An assay to quantify sexual commitment and stage conversion in the human malaria parasite Plasmodium falciparum

Abstract

Sexual commitment and stage conversion are two crucial steps in the blood stage cycle of the human malaria parasite, as they mark its initial decision and subsequent differentiation in preparation for transmission to the mosquito vector. Here we present a Protocol Update that allows tracking and quantification of sexual commitment (in the asexual replicative parent, schizont stage) and conversion (in the nonreplicative sexual progeny, gametocyte stage) rates in the human malaria parasite Plasmodium falciparum. For this purpose, we combine three critical tools we recently established and validated. First, we use a standardized minimal medium to efficiently and reproducibly increase sexual commitment. Second, we use Tubulin Tracker Deep Red to label developing gametocytes and quantify the sexual conversion rate in reporter and reporter-free lines. Third, we use a fluorescent transgenic reporter line for consecutive quantification of sexual commitment and stage conversion in the same experimental procedure. These tools enable multiple experimental readouts including flow cytometry, fixed and live cell microscopy (including high content imaging) and other applications such as fluorescence-activated cell sorting. We anticipate that the presented sexual commitment–conversion assay will enable novel screening approaches for the identification of transmission blocking antimalarials and critical knowledge gaps in the Plasmodium transmission cycle to be closed. The Protocol can be completed in 7 d from start to finish. This Protocol Update uses new transgenic reporter parasite lines and synchronized gametocyte production by shifting cultures to a defined medium. Tubulin Tracker Deep Red in combination with the reporter line enables the quantification of both sexual commitment and conversion in the same experiment.