PyNOT1-G is essential to the formation of male gametes.

(A) The blood stage growth of Py17XNL wild-type parasites and 2 independent clones of pynot1-g⁻ parasites was compared over the entire course of infection in biological triplicate, with each replicate done in technical triplicate. Maximal parasitemia of pynot1-g⁻ parasites was lower and resulted in slightly faster clearance by the mouse. (B) The number of gametocytes produced by Py17XNL WT-GFP parasites and 2 independent clones of pynot1-g⁻ parasites was measured by flow cytometry using the presence of GFP fluorescence in biological duplicate, with each replicate done in technical triplicate. Average values for each biological replicate, their median, and range are provided. Statistical comparisons used an unpaired t test with Welch correction. (C) Male gametogenesis was measured by DIC microscopy to count the number of exflagellation centers (“centers of movement”) per microscopic field using a 10× eyepiece and 40× objective lens. The same mice and time points were used as in panel A and were assessed in biological triplicate, with each replicate done in technical triplicate. A cross symbol (A, C) indicates that a single mouse infected with Py17XNL wild-type parasites was euthanized on day 15 due to parasitemia more than 10% as required by our approved vertebrate animal protocols. Error bars indicate the standard deviation. Underlying data are provided in S1 Data. DIC, differential interference contrast.