2.2 Mosquito rearing and experimental infections
Both experiments were conducted with a wild lineage of Culex pipiens mosquitoes, the main vector of Plasmodium relictum in Europe, collected in Lausanne (Switzerland) and maintained in insectary since August 2017. Mosquito eggs used in the experiments were obtained by feeding 30-40 females on one healthy bird (Serinus canaria ). All eggs were then placed in the same plastic tank. One day post-hatching, larvae were haphazardly collected and randomly assigned to the different treatment groups (see below). Larvae were reared individually in plastic tube (30mL) filled with 6 mL of solution (see below). Larval mortality and development time were daily recorded until adult emergence. Immediately after emergence, mosquitoes belonging to the same treatment were placed inside an adult rearing cage. Males and females were kept together and fed ad libitum on a 10% glucose water solution during 5 ± 2 days.
Afterwards, fifteen females from each experimental treatment were placed together inside a feeding cage (2 and 3 different cages for experiment 1 and 2, respectively) and allowed to feed from aPlasmodium -infected bird for 3 hours. Engorged females were then taken out from the cages, briefly anesthetized with CO2 and placed individually into numbered dry 30ml plastic tubes covered with a mesh. Food was provided in the form of a cotton pad soaked in a 10% glucose solution placed on top of each tube. To distinguish between the treatments, mosquitoes were previously marked using different fluorescent color powders and the color allocated to each treatment changed in each of the cages (for details see Vézilier et al., 2010). Five days post blood meal, females were taken out of the tubes and placed in new plastic tubes filled with 4mL of water to encourage them to lay eggs. The amount of haematin excreted at the bottom of each first tube was quantified as an estimate of the blood meal size (Vézilier et al., 2010). Three days later (day 8 post blood meal), females were taken out of the tubes. The egg-rafts were collected and the number of eggs was counted under a binocular microscope. One wing was removed from each female and measured under a binocular microscope along its longest axis as an index of body size (Van Handel and Day, 1989). Females were then dissected and the number of Plasmodium parasite (oocyst stage) presents in their midguts were counted under a binocular microscope. The wing size was also measured for all males.