Microfuge tubes

Upon completion of each KD experiment, individual whole mosquitoes were anesthetized by chilling and placed in 1.5-ml microfuge tubes (Sarstedt, Nümbrecht, Germany) containing 300 μl of TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and a 2.8-mm ceramic bead. Samples were homogenized on a Bead Ruptor Elite (Omni International, USA) and then frozen at −80°C. Total RNA was extracted with the Direct-zol RNA 96 Magbead Zymo kit (Zymo Research, Irvine, CA) according to the manufacturer’s protocol. Following this step, the samples were processed using an automatic magnetic bead purification system (MagMAX Express 96 system, Applied Biosystems). RNA was eluted in 50 μl RNase free water. RNA was then treated with 5 units of DNase I (Sigma-Aldrich) at room temperature for 15 min, followed by inactivation with 50 mM EDTA at 70°C for 10 min. To measure Wolbachia loads, extractions for both RNA and DNA were performed using the column-based Direct-zol DNA/RNA Miniprep kit. RNA was eluted in 50 μl RNase free water, followed by DNA elution in 50 μl of Direct-zol DNA Elution Buffer. Total RNA and DNA concentrations were determined with a NanoDrop model 2000/2000C (Thermo Scientific, Waltham, MA).

Blood samples were collected pre- and post-exposure, consisting of a serum separator tube (SST, BD).The SST tubes were allowed to clot at room temperature for 30 min (19 (link)). The clot was separated from the serum following the centrifugation of the tubes at 1,500 g for 15 min at room temperature. The serum was aliquoted into 3 microfuge tubes (Sarstedt) using a 1,000 μL pipette, such that each contained ~500 μL of sample. The tubes were frozen and stored at −80°C.