the 50% lethal concentration (LC50) of each generation. For the DS- and DR-strains, the LC50 values were 0.04 mg/L and 8.5 mg/L, respectively.
Genomic DNA Extraction, pre-miR-4448 Amplification and cloning
Genomic DNA (gDNA) was extracted from 3-d post-eclosion (3 d PE) female adult mosquitoes using a MiniBEST Universal Genomic DNA Extraction Kit Ver. 5.0 (Takara, Dalian, China) following the manufacturer’s instructions. gDNA quantity and quality were checked using a Thermo Scientific™ NanoDrop 2000 instrument (Thermo Fisher Scientific, Waltham, MA, USA).
Using the gDNA as a template, PCR was performed using primers (Table 1) designed according the Cx. pipiens pallens pre-miRNA sequence with the following conditions: 94 °C for 5 min; followed by 35 cycles of 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 10 s; and a final extension step at 72 °C for 10 min. The PCR products subjected to electrophoresis through a 2.0% agarose gel. A PCR fragment of around 80 bp was isolated from the gel and purified using a MiniBEST Agarose Gel DNA Extraction Kit Ver. 4.0 (Takara), and then cloned into vector pMD 18-T (Takara). The resultant plasmid was transferred into Escherichia coli Top10 cells for amplification and sequencing.
Quantitative real-time reverse transcription PCR (qRT-PCR) analyses
At 3 d PE, DS-strain and DR-strain female adult mosquitoes were subjected to total RNA extraction using the RNAiso Plus reagent (Takara). The total RNA purity and concentration were checked using a NanoDrop spectrophotometer. The cDNA was synthesized from 1 μg of total RNA using a PrimeScript RT reagent Kit (Takara) and PrimeScript™ RT Master Mix (Takara) according to the manufacturer’s protocol. The cDNA was diluted 1:10 and 4 μl of the diluted cDNA solution was used as template for quantitative real-time PCR (qPCR) using the Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA). PCR was performed in a 20-μl reaction mix containing 10 pmol of forward and reverse PCR primers (designed using Primer Premier 6.0 software (PREMIER Biosoft International, San Francisco, CA, USA)) for miR-4448 and CYP4H31 (Table 1). MiR-4448 expression was measured using the Stem-loop RT-PCR method [23] using the following reaction conditions: 50 °C for 2 min and 95 °C for 10 min; 40 cycles of 95 °C for 15 s and 60 °C for 1 min, followed by melting-curve analysis on an ABI Prism 7300 real-time PCR Instrument (Applied Biosystems). The relative expression level of miR-4448 was normalized to the internal control U6 small nuclear (U6), and the CYP4H31 expression level was normalized to that of β-actin from the DS- and DR-strains. The DS-strain expression level was designated as 1. For each experiment, RNA from three biological replicates was used and PCR amplification of each cDNA sample was performed in triplicate. The 2-ΔΔCt method was used to calculate the relative expression levels [24].
pMIR-REPORT vector construction, cell culture, and Dual-Luciferase Reporter Assay
We identified the region of the CYP4H31 3′ UTR that included the complementary sequences predicted to bind miR-4448. To mutate this region, the binding site complementary region (AUCGAGC) was replaced by UUGGUGG (3′ UTR-Δ). Two pairs of primers were designed according to the transcript sequences from Cx. quinquefasciatus to amplify the wild-type (WT) 3′ UTR and 3′ UTR-Δ of CYP4H31 (Table 1). Luciferase constructs were made by amplifying and sequencing the Cx. quinquefasciatus putative target 3′ UTR-WT/3′ UTR-Δ sequence of the CYP4H31 mRNA (containing the putative seed region of the miR-4448 binding sites) and using the T/A Cloning method to insert them into the HindIII and XbaI sites located downstream of the Renilla translational stop codon within the pMIR-REPORT miRNA Expression Reporter Vector (Promega, Madison, WI, USA) [17].
At 48 h after transfection of the pMIR-REPORT constructs, assays were performed using the Dual‑Luciferase reporter assay System (Promega). 293T cells were cultured in Dulbecco’s modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal calf serum (FCS) in a 5% CO2-humidified incubator at 37 °C [18]. Then, 6 × 104 cells/well in 2.5 ml of complete growth medium were seeded and incubated in a 6-well plate for 24 h, until they reached >80% confluency. Then, 6 ng of pMIR-REPORT-UTR-WT or pMIR-REPORT- UTR-Δ treated with 6 µl of miR-4448 mimic and miRNA negative control (NC1) (GenePharma, Shanghai, China) along with 6 ng of PGL4.7 (Promega) were cotransfected using the FuGENE HD transfection reagent (Promega). Vector PGL4.7, which constitutively expresses Renilla luciferase, was cotransfected as an internal control to correct for differences in the efficiency of transfection and harvest between the groups. In each sample, Renilla luciferase was normalized using Firefly luciferase expression [19]. An M200 microplate fluorescence reader (Tecan, Lyon, France) was used to detect the luciferase activity. Cells were treated in in triplicate, and the transfections were repeated three times.
Oral feeding
For the oral feeding experiments, all of the materials (e.g., water, glucose, and sponges) were treated with diethyl pyrocarbonate (DEPC) to remove RNase. DR-strain pupae of Cx. pipiens pallens were collected and placed in three cages until eclosion, and then starved for 12 h. The 12 h eclosion (12 h PE) mosquitoes of blank group (WT) were treated with 5% glucose water, while the negative control group (NC1) were given the miRNA mimic control dissolved in 5% glucose water at a final dose of 100 nmol/L. The experimental group (miR-4448 mimic) was supplied with the miR-4448 mimic dissolved in 5% glucose water at final a dose 100 nmol/L. At 48 h after treatment, RNA was extracted from female adult mosquitoes to validate the expression of miR-4448 and its target gene CYP4H31. The miR-4448 mimic and miRNA control mimic were obtained from GenePharma (Table 2).
Microinjection of miR-4448 mimic and CYP4H31 siRNA (si-CYP4H31)
GenePharma deigned and synthesized a small interfering RNA targeting the open reading frame (ORF) of CYP4H31 (si-CYP4H31) (Table 2). For the microinjection of miRNA, DR-strain 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 injected in the thorax with different moieties. The negative control group (NC1) was injected with 0.5 μl of miRNA control mimic at a dose of 20 nmol/l and the experimental group (miR-4448 mimic) was injected with 0.5 μl of the miR-4448 mimic under the same conditions at a final dose of 20 nmol/L. For the microinjection of siRNA, the negative control group (NC2) was injected with 69 nl of control at a dose of 5 μg/μl, and experiment group (si-CYP4H31) was injected with 69 nl of si-CYP4H31 under the same conditions at a final dose of 5 μg/μl. Thereafter, the mosquitoes were transferred to holding tubes and maintained in our laboratory with a constant light/dark cycle (14 h: 10 h) at 28 °C with 70–80% humidity. After 72 h, the expression levels of miR-4448 and its target gene CYP4H31 were validated using qRT-PCR. Three biological replicates, each containing 20 female mosquitoes, with three technical replicates, were performed.
Eukaryotic expression vector pIB/V5-His construction, cell culture, and transfection
Standard molecular biology procedures were used for plasmid construction. Overlap PCR was performed to amplify the ORF of CYP4H31 using corresponding primer pairs (Table 1) from Cx. quinquefasciatus, which was inserted between unique restriction enzyme sites (SpeI/XhoI) of the eukaryotic expression vector, pIB/V5-His. The positive recombinant plasmid, named pIB/V5‑His‑CYP4H31 was confirmed using DNA sequencing.
Aedes albopictus C6/36 cells (CRL-1660; ATCC) were cultured in DMEM supplemented with 10% (v/v) FCS. The C6/36 cells were grown in a 6-well plate at 28 °C in a 5% CO2-humidified incubator at. The cells were then plated at 5 × 105 cells/well and incubated for 24 h in a 6-well in 2.5 ml of complete growth medium. The cells were transfected when they reached 60–80% confluence. The transfection protocol was as follows: The plasmid DNA (pIB/V5-His-CYP4H31) was diluted to 1.5 ng per 100 µl in complete growth medium, followed by the addition of 5 µl of FuGENE HD transfection reagent. The DNA mixture was incubated at room temperature for 25 min and then added to the medium below the surface. The plate was rocked back-and-forth and from side-to-side to ensure distribution over the entire plate surface. Meanwhile, C6/36 cells transfected with pIB/V5-His were used as controls. Three biological replicates with three technical replicates were performed.
qRT-PCR and Western blotting analysis of CYP4H31 in the transfection cells
At 48 h after transfection, the transiently transfected C6/36 cells were subjected to western blotting and qRT-PCR. To evaluate the CYP4H31 transfection efficiency, total RNA was isolated from the transfected cells, and qRT-PCR was performed, as described above, to check the expression level of CYP4H31.
Transfected cells were washed with phosphate-buffered saline (PBS). Protein was extracted from the cells after digestion with trypsin solution and lysis using Radioimmunoprecipitation assay (RIPA) buffer (Beyotime, Shanghai, China). Protein concentrations were tested using a bicinchoninic acid (BCA) Protein Assay kit (Pierce, Rockford, IL, USA). Soluble protein (50 µg) was denatured and subjected to 10% SDS-PAGE. Proteins were transferred to a nitrocellulose membrane using Trans-Blot SD Cell and Systems for 60 min at 300 mA (Bio-Rad, Hercules, CA, USA). The membrane was washed twice in 1´ Tris-buffered saline-Tween 20 (TBS-T), and then blocked for 60 min at 37 °C in 5% DifcoTM Skin Milk (BD Biosciences, San Jose, CA, USA). The membrane was incubated with anti-His-Tag monoclonal primary antibodies (1:1000, NovaGen, Madison, WI, USA) and β-actin monoclonal primary antibodies (1:2000, ABGENT, Suzhou, China), with shaking overnight at 4 °C. The membranes were then washed with TBS-T, and incubated with horseradish peroxidase (HRP)-conjugated Goat Anti-mouse secondary antibodies (1:2000, Bioworld, Shenzen, China) in blocking buffer at 37 °C for 2 h. The membranes were washed thoroughly with TBS-T before imaging using BIO-RAD UNIVERSAL HOOD II and Pierce™ ECL Western Blotting Substrate, according to the manufacturer's instructions.
Cell viability assay using a Cell Counting Kit-8
CYP4H31 overexpression in relation to deltamethrin resistance was assessed by measuring cell viability using a Cell Counting Kit-8 (CCK-8; Dojindo, Japan) [25]. Cells (100 µl) were added to each well of a 96-well plate 5 × 103 cells/well and incubated in a 5% CO2-humidified incubator at 28 °C for 24 h. Then, the cells were treated with various concentrations of deltamethrin in 100 µl (0, 100.5, 101, 101.5, 102, and 102.5 mg/L) [26]. Twenty-four hours later, CCK-8 solution (10 µl) was added to the wells and incubated for 28 °C for 3 h. The absorbance was then detected using dual wavelength spectrophotometry at 450 nm and 630 nm in a microplate reader. Dimethyl sulfoxide (DMSO, Sigma, St, Louis, MO, USA) was used to dissolve deltamethrin and the final concentration of DMSO was 0.5% (v/v) for the different concentrations of deltamethrin [22]. Three biological replicates with three technical replicates were performed.
American CDC Bottle Bioassay
According to published guidelines, American Centers for Disease Control and Prevention (CDC) bottle bioassays were conducted to detect the sensitivity of mosquitoes injected with the miR-4448 mimic and si-CYP4H31 to deltamethrin [27]. Each 250-ml bottle and its cap were coated with 1 ml of deltamethrin solution using inversion and rolling of the bottles. Control bottles were coated using 1 ml of acetone. A sheet was used to cover all bottles, which were left to dry in the dark. Twenty mosquitoes were placed in each bottle and exposed to deltamethrin or acetone for 120 min. Following exposure, knockdown mosquitoes were monitored at 15 min intervals up to 2 h and the percent mortality (Y axis) was plotted against time (X axis) using a linear scale.
Statistical Analysis
Statistical significance was accepted at P < 0.05. Qualitative variables were detected using the chi‑square test, while quantitative variables were detected using analysis of variance [28, 29].