Graduated glass capillary tube

At 14 dpi, DENV-4 infected mosquitoes were starved overnight. To collect saliva, mosquitoes were cold-anesthetized, and their wings and legs were removed. Each mosquito was then fastened to a glass microscope slide with tape, and their proboscis was inserted into a graduated glass capillary tube (Drummond, Broomall, PA, United States) filled with 3μl of warmed human O⁺ blood (1,1 O⁺ human red blood cells: heat inactivated human serum) to initiate feeding cues and facilitate saliva collection (Stephenson et al., 2021 (link)). The mosquitoes were placed in a lit rearing chamber at 28°C with 80% relative humidity for 45min or until they ingested approximately 2μl of blood. Each proboscis was then removed from its capillary tube, and the remaining blood from each capillary tube was aspirated into 1.5ml microcentrifuge tubes with 200μl of reduced (3% FBS) DMEM. Mosquitoes were then surface sterilized in 70% ethanol and rinsed twice in 1x PBS before being dissected in PBS to produce paired ovary and midgut samples. All samples were immediately stored at −80°C until use.

ORL and COL mosquitoes were sugar starved overnight, and 14 days after the infectious blood feed, saliva was then collected from mosquitoes in a manner similar to previously published methods (17 (link), 26 (link), 33 ). In brief, ORL and COL mosquitoes were cold anesthetized, and their wings and legs were removed with sterile forceps. Each mosquito was then fastened to a glass microscope slide with tape, and its proboscis was inserted into a graduated glass capillary tube (Drummond, Broomall, PA) filled with 3 μl of mineral oil (ORL only) or 1:1 hematocrit-HI human serum (ORL and COL) (Fig. 5A and C). Mosquitoes were then placed in a rearing chamber under the conditions described above for 45 min or until they ingested approximately 2 μl of blood. Each proboscis was then removed from its capillary tube, and the blood from each capillary tube was aspirated into 1.5-ml microcentrifuge tubes with 200 μl of reduced DMEM. All samples were immediately stored at −80°C until use.