2.5. Sampling/Processing Mosquitoes

To determine whether mosquitoes that ingested ZIKV became infected, and whether the infections disseminated from the midgut into the hemocoel, they were anesthetized on ice fourteen days post-bloodmeal, and legs of individual mosquitoes were removed and placed into microfuge tubes containing sterilized steel ball bearings and 500 μL of mosquito collection medium (MCM) (DMEM, 2% FBS, 1% Pen–Strep, and 2.5 μg/mL of amphotericin B). The individual carcasses were placed into separate tubes with 500 μL of MCM and processed by trituration for 5 min at 26 Hz in TissueLyser II (QIAGEN, Hilden Germany), followed by centrifugation at 200× g for 5 min [35,40,41,42,43].