Asexual growth rates were determined in groups of six BALB/c mice injected intravenously with 1000 parasites. Parasite growth was monitored on Giemsa-stained thin blood films, counting parasites daily in 1,000–40,000 erythrocytes until mice developed severe disease.
To screen mutants for their ability to undergo sexual development and sporogony, groups of 3–4 outbred Theiler's Original mice were infected with 5 × 106 mutant or wild-type parasites intraperitoneally and monitored for gametocyte production on Giemsa-stained thin blood films. On day 3–4 p.i., blood samples from each mouse were taken from the tail and mixed with gametocyte-activating medium. Short-term cultures were set up to quantify exflagellation of microgametocytes (after 10 min by phase contrast microscopy) and macrogamete-to-ookinete conversion (at 24 hr after fluorescence labeling of the activation marker P28). Each mouse was then anesthetized, and ∼100 female Anopheles stephensi mosquitoes were allowed to feed for 20 min. Unfed mosquitoes were removed the following day. Batches of infected mosquitoes were dissected at different time points after feeding to monitor parasite development. On day 10, around 20 midguts were individually inspected by phase contrast microscopy, and oocysts were counted. On day 14, around 20 midguts were homogenized gently to release sporozoites, which were then counted in a hemocytometer and expressed as average per mosquito. On day 21 after feeding, this procedure was repeated with salivary glands. After 21 days, any remaining infected mosquitoes were allowed to feed on 3 or 4 naive C57BL/6 mice in groups of 5–10. These mice, which are highly susceptible to sporozoite challenge, were then monitored daily from day 4 to day 15 after feeding for blood infections to determine if mutants could complete the life cycle. SeeSupplemental Experimental Procedures for details of phenotyping assays.
To screen mutants for their ability to undergo sexual development and sporogony, groups of 3–4 outbred Theiler's Original mice were infected with 5 × 106 mutant or wild-type parasites intraperitoneally and monitored for gametocyte production on Giemsa-stained thin blood films. On day 3–4 p.i., blood samples from each mouse were taken from the tail and mixed with gametocyte-activating medium. Short-term cultures were set up to quantify exflagellation of microgametocytes (after 10 min by phase contrast microscopy) and macrogamete-to-ookinete conversion (at 24 hr after fluorescence labeling of the activation marker P28). Each mouse was then anesthetized, and ∼100 female Anopheles stephensi mosquitoes were allowed to feed for 20 min. Unfed mosquitoes were removed the following day. Batches of infected mosquitoes were dissected at different time points after feeding to monitor parasite development. On day 10, around 20 midguts were individually inspected by phase contrast microscopy, and oocysts were counted. On day 14, around 20 midguts were homogenized gently to release sporozoites, which were then counted in a hemocytometer and expressed as average per mosquito. On day 21 after feeding, this procedure was repeated with salivary glands. After 21 days, any remaining infected mosquitoes were allowed to feed on 3 or 4 naive C57BL/6 mice in groups of 5–10. These mice, which are highly susceptible to sporozoite challenge, were then monitored daily from day 4 to day 15 after feeding for blood infections to determine if mutants could complete the life cycle. See
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