Fig 3. Phenotypic analyses of blood stage mutant P. berghei parasites.

A. in vivo growth assay of mutants in mixed infections in competition with wild type parasites over 12 days. Coloured lines represent non-linear fit of percentage of mutant parasites in total parasite population. Data representative of n = 3 independent biological replicates. (Also see Fig C- A, B and C in S1 Text.) B. Lethality experiment in C57/B6 mice by wt and mutant P. berghei parasites. 10⁴ parasites were injected intra-peritoneally in mice (n = 5) on day 0 and they were monitored for 21 days. The mice were culled humanely when they showed severe malaria pathology. All mutant parasites were found to be lethal to mice. C. Gametocyte conversions during blood stages in mutant P. berghei parasites over 5 days post infection. Data from 2 independent observed gametocyte conversion experiments are shown ± S.D. Gametocyte conversion was observed using a wt parent line which expresses GFP in male gametocytes and RFP in female gametocytes (RMgm-164). P. berghei mutants were generated in the same genetic background and analysed using FACS determining the number of gametocytes in infected blood. P-values: *p<0.05, **p<0.005, ***p<0.0005, paired two tailed t-test. D. Exflagellation (male gamete formation) in mutant P. berghei parasites normalised to wt in in vitro activation assay. The error is given as the SD of n = 3 independent biological replicates. P-values: **p<0.005, ***p<0.0005, paired two tailed t-test. E. Determination of DNA content of male gametocytes over 20 minutes post activation by FACS analysis in mutant P. berghei parasites normalised to wt. DNA content was determined in Hoechst-33258-stained MACS purified gametocytes. Before activation (0minutes) males show low DNA content with increasing amounts post activation reaching maximum levels between 8 to 12 minutes in wt. Data from 3 independent biological replicates are given ± S.D. P-values: **p<0.005, ***p<0.0005, unpaired two tailed t-test (also see Fig C- D in S1 Text).