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

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.

In the original protocol published in 2015, a transgenic reporter parasite line was used. Gametocyte production was increased by shifting parasite cultures to a spent ‘conditioned’ medium. In this Protocol Update, we use newly developed transgenic reporter parasite lines and gametocyte production is increased via nutrient depletion by shifting the parasite cultures to a defined minimal fatty acid medium.

We have established the use of Tubulin Tracker Deep Red in combination with the reporter line as versatile tools for quantification of both sexual commitment and conversion in the same experimental setup.

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All data shown in figures were generated or analyzed for this Protocol, and source data are included as Supplementary information. Source data are provided with this paper.

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This work was supported by Swiss National Science Foundation grant no. 310030-207789 (M.M. and K.V.), European Research Council Consolidator award BoneMalar (M.M. and K.V.), Royal Society Wolfson Merit Award (M.M.), Wellcome Trust Center award 104111 (M.M. and K.V.), Swiss National Science Foundation grant no. 310030_200683 (N.M.B.B. and B.T.T.) and the Secretaría Nacional de Ciencia, Tecnología e Innovación (SENACYT) grant no. DDCCT 204-2023 (E.D.A.B. and L.C.).

Institute of Parasitology, Vetsuisse and Medical Faculties, University of Zurich, Zurich, Switzerland

Kannan Venugopal, Eveline D. A. Borbon & Matthias Marti

School of Infection and Immunity, University of Glasgow, Glasgow, UK

Institute of Scientific Research and High Technology Services of Panama (INDICASAT), Panama City, Panama

Swiss Tropical and Public Health Institute, Allschwil, Switzerland

Basil T. Thommen & Nicolas M. B. Brancucci

K.V.: conceptualization, data curation, methodology, investigation and writing—original draft and editing. E.D.A.B.: investigation. B.T.T.: investigation and writing—review and editing. N.M.B.B.: supervision and writing—review and editing. L.C.: supervision and funding acquisition. M.M.: conceptualization, supervision, funding acquisition and writing—original draft and editing.

Correspondence to Kannan Venugopal or Matthias Marti.

The authors declare no competing interests.

Nature Protocols thanks Felix Müller, Gabriele Pradel and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Venugopal, K. et al. Mol. Cell. Proteomics 25, 101505 (2026): https://doi.org/10.1016/j.mcpro.2025.101505

Watzlowik, M. T. et al. Nature 639, 1069–1075 (2025): https://doi.org/10.1038/s41586-025-08595-x

Brancucci, N. M. B. et al. Cell 171, 1532–1544.e15 (2017): https://doi.org/10.1016/j.cell.2017.10.020

This protocol is an update to Nat. Protoc. 10, 1131–1142 (2015): https://doi.org/10.1038/nprot.2015.072

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Venugopal, K., Borbon, E.D.A., Thommen, B.T. et al. An assay to quantify sexual commitment and stage conversion in the human malaria parasite Plasmodium falciparum. Nat Protoc (2026). https://doi.org/10.1038/s41596-026-01378-2

DOI: https://doi.org/10.1038/s41596-026-01378-2

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