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.