Blood containing ~50,000 parasites of the PP1PTD line was injected intraperitoneally (i.p.) into mice to initiate infections. Asexual stages and gametocyte production were monitored by microscopy on Giemsa-stained thin smears. Four to five days post infection, exflagellation and ookinete conversion were examined with a Zeiss AxioImager M2 microscope (Carl Zeiss, Inc) fitted with an AxioCam ICc1 digital camera70 (link). To analyse mosquito transmission, 30–50 Anopheles stephensi SD 500 mosquitoes were allowed to feed for 20 min on anaesthetised, infected mice with an asexual parasitaemia of 15% and a comparable number of gametocytes as determined on Giemsa-stained blood films. To assess mid-gut infection, ~15 guts were dissected from mosquitoes on day 14 post feeding, and oocysts were counted on an AxioCam ICc1 digital camera fitted to a Zeiss AxioImager M2 microscope using a ×63 oil immersion objective. On day 21 post-feeding, another 20 mosquitoes were dissected, and their guts crushed in a loosely fitting homogenizer 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.
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