Plasmodium Transmission Dynamics Evaluation

Blood samples containing approximately 50,000 parasites of the ark2-knockdown/eb1 knockout lines were injected intraperitoneally (i.p.) into mice to initiate infection. Asexual stages and gametocyte production were monitored by microscopy on Giemsa-stained thin smears. Four to 5 days post infection, exflagellation and ookinete conversion were examined as described previously^{52 (link)} with a Zeiss AxioImager M2 microscope (Carl Zeiss, Inc.) fitted with an AxioCam ICc1 digital camera. To analyse mosquito infection and transmission, 30 to 50 Anopheles stephensi SD 500 mosquitoes were allowed to feed for 20 min on anaesthetised, infected mice with at least 15% asexual parasitaemia and carrying comparable numbers of gametocytes as determined on Giemsa-stained blood films. To assess midgut infection, ~15 guts were dissected from mosquitoes on days 7 and 14 post feeding and oocysts were counted using a ×63 oil immersion objective. On day 21 post feeding, another 20 mosquitoes were dissected, and their guts and salivary glands crushed separately in a loosely fitting homogeniser to release sporozoites, which were then quantified using a haemocytometer or used for imaging. Mosquito bite-back experiments were performed 21 days post feeding using naive mice, and blood smears were examined after 3–4 days.