Figure 4. Effect of a dihydroartemisinin (DHA) pulse at the ring stage on sexual conversion by the same cycle conversion (SCC) route.

(A) Schematic representation of the assay. Tightly synchronized cultures of the NF54-gexp02-Tom line under non-inducing (+ choline) or inducing (– choline) conditions were exposed to a 3 hr DHA pulse at subcurative doses at the early ring stage (0–10 hpi). Sexual conversion was measured by flow cytometry (FACS) within the same multiplication cycle (~30–40 hpi) to determine the effect of the drug pulse only on production of new gametocytes by the SSC route. (B) Sexual conversion rate determined by flow cytometry. No significant difference (p<0.05) with the control (no drug) was observed for any treatment condition. (C) Distribution of absolute parasitemia of asexual and sexual parasites (from the same flow cytometry measurements as in panel B). In all panels, data are presented as the average and s.e.m. of three independent biological replicates.

Figure 4—figure supplement 1. Effect of a dihydroartemisinin (DHA) pulse at the ring stage on sexual conversion by the same cycle conversion (SCC) route, determined using MitoTracker to identify viable parasites.

(A) Schematic representation of the assay. Tightly synchronized cultures of the NF54-gexp02-Tom line under non-inducing (+ choline) or inducing (– choline) conditions were exposed to a 3 hr DHA pulse at subcurative doses at the early ring stage (0–10 hpi). Sexual conversion was measured by flow cytometry (FACS) within the same cycle (~30–40 hpi) to determine the effect of the drug pulse only on production of new gametocytes by the SSC route. (B) Sexual conversion rates determined by flow cytometry, calculated using MitoTracker-positive cells only. No significant difference (p<0.05) with the control (no drug) was observed for any treatment condition. (C) Distribution of absolute parasitemia of asexual and sexual parasites (from the same flow cytometry measurements as in panel B). In all panels, data are presented as the average and s.e.m. of three independent biological replicates.

Figure 4—figure supplement 2. Effect of a dihydroartemisinin (DHA) pulse at the ring stage on sexual conversion by the same cycle conversion (SCC) route in the NF54-10.3-Tom line, determined by three different methods.

(A) Schematic representation of the assay. Tightly synchronized cultures of the NF54-10.3-Tom line under non-inducing (+ choline) or inducing (– choline) conditions were exposed to a 3 hr DHA pulse at subcurative doses at the early ring stage (1–6 hpi). Sexual conversion was measured by: (i) flow cytometry analysis (FACS) on D1 (D0 is the first day of Generation 0, that is, the day of drug treatment) to determine the initial parasitemia (using the SYTO 11 stain), and on D3 to determine the gametocytemia (SYTO 11 and TdTomato signal) in cultures treated with N-acetylglucosamine (GlcNAc); (ii) immunofluorescence assay (IFA) analysis of cultures treated with ML10 using the Pfs16 marker; (iii) flow cytometry analysis on D1 to determine the initial parasitemia, and on D4 light microscopy analysis of Giemsa-stained blood smears (Giemsa) to determine the gametocytemia in cultures treated with GlcNAc. (B) Sexual conversion rates as determined by FACS, IFA, and Giemsa-stained blood smears. The p-value is indicated only for treatment versus control (no drug) significant differences (p<0.05). (C) Distribution of absolute parasitemia of asexual and sexual parasites, determined by flow cytometry (from the same flow cytometry measurements as in panel B). In all panels, data are presented as the average and s.e.m. of two independent biological replicates.