Fig. 4.

Fertility of Δfact-l_gam male gametes is strongly reduced. A. Fertility of Δfact-l_gam gametocytes as measured by the capacity to form ookinetes in vitro and by crossing Δfact-l_gam gametes with fertile female gametes of ΔP48/45 and fertile males of ΔP47. Δfact-l_gam gametocytes show strongly reduced capacity to form ookinetes. Δfact-l_gam female gametes are fertilized by fertile ΔP47 male gametes at wild-type levels, whereas Δfact-l_gam males show a strongly reduced capacity to form ookinetes after fertilization with the fertile ΔP48/45 females. The observations shown are from three independent experiments each performed in triplicate. The Δfact-l 1b and 2b mutant lines are in a GFP+ background. B. Fertility of Δfact-l_gam gametes as measured by the capacity to form 4N zygotes in vitro by FACS analysis of the fluorescence intensity (DNA content) of Hoechst-33258-stained, unfertilized female gametes (1N) and zygotes (4N) in the Fluofrmg background, which generates GFP+ve male gametocytes and RFP+ve female gametocytes. Female gametes and zygotes were selected for analysis of DNA content on basis of their RFP expression as shown in the upper, left dot plot. DNA content is measured 4 h after activation when meiosis should be completed in Hoechst-stained cells and the fertilization rate calculated (upper right dot plot). Analyses of cultures of Δfact-l_gam gametocytes show a strongly reduced capacity to form zygotes (middle panel right dot plots). C. Crossing of Δfact-l_gam gametes with fertile female gametes of ΔP48/45 and fertile males of ΔP47 (lower panel) show a strongly reduced capacity of male Δfact-l_gam gametes to produce zygotes after fertilization with the fertile female gametes of ΔP48/45. The graph shows the mean fertilization rates and standard deviations, defined as the percentage of females that develop into zygotes, as determined in triplicate in three independent crossing experiments. These Δfact-l 1c and 2c mutant lines express GFP and RFP in males and females respectively.