Nanoject 2 injection system

Newly emerged adult females were placed in a cup and were fed with 10% sterile sucrose solution containing 20 mM of apoptosis inducer PAC-1 (Selleck), apoptosis inhibitor Z-VAD-FMK (Selleck), autophagy inducer rapamycin (Millipore), autophagy inhibitor 3-MA (Selleck), or histone deacetylase (HDAC) inhibitor Scriptaid (Millipore). An equivalent amount of DMSO was used as a control. All chemicals were changed daily. On the fifth day of pharmacological treatment, mosquitoes were infected with the various viruses through blood feeding.
The viruses were injected intrathoracically into the mosquitoes by using the Nanoject II injection system (Drummond Scientific) with 69 nl of MAYV (1.8 × 10⁸ pfu/ml), DENV2 (10⁷ pfu/ml) or ZIKV (10⁷ pfu/ml) per mosquito. An equivalent volume of C6/36 cell culture medium was injected as a control. Each experiment had at least two biological replicates, and each replicate included at least 20 individual females. The number of dead mosquitoes in each cup was recorded daily. Statistical significance was determined by Kaplan-Meier survival analysis with pooled data from different replicates by using GraphPad Prism software, and the P-values were determined by the logrank (Mantel-Cox) test.

To silence expression of the TcGr20 gene in adults, we synthesized a double-stranded RNA (dsRNA) in vitro using a MEGAscript^® T7 RNAi kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. The primers sequences are shown in S4 Table. The dsRNA (1 μg/μL) was kept at –80°C until use. A sample of dsRNA (150–200 nL) was injected between the internode of the head and thorax of adult beetles using a Nanoject II injection system (Drummond Scientific Company, Broomall, PA) on a cooling block at –10°C. Emerald luciferase (Eluc, TOYOBO) was used as a negative control. The NCBI Tribolium genome (ID:216) database was searched for similar sequences to Eluc. Since to Eluc and TcGr20 of more than 22 bp could potentially function as false targets, these sequences were trimmed from the dsRNA regions. The dsRNA-injected adults were kept at 25°C. Adult beetles were used for the TribUTE assays and quantitative RT-PCR at 48 h after injections.

For each target gene, DNA templates of around 500 bp were amplified from midgut cDNA using forward and reverse primers encoding T7 promoter sequences at their 5' ends. Using the MEGAscript T7 kit (Ambion, Austin, TX), 1 μg of template cDNA was in vitro transcribed at 37°C for 4 h to generate dsRNA. The generated dsRNA was treated with turbo DNAse I at 37°C for 15 min, purified using the MEGAclear kit (Ambion), and adjusted with nuclease-free water to a final concentration of 2 μg/μl. Around 140 ng dsRNA was injected intrathoracically into one-week old females using the Nanoject II injection system (Drummond Scientific). Two days after dsRNA injection, mosquitoes received a CHIKV-containing or a non-infectious bloodmeal. Midguts and carcasses were dissected and processed for qRT-PCR and/or plaque assays as described before [7 (link),27 (link)] using specific primers listed in S1 Table.