Figure 1. Gametocyte development in the transgenic 164/TdTomato line.

Shown are representative cells for each developmental stage as defined previously by Hawking et al (Hawking et al., 1971) and Sinden (Sinden, 1982). Stage I emerges 30 hours post invasion (pi). It is indistinguishable from the young asexual trophozoite by light microscopy. We defined Stage I parasite by shape and the presence of TdTomato fluorescence. Stage II emerges 48 –72 hours pi. We defined early Stage II parasites by the oat grain like morphology and later Stage II by the D-shaped morphology. Stage III emerges 3–5 days pi. We defined Stage III parasites by the relative elongation/flattened D-shape compared to late Stage II. Later Stage III parasites were defined by the presence of lightly pointed ends. Stage IV emerges 6–8 days pi. These parasites were defined by the spindle shape and axial symmetry with two pointed ends. Stage V emerges 10–14 days pi. This stage is mainly defined by the crescent female morphology, while the male Stage V gametocyte requires additional markers for unambiguous staging. Since male Stage V gametocytes only make up 15–20% of all Stage V gametocytes, we defined Stage V by the easily recognizable crescent shape of the female Stage V. For each cell an epifluorescence image with TdTomato distribution, differential interference contrast (DIC) and a merge of the two are shown. Images were collected at ambient temperature with a Zeiss Axioscope microscope using a 100×/1.4 immersion oil lens, and captured using a Hamamatsu Orca C4742-95 camera. Acquisition software Zeiss Axiovision was used.