Mosquito Rearing
The C. pipiens pallens were reared at 28 ± 1 °C and 70–80% relative humidity with a 12 h light and 12 h dark photoperiod. The larvae were fed with rat chow. Adult mosquitoes were maintained on 7% sucrose solution. The female mosquitoes were fed fresh mouse blood to induce egg laying. Mosquitoes were not treated with insecticides or other chemicals.
CpCHSA cDNA cloning
The RNAiso Plus reagent (Takara, Tokyo, Japan) was used to isolate RNA. The full‑length sequence of CpCHSA cDNA was determined from seven overlapping PCR fragments (Additional file 1: Table S1). The 5'- and 3'- end fragments were obtained using rapid amplification of cDNA ends (RACE) using a SMARTer RACE 5'/3' Kit (Takara). PCR amplification products were analyzed using agarose gels and then purified (TIANGEN, Beijing, China). Purified DNA was ligated into vector pClone007 (TSINGKE, Nanjing, China) and sequenced. The obtained full-length cDNA of CpCHSA was submitted to the NCBI and received the accession number MH013352.
Analysis of gene expression patterns
Total RNA was isolated from the larval stage (1st, 2nd, 3rd, and 4th), pupal stage (0 and 24 h) and adult stage (1–3 posteclosion (PE) and 1–3 post-blood meal (PBM)) to investigate the C. pipiens pallens developmental expression profile. The head, foregut, midgut, hindgut, Malpighian tubules, and carcass were dissected from 4th larvae or 3‑d old adult mosquitoes for tissue-specific expression analysis. Total RNA was isolated from the whole bodies of five mosquitoes and the tissues of ten mosquitos for biological replicates. First stand cDNA was synthesized using PrimeScript RT Master Mix (Takara). A LightCycler® 96 Instrument was used for quantitative real-time PCR (qPCR) analysis (Roche, Basel, Switzerland) with the BrightGreen 2*qPCR MasterMix-No Dye (Applied Biological Materials, Vancouver, Canada). The specific primers are shown in Additional file 2: Table S2. The qPCR reaction volume (10 μL) contained the Power SYBR Green PCR Master Mix, specific primer sequences, and diluted cDNA (1 mg/mL). The relative expression levels were normalized to the internal control ACTB (encoding β-actin) using the 2−ΔΔCt method [36]. All experiments were performed with three biological replicates.
Microinjection
RNAi was used to knockdown the expression of CpCHSA. The small interfering RNA sequences used to silence the CpCHSA gene (siCHSA) and negative control (NC) are shown in Additional file 3: Table S3, both of which were designed and manufactured by Gene Pharma (Shanghai, China). siCHSA or NC (0.30 μg) was injected into the 3rd larvae abdomen and the adult female mosquitoe thorax. In the pupal stage, they were injected into the dorsal cuticle between the thorax and the abdomen. Both the wild-type (WT) group and NC group served as controls. Total RNA was isolated from whole mosquitoes (n ≥ 5), and CpCHSA transcript levels were analyzed after RNAi using qPCR.
Immunofluorescence staining
The chitin content of the abdominal integuments was quantified after siCHSA or NC injection using chitin staining. Pupae (0–1 h) were selected to be injected with siRNA, and at 24 h after injection, the pupal abdomen was immediately dissected. Slides of paraffin-embedded tissue were deparaffinized in xylene and rehydrated using an ethanol gradient. Fluorescent Brightener 28 (Sigma-Aldrich, Hamburg, Germany) was used to stain the sample, propidium iodide was used as a counterstain, anti-fluorescent stain was added, and the samples were observed under an AXIO confocal fluorescence microscope (Zeiss, Oberkochen, Germany)[37].
For the immunofluorescence experiment, rabbit polyclonal antibodies were prepared against CpCHSA. We designed the peptide antigen of CpCHSA by analyzing the cDNA and protein sequences (Additional file 4: Table S4). The peptide was synthesized, and then subcloned into pET-28a-sumo and PGEX-4T-AB1 transfer plasmids. (ABclonal Wuhan, China) synthesized the gene and produced polyclonal antibody. The acquired antibodies were tested to ensure that they met the experimental requirements.
To analyze the localization of the CpCHSA protein, paraffin sections were made from pupae treated with siCHSA or NC. The tissues were fixed in 4% paraformaldehyde at 4 °C overnight. Sections were acquired and then deparaffinized using xylene with two washes of 15 min, rehydrated through successive baths of ethanol (100, 96, and 70% in water, 15 min each), washed twice for 5 min, and then once with PBST (0.01 M phosphate-buffered saline (PBS), pH 7.4 containing 0.1% Tween 20) for 10 min. The sections were blocked with 2% bovine serum albumin for 30 min, followed by incubation by anti-CpCHSA antibodies (1:100) at 4 °C overnight. The sections were washed and then incubated with Alexa Fluor@ 594-conjugated donkey anti-rabbit IgG (Abcam, Cambridge, UK) secondary antibody (1:200 in blocking buffer) for 50 min, in the dark. After three washes in PBST for 5 min each, the nuclei were stained with 4' ,6‑diamidino-2-phenylindole (DAPI) for 10 min in the dark. The sections were then wash with PBST and observed under a fluorescence microscope (Zeiss)[26].
Electron microscopy
Pupae (0–1 h) and adults (12–24 h PE) were injected with siCHSA or NC. At 24 h after injection of pupae and at 72 h after injection of the PE adults, tissues were collected and fixed in 4% paraformaldehyde at 4 °C, and then washed with PBS three times for 15 min each. 1% OsO4 in 0.1 M PBS (pH 7.4) was used to post-fix the samples for 2 h at room temperature. The OsO4 was removed and the samples were rinsed in PBS (0.1 M, pH 7.4) three times for 15 min each. The samples were then dehydrated through successive concentrations of ethanol (50, 70, 80, 90, 95, and 100%, for 15 min), and finally through two changes of acetone for 15 min, and then infiltrated with 1:1 acetone: EMBed 812 for 3 h, 2:1 acetone: EMBed 812 overnight, and pure EMBed 812 for 7 h. The samples were kept at 37 °C overnight and then baked at 60 °C for 48 h. Sections were cut and stained with uranyl acetate for 15 min followed by lead citrate staining for 15 min. The sections were air dried overnight. The ultrastructure of the cuticles was then analyzed using transmission electron microscopy (TEM)[38].
Western blotting
Western blotting was used to evaluate the specificity of the anti-CpCHSA antiserum and to verify the knockdown efficiency of RNAi. Total proteins were extracted from the whole body of larval, pupal, or adult mosquitoes using Radioimmunoprecipitation assay (RIPA) buffer containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and a protease inhibitor cocktail (Thermo, Rockford, IL, USA), and then centrifuged at 12,000 × g at 4 °C for 30 min. A bicinchoninic acid (BCA) protein assay kit (Beyotime, Shanghai, China) was used for protein quantification. The proteins were fractionated using 5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to nitrocellulose membranes. The membranes were probed using anti‑CpCHSA (1:1000) and β-actin (Abclonal 1:7000) antibodies, followed by incubation with labeled secondary antibodies and visualization of the immunoreactive protein bands. The bands were analyzed using Image J software (NIH, Bethesda, MA, USA).
Statistical analysis
SPSS 23.0 (IBM Corp., Armonk, NY, USA) and GraphPad Prism 6.0 software (GraphPad Software Inc., La Jolla, CA, USA) were used for statistical analyses[37]. The statistical significance of the RNAi knockdown efficiency and the survival rate were analyzed using an unpaired Student’s t test. *p < 0.05, **p < 0.01, and ***p <0.001. All experiments were performed using at least three independent cohorts.