Mosquito Saliva Collection Protocol

Fourteen days post-infection, mosquito saliva was collected by forced salivation as described earlier [23 (link)]. Mosquitoes were first immobilized using 100% CO₂, and their legs and wings were removed. To collect mosquito saliva, the mosquito proboscis was inserted in a 200 µl pipette tip holding 5 µl of a 1:1 mixture of FBS and 50% sugar in autoclaved tap water. After 45 min, the samples containing mosquito saliva were mixed with 55 μl DMEM HEPES complete and stored at −80 °C. The mosquito bodies were collected in SafeSeal micro tubes (Sarstedt) containing 0.5 mm zirconium oxide beads (Next Advance) and also stored at −80 °C.