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BackgroundMalaria elimination/eradication campaigns emphasize interruption of parasite transmission as a priority strategy. Screening for new drugs and vaccines against gametocytes is therefore urgently needed. However, current methods for sexual stage drug assays, usually performed by counting or via fluorescent markers are either laborious or restricted to a certain stage. Here we describe the use of a transgenic parasite line for assaying drug sensitivity in all gametocyte stages.MethodsA transgenic parasite line expressing green fluorescence protein (GFP) under the control of the gametocyte-specific gene α-tubulin II promoter was generated. This parasite line expresses GFP in all gametocyte stages. Using this transgenic line, we developed a flow cytometry-based assay to determine drug sensitivity of all gametocyte stages, and tested the gametocytocidal activities of four antimalarial drugs.FindingsThis assay proved to be suitable for determining drug sensitivity of all sexual stages and can be automated. A Z' factor of 0.79 ± 0.02 indicated that this assay could be further optimized for high-throughput screening. The daily sensitivity of gametocytes to three antimalarial drugs (chloroquine, dihydroartemisinin and pyronaridine) showed a drastic decrease from stage III on, whereas it remained relatively steady for primaquine.ConclusionsA drug assay was developed to use a single transgenic parasite line for determining drug susceptibility of all gametocyte stages. This assay may be further automated into a high-throughput platform for screening compound libraries against P. falciparum gametocytes.

In vitro culture of Plasmodium falciparum gametocytes is essential for studying sexual development of the parasite. Here we describe a simple method for producing synchronous gametocyte culture without contamination of asexual stages. This method employs heparin's activity in blocking merozoite invasion of erythrocytes to eliminate asexual stage parasites from gametocyte culture. We show that following induction of gametocyte formation, addition of heparin in culture medium for four days effectively eliminates asexual stages and produces pure, synchronous cultures of gametocytes. Compared with the commonly used N-acetylglucosamine treatment method, heparin treatment requires shorter time to eliminate asexual stages and causes significantly less hemolysis in late stage gametocyte cultures.

Translational control of gene expression plays an essential role in development. In malaria parasites, translational regulation is critical during the development of specialized transition stages between the vertebrate host and mosquito vector. Here we show that a Pumilio/FBF (Puf) family RNA-binding protein, PfPuf2, is required for the translation repression of a number of transcripts in gametocytes including two genes encoding the transmission-blocking vaccine candidates Pfs25 and Pfs28. Whereas studies to date support a paradigm of Puf-mediated translation regulation through 3' untranslated regions (UTRs) of target mRNAs, this study, for the first time, identifies a functional Puf-binding element (PBE) in the 5'UTR of pfs25. We provide both in vitro and in vivo evidence to demonstrate that PfPuf2 binds to the PBEs in pfs25 and pfs28 to mediate translation repression. This finding provides a renewed view of Pufs as versatile translation regulators and sheds light on their functions in the development of lower branches of eukaryotes.