Figure 1. Purification of early gametocyte stages from cultures of 3D7a parasites. The top panel shows stage I gametocytes stained with ethidium bromide (blue) and the sexual parasite-specific anti-Pfs16 antibody (green). The majority of nucleated erythrocytes are also Pfs16 positive and therefore gametocytes. The lower panel shows a Giemsa– stained thin film of the same culture 2 days later, when all parasites have reached Stage IIb of gametocyte development. Asexual parasite contamination was estimated at less than 1%. doi:10.1371/journal.pone.0002280.g001 Gametocyte Surface Antigens Recognition of gametocyte-infected erythrocytes by plasma antibodies Serum from a Dutch adult individual, previously established to have transmission blocking immunity against P. falciparum, was incubated with a culture of mature asexual stages of P. falciparum 3D7a (late stage trophozoites and schizonts) and the proportion of ethidium-bromide positive cells that were recognized by antibodies present in the serum were counted, using flow cytometry. We then performed similar analyses on 4 sequential stages of gametocyte development, each derived from the culture shown in Figure 1. The serum from this individual did not recognize erythrocytes infected with asexual stages, nor those infected with any of the immature gametocyte stages. However, this serum strongly recognised the surface of erythrocytes infected with mature stage V gametocytes (Fig. 2), suggesting the presence of GSA in the form of parasite-encoded molecules, altered host membrane compo- nents, or both on the surface of these cells. We then tested plasma from 200 Gambian children, to investigate further the kinetics and nature of development of the antibodies to surface antigens on RBCs infected with asexual parasites, with developing sexual stages, or with mature gameto- cytes of P. falciparum. These children donated blood for membrane feeding experiments (DMFA) 7, 10 or 14 days after treatment as previously described [23–25]. The mean age of the children was 5.0 years (95% C.I. 4.71–5.38), and 49 of them (24.4%) had carried gametocytes at presentation prior to treatment (day 0). Transmission blocking activity was assessed for each of the plasma using results from the DMFA. Mosquito infections occurred in 71 of the 199 experiments in which mosquitoes survived until dissection, 62 of these experiments generated interpretable data in both cages due to adequate mosquito survival and in 18 of these (29.0%) there was evidence of transmission-blocking activity by one of the criteria given in Materials and Methods. There was no recognition of any immature (stage I–IV) 3D7 gametocytes among the 194 plasma for which data were obtained (not shown). Sixty-six (34.0%) of these plasma recognized GSA on the surface of RBCs infected with mature stage V gametocytes, with a mean proportion of 39.5% of cells in the RUQ (inter-quartile range 33.0 to 44.6% ; positivity cut-off was 26.1%). Examination of material after flow cytometry confirmed that gametocytes remained Figure 2. Serum antibodies recognise the surface of mature gametocyte-infected RBCs. Serum from a Dutch individual with previously demonstrated transmission-blocking antibodies was incubated with mature asexual parasites or stage V gametocytes and analysed by flow cytometry. Parasites were dual labeled with FITC conjugate, indirectly recognising human IgG, and EB staining nuclear DNA. Axes denote the number of number of cells counted (events) in each dimension. doi:10.1371/journal.pone.0002280.g002 PLoS ONE | www.plosone.org 4 May 2008 | Volume 3 | Issue 5 | e2280