(A) Schematic representation of the assay. Sorbitol-synchronized cultures of the NF54-gexp02-Tom line were maintained under control non-inducing conditions (+ choline, CTL), exposed to a 3 hr 5 nM DHA pulse (in the presence of choline) at the trophozoite stage, or maintained in the absence of choline (– choline, used as a positive control for a gametocyte-inducing condition). On the first day of Generation 1, N-acetylglucosamine (GlcNAc) was added and maintained for 4 d to eliminate asexual parasites and obtain pure gametocyte cultures. The different cultures were used to infect Anopheles mosquitoes by standard membrane feeding. (B) Gametocytemia at the time of mosquito infection (10–13 d after DHA treatment), exflagellation levels (after 10 min of activation with fetal calf serum), number of oocysts/mosquito (n = 53 for CTL, n = 103 for DHA 5 nM, and n = 79 for – choline, data for all individually dissected mosquitoes from all replicates is shown) and average number of sporozoites/mosquito in each independent biological replicate (obtained from pooled dissections; in total, n = 65 for CTL; n = 111 for DHA; n = 123 for – choline). Results are from three independent biological replicates, but in one experiment the CTL culture was lost and in another one the – choline control was not included. Data are presented as the average and s.e.m. of the independent biological replicates, except for oocysts/mosquito results that are presented as standard box and whisker plots. The p-value is indicated only for significant differences (p<0.05) between conditions. (C) Representative images of gametocytes at different stages and activated gametes from DHA-treated cultures, showing no apparent abnormality. Images from live cell fluorescence analysis (Hoechst stains nuclei; TdTomato is expressed under the control of the gexp02 promoter) and Giemsa-stained smears are shown. (D) Representative images of mosquito midguts (transparent, circular structures are oocysts), oocysts, and sporozoites from DHA-treated and – choline cultures, showing no apparent abnormality.