Fig 1: Functional characterization of a gene critical for gametocyte development. (A) Gametocytemia was determined in Giemsa-stained blood films on day 3 p.i. Compared with wild-type (WT) 17XNL parasites, the Pyap2-g3 KO parasites produced significantly fewer male and female gametocytes. Mean values and standard errors of the means (SEM), shown by error bars, were calculated from the results for three infected mice. (B) Total numbers of oocysts per mosquito on day 8 postfeeding with WT or Pyap2-g3 KO parasites. Mean values and SEM were calculated from the results for 30 mosquito midguts. (C) Numbers of salivary gland (SG) sporozoites per infected mosquito on day 14 postfeeding. Mean values and SEM were calculated from the results of three independent experiments with 20 to 25 mosquitos each. (D) Levels of parasitemia caused by WT and Pyap2-g3 KO parasites on days 2 to 10 postinfection. (E and F) Relative transcript levels in blood stages of WT, Pyap2-g3 KO, or Pyap2-g KO parasites detected using quantitative reverse transcription (qRT)-PCR; signals were normalized against those for the 18S rRNA gene. The results are representative of two independent Pyap2-g3 KO clones. (A to C and E to F) The results are representative of three independent experiments. (D) The data are representative of three mice. P values were determined using two-sided unpaired t test (B and C) or Mann-Whitney test (A and E to F). **, P < 0.01; ***, P < 0.001. (G) Immunofluorescence assay (IFA) of PyAP2-G3 protein expression in various developmental stages detected using anti-HA antibody (rabbit monoclonal antibody 3724; Cell Signaling Technology, Inc., Danvers, MA). DIC, differential interference contrast. Scale bar = 5 µM. (H) Detection of HA-tagged PyAP2-G3 expressed in lysates of blood stages. G3:HA, 6×HA-tagged PyAP2-G3. Anti-BiP antibody was included as the protein loading control.