Protocol | Published:

An assay to probe Plasmodium falciparum growth, transmission stage formation and early gametocyte development

Nature Protocols volume 10, pages 11311142 (2015) | Download Citation


Conversion from asexual proliferation to sexual differentiation initiates the production of the gametocyte, which is the malaria parasite stage required for human-to-mosquito transmission. This protocol describes an assay designed to probe the effect of drugs or other perturbations on asexual replication, sexual conversion and early gametocyte development in the major human malaria parasite Plasmodium falciparum. Synchronized asexually replicating parasites are induced for gametocyte production by the addition of conditioned medium, and they are then exposed to the treatment of interest during sexual commitment or at any subsequent stage of early gametocyte development. Flow cytometry is used to measure asexual proliferation and gametocyte production via DNA dye staining and the gametocyte-specific expression of a fluorescent protein, respectively. This screening approach may be used to identify and evaluate potential transmission-blocking compounds and to further investigate the mechanism of sexual conversion in malaria parasites. The full protocol can be completed in 11 d.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


  1. 1.

    et al. A research agenda to underpin malaria eradication. PLoS Med. 8, e1000406 (2011).

  2. 2.

    , , , & Effects of pyrimethamine-sulphadoxine, chloroquine plus chlorpheniramine, and amodiaquine plus pyrimethamine-sulphadoxine on gametocytes during and after treatment of acute, uncomplicated malaria in children. Mem. Inst. Oswaldo Cruz 101, 887–893 (2006).

  3. 3.

    & Plasmodium falciparum gametocytaemia in Nigerian children: before, during and after treatment with antimalarial drugs. Trop. Med. Int. Health 8, 783–792 (2003).

  4. 4.

    , & Intermittent preventive treatment during pregnancy with sulphadoxine-pyrimethamine may promote Plasmodium falciparum gametocytogenesis. Med. Princ. Pract. 21, 63–67 (2012).

  5. 5.

    , , & Chloroquine increases Plasmodium falciparum gametocytogenesis in vitro. Parasitology 118 (Part 4), 339–346 (1999).

  6. 6.

    et al. Risk factors for gametocyte carriage in uncomplicated falciparum malaria. Am. J. Trop. Med. Hyg. 60, 1019–1023 (1999).

  7. 7.

    & Effects of antimalarial molecules on the gametocyte stage of Plasmodium falciparum: the debate. J. Med. Chem. 55, 10328–10344 (2012).

  8. 8.

    et al. Enhanced transmission of drug-resistant parasites to mosquitoes following drug treatment in rodent malaria. PLoS ONE 7, e37172 (2012).

  9. 9.

    World Health Organization. WHO Malaria Report 2014. (2014).

  10. 10.

    et al. A high-throughput screen targeting malaria transmission stages opens new avenues for drug development. J. Infect. Dis. 203, 1445–1453 (2011).

  11. 11.

    et al. Torins are potent antimalarials that block replenishment of Plasmodium liver stage parasitophorous vacuole membrane proteins. Proc. Natl. Acad. Sci. USA 110, E2838–E2847 (2013).

  12. 12.

    et al. Drug screen targeted at Plasmodium liver stages identifies a potent multistage antimalarial drug. J. Infect. Dis. 205, 1278–1286 (2012).

  13. 13.

    et al. A transcriptional switch underlies commitment to sexual development in malaria parasites. Nature 507, 248–252 (2014).

  14. 14.

    et al. A cascade of DNA-binding proteins for sexual commitment and development in Plasmodium. Nature 507, 253–257 (2014).

  15. 15.

    et al. A Plasmodium falciparum histone deacetylase regulates antigenic variation and gametocyte conversion. Cell Host Microbe 16, 177–186 (2014).

  16. 16.

    , , & A var gene upstream element controls protein synthesis at the level of translation initiation in Plasmodium falciparum. PLoS ONE 9, e100183 (2014).

  17. 17.

    & The role of extracellular vesicles in Plasmodium and other protozoan parasites. Cell. Microbiol. 16, 344–354 (2014).

  18. 18.

    et al. Cell-cell communication between malaria-infected red blood cells via exosome-like vesicles. Cell 153, 1120–1133 (2013).

  19. 19.

    & Evidence for environmental modulation of gametocytogenesis in Plasmodium falciparum in continuous culture. Bull. World Health Organ. 57 (suppl. 1), 37–52 (1979).

  20. 20.

    & Expression of Plasmodium falciparum trimeric G proteins and their involvement in switching to sexual development. Mol. Biochem. Parasitol. 108, 67–78 (2000).

  21. 21.

    , , & Temporal evaluation of commitment to sexual development in Plasmodium falciparum. Malar. J. 12, 134 (2013).

  22. 22.

    & Plasmodium falciparum gametocyte formation in vitro: its stimulation by phorbol diesters and by 8-bromo cyclic adenosine monophosphate. J. Protozool. 36, 451–454 (1989).

  23. 23.

    Stage-specific inhibitory effect of cyclic AMP on asexual maturation and gametocyte formation of Plasmodium falciparum. J. Parasitol. 69, 592–597 (1983).

  24. 24.

    & Comparison of adenylate cyclase and cAMP-dependent protein kinase in gametocytogenic and nongametocytogenic clones of Plasmodium falciparum. J. Parasitol. 77, 346–352 (1991).

  25. 25.

    , & Endoplasmic reticulum stress triggers gametocytogenesis in the malaria parasite. J. Biol. Chem. 289, 16662–16674 (2014).

  26. 26.

    et al. Stress and sex in malaria parasites: why does commitment vary? Evol. Med. Public Health 2013, 135–147 (2013).

  27. 27.

    , , , & Sex: how malaria parasites get turned on. Curr. Biol. 24, R368–R370 (2014).

  28. 28.

    , , , & Flow cytometry for the evaluation of anti-plasmodial activity of drugs on Plasmodium falciparum gametocytes. Malar. J. 9, 49 (2010).

  29. 29.

    , , , & Gametocytocidal screen identifies novel chemical classes with Plasmodium falciparum transmission blocking activity. PLoS ONE 9, e105817 (2014).

  30. 30.

    , , , & A high-throughput assay for the identification of drugs against late-stage Plasmodium falciparum gametocytes. Mol. Biochem. Parasitol. 180, 127–131 (2011).

  31. 31.

    et al. Activity of clinically relevant antimalarial drugs on Plasmodium falciparum mature gametocytes in an ATP bioluminescence 'transmission blocking' assay. PLoS ONE 7, e35019 (2012).

  32. 32.

    et al. A quantitative high throughput assay for identifying gametocytocidal compounds. Mol. Biochem. Parasitol. 188, 20–25 (2013).

  33. 33.

    et al. A Plasmodium falciparum screening assay for anti-gametocyte drugs based on parasite lactate dehydrogenase detection. J. Antimicrob. Chemother. 68, 2048–2058 (2013).

  34. 34.

    , , , & Identification of MMV malaria box inhibitors of Plasmodium falciparum early-stage gametocytes using a luciferase-based high-throughput assay. Antimicrob. Agents Chemother. 57, 6050–6062 (2013).

  35. 35.

    et al. A flow cytometry-based quantitative drug sensitivity assay for all Plasmodium falciparum gametocyte stages. PLoS ONE 9, e93825 (2014).

  36. 36.

    et al. Genome-wide identification of genes upregulated at the onset of gametocytogenesis in Plasmodium falciparum. Mol. Biochem. Parasitol. 143, 100–110 (2005).

  37. 37.

    et al. Host cell deformability is linked to transmission in the human malaria parasite Plasmodium falciparum. Cell. Microbiol. 14, 983–993 (2012).

  38. 38.

    & Age-dependent increase in green autofluorescence of blood erythrocytes. J. Biosci. 32, 1139–1145 (2007).

  39. 39.

    et al. Antibody recognition of heterologous variant surface antigens after a single Plasmodium falciparum infection in previously naive adults. Am. J. Trop. Med. Hyg. 76, 860–864 (2007).

  40. 40.

    et al. Evaluation of the antigenic diversity of placenta-binding Plasmodium falciparum variants and the antibody repertoire among pregnant women. Infect. Immun. 78, 1963–1978 (2010).

  41. 41.

    , & A simple statistical parameter for use in evaluation and validation of high-throughput screening assays. J. Biomol. Screen. 4, 67–73 (1999).

  42. 42.

    , , , & Methods in Malaria Research 6th edn. (MR4/ATCC, 2013).

  43. 43.

    et al. Plasmodium falciparum: parasites defective in early stages of gametocytogenesis. Exp. Parasitol. 81, 227–235 (1995).

  44. 44.

    , , , & Histone deacetylases play a major role in the transcriptional regulation of the Plasmodium falciparum life cycle. PLoS Pathog. 6, e1000737 (2010).

Download references


We thank D. Ravel for critical reading of the manuscript. This work has been supported through a career development grant from the Burroughs Wellcome Fund and by US National Institutes of Health (NIH) grants R21 AI105328 and RC1AI086222 (M.M.). N.M.B.B. is supported by a postdoctoral fellowship from the Swiss National Science Foundation and K.B. was supported through a Feodor Lynen postdoctoral fellowship from the Alexander von Humboldt Foundation.

Author information

Author notes

    • Kathrin Buchholz

    Present address: Biochemistry and Molecular Biology, Justus Liebig University Giessen, Giessen, Germany.

    • Nicolas M B Brancucci
    •  & Ilana Goldowitz

    These authors jointly directed this work.


  1. Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, Boston, Massachusetts, USA.

    • Nicolas M B Brancucci
    • , Ilana Goldowitz
    • , Kathrin Buchholz
    • , Kristine Werling
    •  & Matthias Marti


  1. Search for Nicolas M B Brancucci in:

  2. Search for Ilana Goldowitz in:

  3. Search for Kathrin Buchholz in:

  4. Search for Kristine Werling in:

  5. Search for Matthias Marti in:


I.G., N.M.B.B. and M.M. wrote the manuscript, with input from all coauthors. K.B. and M.M. conceived and developed the originally published protocol. I.G. conceived and performed preliminary experiments and N.M.B.B. conceived and performed later experiments leading to improvements in the protocol. N.M.B.B. and K.W. generated the data shown in the figures. All authors revised the manuscript and contributed to interpretation of the results.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Matthias Marti.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1 and 2

About this article

Publication history



Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.