Intraerythroblastic gametocyte development occurs from the polychromatic stage. (A) Morphological analysis of developing erythroblasts was performed by May-Grünwald Giemsa staining from 0 dod to 8 dod; a representative experiment is shown. Erythroblasts were primarily at the proerythroblast stage (PRO-E) at 0 dod, the basophilic stage (BASO) at 2 dod, the polychromatic stage (POLY) at 4 dod, the orthochromatic stage (ORTHO) at 6 dod, and the reticulocyte stage (RETIC) at 8 dod. The red boxes (upper panels) are shown at higher magnification (lower panels). Scale bars, 5 μm. (B) Representative experiment of erythroblast differentiation monitored by flow cytometry from 0 dod to 8 dod by cell surface expression of Band 3 and α-4-integrin. (C) Evolution of cell surface markers CD71, Band 3, GPA, and α-4-integrin and of reticulocyte rate measured by flow cytometry during terminal erythropoiesis. (D) Diagram illustrating the infection protocol of synchronized erythroblasts. At 40 hpi, synchronized schizonts were added to proerythroblasts, as well as basophilic, polychromatic, and orthochromatic erythroblasts, at an MOI of 2. Infected erythroblasts were analyzed by flow cytometry at 2, 4, and 6 dpi. Total parasitemia (E) and gametocytemia (F) in erythroblasts was evaluated by flow cytometry at 2 dpi, 4 dpi, and 6 dpi after infection of erythroblasts from each stage with the Hsp70-GFP and Pfs16-GFP lines. Circles indicate the number of independent experiments, and error bars show the standard error of the mean.