Mosquito Infection Profiling Protocol

For the expression profiling experiments, two groups of mosquitoes from the same rearing culture were used per replicate: the test group fed on blood donated by a gametocyte carrier, whereas the control group fed on the same blood which was previously incubated at 42–43°C for 12 min under constant shaking at 500 rpm for gametocyte inactivation. For the gene silencing experiments, we also used two groups of freshly emerged female mosquitoes per gene and per replicate, also separated randomly from the same rearing culture in small containers. The first group was subjected to silencing of the examined gene whereas the second control group was injected with dsRNA of the LacZ gene. In both cases, the two groups were housed under the same microclimate and treated identically, both before and after the blood feeding.
Mosquitoes were allowed to feed via a membrane on blood donated by P. falciparum gametocyte carries. To eliminate transmission blocking immunity factors, the carrier serum was replaced by non-immune AB serum [51] (link). Blood samples (700 µl each) were transferred into pre-warmed (37°C) artificial membrane feeders and exposed to mosquitoes that were previously starved for 12 hours, according to standard procedures [21] (link). To determine the levels of infection, mosquito midguts were dissected 8–10 days post blood feeding and stained with 2% mercurochrome before microscopic examination.