- Introduction
- High-Yield Ookinete Production
- Purification of P. Falciparum Sexual Stage Parasites
- Pfs25 and Chitinase (PfCHT1) Detection in Gametocytes and Ookinetes
- Ultrastructural Analysis of P. Falciparum Sexual Stage Parasites
- P. falciparum gametocyte cultures.
- Exflagellation and emergence assays.
- Leukostat-stained light microscopy.
- Immunofluroescence of cultured parasites.
- TEM of cultured parasites.
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In Vitro Generation of Plasmodium falciparum Ookinetes
Plasmodium transmission from the human host to the mosquito depends on the ability of gametocytes to differentiate into ookinetes, the invasive form of the parasite that invades and establishes infection in the mosquito midgut. The biology of P. falciparum ookinetes is poorly understood, because sufficient quantities of this stage of this parasite species have not been obtained for detailed study. This report details methods to optimize production of P. falciparum sexual stage parasites, including ookinetes. Flow cytometric sorting was used to separate diploid/tetraploid zygotes and ookinetes from haploid gametetocytes and unfertilized gametes based on DNA content. Consistent production of 106–107 P. falciparum ookinetes per 10 mL culture was observed, with ookinete transformation present in 10–40% of all parasite forms. Transmission electron micrographs of cultured parasites confirmed ookinete development.
Author Notes
Authors' addresses: Viengngeun Bounkeua, Fengwu Li, and Joseph M. Vinetz, Division of Infectious Diseases, Department of Medicine, University of California San Diego, La Jolla, CA.