Figure 5: gABCG2-knockout parasite lines show increased gametocyte production.

(a) Numbers of RBCs infected with 3D7-wild-type and clonal-knockout (ΔgABCG2-07 and ΔgABCG2-42) parasites at different stages were counted using a flow cytometer. At least 1,000 infected RBCs per experiment were counted. Knockout parasite numbers relative to the wild-type numbers are shown. III–V, gametocytes stage 3 to stage 5. Gm, gametes. Means and ±s.d. of three independent experiments are shown. ***P<0.001 (unpaired t-test). (b) The sex ratio of gABCG2-07-disrupted parasites is unaltered. Mature male and female gametocytes of 3D7-wild-type and -knockout (ΔgABCG2-07 and ΔgABCG2-42) cell lines were differentiated using morphological characteristics of mature gametocytes in Giemsa-stained thin smears⁶⁹. At least 500 gametocytes per experiment were counted by two independent microscopists from de-identified samples. Means and ±s.d. of three independent experiments are shown. (c) Expression of gABCG2 under the control of the endogenous promoter results in gametocyte production reverting to close to that in wild-type parasites. The numbers of RBCs infected with 3D7-wild-type, -knockout ΔgABCG2-07, untagged endogenous complementation (pLUX-6/endo-ΔPfgABCG2-07) and GFP-tagged endogenous complementation (pGLUX-6/endo-ΔPfgABCG2-07) parasites at different stages were counted by flow cytometer. At least 1,000 infected RBCs per experiment were counted. Transgenic parasite numbers relative to the wild-type numbers are shown. III–V, gametocytes stage 3 to stage 5. Gm, gametes. Means and ±s.d. of three independent experiments are shown. (d) Live-cell fluorescence microscopy of pGLUX-6/endo-ΔPfgABCG2-07 transfectant was performed on late-stage gametocyte. BF, bright field. Scale bar, 5 μm.