The Standard Membrane Feeding Assay: Advances Using Bioluminescence

Part of the Methods in Molecular Biology book series (MIMB, volume 1325)


In preclinical development, the efficacy of agents with putative effects on Plasmodium transmission is determined using the standard membrane feeding assay (SMFA). Because the end-point of the SMFA is normally the enumeration of oocysts on the mosquito midgut, the assays reliance on mosquito dissections and microscopy makes it slow, labor-intensive, and subjective. Below, we describe a novel method of assessing the transmission of a Plasmodium falciparum strain expressing the firefly luciferase protein in the SMFA. The use of a transgenic parasite strain allows for the elimination of mosquito dissections in favor of a simple approach where whole mosquitoes are homogenized and examined directly for luciferase activity. Measuring the mean luminescence intensity of groups of individual or pooled mosquitoes provides comparable estimates of transmission reducing activity at 5–10-fold the throughput capacity of the standard microscopy based SMFA. This high efficiency protocol may be of interest to groups screening novel drug compounds, vaccine candidates, or sera from malaria exposed individuals for transmission reducing activity (TRA).

Key words

SMFA Mosquito feeding assay Oocysts Malaria, mosquitoes Transmission reducing activity Anopheles Plasmodium falciparum Infectivity Luminescence 


  1. 1.
    Moonen B, Cohen JM, Snow RW et al (2010) Operational strategies to achieve and maintain malaria elimination. Lancet 376:1592–1603PubMedCentralCrossRefPubMedGoogle Scholar
  2. 2.
    Vannice K, Brown G, Akanmori B, Moorthy V (2012) MALVAC 2012 scientific forum: accelerating development of second-generation malaria vaccines. Malar J 11:372PubMedCentralCrossRefPubMedGoogle Scholar
  3. 3.
    Okell L, Drakeley C, Ghani A, Bousema T, Sutherland C (2008) Reduction of transmission from malaria patients by artemisinin combination therapies: a pooled analysis of six randomized trials. Malar J 7:125PubMedCentralCrossRefPubMedGoogle Scholar
  4. 4.
    Shekalaghe S, Drakeley C, Gosling R et al (2007) Primaquine clears submicroscopic Plasmodium falciparum gametocytes that persist after treatment with sulphadoxine-pyrimethamine and artesunate. PLoS One 2:e1023PubMedCentralCrossRefPubMedGoogle Scholar
  5. 5.
    Adjalley SH, Johnston GL, Li T et al (2011) Quantitative assessment of Plasmodium falciparum sexual development reveals potent transmission-blocking activity by methylene blue. Proc Natl Acad Sci 108:E1214–E1223PubMedCentralCrossRefPubMedGoogle Scholar
  6. 6.
    The malERA Consultative Group on Drugs (2011) A research agenda for malaria eradication: drugs. PLoS Med 8:e1000402PubMedCentralCrossRefGoogle Scholar
  7. 7.
    Delves M, Plouffe D, Scheurer C et al (2012) The activities of current antimalarial drugs on the life cycle stages of Plasmodium: a comparative study with human and rodent parasites. PLoS Med 9:e1001169PubMedCentralCrossRefPubMedGoogle Scholar
  8. 8.
    Delves MJ, Ruecker A, Straschil U et al (2013) Male and female Plasmodium falciparum mature gametocytes show different responses to antimalarial drugs. Antimicrob Agents Chemother 57:3268–3274PubMedCentralCrossRefPubMedGoogle Scholar
  9. 9.
    Bousema T, Churcher TS, Morlais I, Dinglasan RR (2013) Can field-based mosquito feeding assays be used for evaluating transmission-blocking interventions? Trends Parasitol 29:53–59CrossRefPubMedGoogle Scholar
  10. 10.
    Outchkourov NS, Roeffen W, Kaan A et al (2008) Correctly folded Pfs48/45 protein of Plasmodium falciparum elicits malaria transmission-blocking immunity in mice. Proc Natl Acad Sci 105:4301–4305PubMedCentralCrossRefPubMedGoogle Scholar
  11. 11.
    Chowdhury DR, Angov E, Kariuki T, Kumar N (2009) A potent malaria transmission blocking vaccine based on codon harmonized full length Pfs48/45 expressed in Escherichia coli. PLoS One 4:e6352PubMedCentralCrossRefPubMedGoogle Scholar
  12. 12.
    Wu Y, Ellis RD, Shaffer D et al (2008) Phase 1 trial of malaria transmission blocking vaccine candidates Pfs25 and Pvs25 formulated with montanide ISA 51. PLoS One 3:e2636PubMedCentralCrossRefPubMedGoogle Scholar
  13. 13.
    Ouédraogo AL, Roeffen W, Luty AJF et al (2011) Naturally acquired immune responses to Plasmodium falciparum sexual stage antigens Pfs48/45 and Pfs230 in an area of seasonal transmission. Infect Immun 79(12):4957–4964PubMedCentralCrossRefPubMedGoogle Scholar
  14. 14.
    Bousema T, Sutherland CJ, Churcher TS et al (2011) Human immune responses that reduce the transmission of Plasmodium falciparum in African populations. Int J Parasitol 41:293–300PubMedCentralCrossRefPubMedGoogle Scholar
  15. 15.
    Roeffen W, Mulder B, Teelen K et al (1996) Association between anti-Pfs48/45 reactivity and P. falciparum transmission-blocking activity in sera from Cameroon. Parasite Immunol 18:103–109CrossRefPubMedGoogle Scholar
  16. 16.
    Bousema T, Drakeley C (2011) Epidemiology and Infectivity of Plasmodium falciparum and Plasmodium vivax gametocytes in relation to malaria control and elimination. Clin Microbiol Rev 24:377–410PubMedCentralCrossRefPubMedGoogle Scholar
  17. 17.
    Churcher TS, Blagborough AM, Delves M et al (2012) Measuring the blockade of malaria transmission—an analysis of the standard membrane feeding assay. Int J Parasitol 42:1037–1044CrossRefPubMedGoogle Scholar
  18. 18.
    van der Kolk M, de Vlas SJ, Saul A, van de Vegte-bolmer M, Eling WM, Sauerwein W (2005) Evaluation of the standard membrane feeding assay (SMFA) for the determination of malaria transmission-reducing activity using empirical data. Parasitology 130:13–22CrossRefPubMedGoogle Scholar
  19. 19.
    Miura K, Deng B, Tullo G et al (2013) Qualification of standard membrane-feeding assay with Plasmodium falciparum malaria and potential improvements for future assays. PLoS One 8:e57909PubMedCentralCrossRefPubMedGoogle Scholar
  20. 20.
    Delves M, Sinden R (2010) A semi-automated method for counting fluorescent malaria oocysts increases the throughput of transmission blocking studies. Malar J 9:35PubMedCentralCrossRefPubMedGoogle Scholar
  21. 21.
    Bell AS, Ranford-Cartwright LC (2004) A real-time PCR assay for quantifying Plasmodium falciparum infections in the mosquito vector. Int J Parasitol 34:795–802CrossRefPubMedGoogle Scholar
  22. 22.
    Janse CJ, Franke-Fayard B, Mair GR et al (2006) High efficiency transfection of Plasmodium berghei facilitates novel selection procedures. Mol Biochem Parasitol 145:60–70CrossRefPubMedGoogle Scholar
  23. 23.
    Delves MJ, Ramakrishnan C, Blagborough AM, Leroy D, Wells TNC, Sinden RE (2012) A high-throughput assay for the identification of malarial transmission-blocking drugs and vaccines. Int J Parasitol 42:999–1006CrossRefPubMedGoogle Scholar
  24. 24.
    Vaughan AM, Mikolajczak SA, Camargo N et al (2012) A transgenic Plasmodium falciparum NF54 strain that expresses GFP–luciferase throughout the parasite life cycle. Mol Biochem Parasitol 186:143–147CrossRefPubMedGoogle Scholar
  25. 25.
    Stone WJR, Churcher TS, Graumans W et al (2014) A scalable assessment of Plasmodium falciparum transmission in the standard membrane feeding assay using transgenic parasites expressing GFP-luciferase. J Infect Dis. doi: 10.1093/infdis/jiu271 PubMedGoogle Scholar
  26. 26.
    Stone WJR, Eldering M, van Gemert G-J et al (2013) The relevance and applicability of oocyst prevalence as a read-out for mosquito feeding assays. Sci Rep 3Google Scholar
  27. 27.
    Zollner GE, Ponsa N, Garman GW et al (2006) Population dynamics of sporogony for Plasmodium vivax parasites from western Thailand developing within three species of colonized Anopheles mosquitoes. Malar J 5:68PubMedCentralCrossRefPubMedGoogle Scholar
  28. 28.
    Ponnudurai T, Lensen AHW, Van Gemert GJA, Bensink MPE, Bolmer M, Meuwissen JHET (1989) Infectivity of cultured Plasmodium falciparum gametocytes to mosquitoes. Parasitology 98:165–173CrossRefPubMedGoogle Scholar
  29. 29.
    Ponnudurai T, Lensen AHW, Leeuwenberg ADEM, Meuwissen JHET (1982) Cultivation of fertile Plasmodium falciparum gametocytes in semi-automated systems. 1. Static cultures. Trans R Soc Trop Med Hyg 76:812–818CrossRefPubMedGoogle Scholar
  30. 30.
    Molina-Cruz A, Garver LS, Alabaster A et al (2013) The human malaria parasite Pfs47 gene mediates evasion of the mosquito immune system. Science 340:984–987CrossRefPubMedGoogle Scholar
  31. 31.
    Lensen AHW, van Druten J, Bolmer M, van Gemert GJA, Eling WM, Sauerwein R (1996) Measurment by membrane feeding of reduction in Plasmodium falciparum transmission induced by endemic sera. Trans R Soc Trop Med Hyg 90:20–22CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of Medical MicrobiologyRadboud University Medical CenterNijmegenThe Netherlands
  2. 2.Department of Immunology and InfectionLondon School of Hygiene and Tropical MedicineLondonUK

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