Determination of infection and dissemination

Dengue infection in mosquitoes can be assessed through three key stages: infection, dissemination, and infectiousness. Per parental convergence group and replicate, 20 mosquitoes were processed on days 3, 6, 9, 12, 15, and 18 post-infection (n = 120 females per time point, n = 760 females in total). To do this, females were immobilized on ice and then transferred to a chill table (Bioquip, Rancho Dominguez, CA). Legs and wings were removed, and the proboscis inserted into a 200 µl pipette tip with the end cut-off and containing 30 µl of salvation mix (1900 µl FBS, 20 µl 300 mM ATP, 80 µl red food dye). Legs were kept for analysis of dissemination and transferred into 2 ml tubes containing DMEM with 1× antibiotic/antimycotic. Females were allowed to salivate for 40 min on a heat plate. Successful salivation was confirmed by the presence/absence of red food dye in female abdomens. Afterward, the saliva was transferred into Eppendorf tubes containing 700 µl of DMEM with 1× antibiotic/antimycotic to test for infectiousness. Heads of females were then cut off and added to the tubes containing the leg samples to test for dissemination. The remaining body was transferred into Eppendorf tubes containing 700 µl DMEM with 1× antibiotic/antimycotic to test for infection.