Artificial feeder

Most experiments were carried out with Aedes aegypti mosquitoes derived from a wild population originally sampled in 2013 in Thep Na Korn, Thailand and took place within 10 generations of laboratory colonization. One experiment was carried out with Ae. aegypti mosquitoes derived from a wild population originally sampled in 2015 in Phnom Penh City, Cambodia and took place 8 generations after laboratory colonization. Experimental infections were carried out as previously described [60 (link)]. Briefly, four- to seven-day-old females were offered a washed rabbit erythrocyte suspension mixed 2:1 with pre-diluted DENV-1 KDH0030A viral stock and supplemented with 10 mM ATP (Sigma), to reach an expected titer of 10⁷ FFU/mL. A control blood meal was prepared with the supernatant of mock-inoculated C6/36 cells. Mosquitoes were allowed to blood feed for 30 min through a pig-intestine membrane using an artificial feeder (Hemotek Ltd, Blackburn, UK) set at 37°C. Samples of the blood meals were saved and stored at -80°C for further titration. Fully engorged females were incubated at 28°C, 70% relative humidity and under a 12-hour light-dark cycle in 1-pint cardboard cups (20–30 females per cup, at least 2 cups/condition) with permanent access to 10% sucrose.

Three-day-old female sand flies and private of sugar solution food for a period of 24h were used in this study. The insects were fed with heparinized blood through chick skin membrane attached in an artificial feeder (Hemotek). The artificial infection was performed with blood containing VSV or a control without virus at 37°C. Fully engorged females were selected and harvested individually at 2, 4 and 6 dpf. Insects were anesthetized with carbon dioxide and directly ground in TRIzol (Invitrogen) using glass beads as previously described [23 (link)].

Adult mosquitoes were kept in cages with free access to sugar solution (10%) until they were 5–6 days old. One day before virus experimental infection, female mosquitoes were deprived from sugar solution and transferred to infection cages (8 cm height, 6 cm diameter). The blood meal (1 mL of erythrocytes, 1 mL of virus suspended in L15 medium for infected groups; 1 mL of erythrocytes, 1 mL of L15 medium for uninfected groups) was offered using an artificial feeder (Hemotek, Great Hardwood, UK) at 37 °C for approximately 30 min. Only mosquitoes which were visually blood-engorged proceeded to further investigations. Aedes aegypti used in fitness experiments (longevity, fecundity and fertility evaluation) were individualized in plastic vials (6.5 cm height, 2.5 cm diameter) [33 (link),36 (link),37 (link)]. Females used for the egg quiescence assessment were transferred to cylindrical cages (25 cm height, 20 cm diameter) with oviposition cups. In both experiments, mosquitoes were kept inside an incubator (26 ± 1 °C, 75% ± 5 humidity) with free access to sugar solution (10%).