Fig. 2: GEP1 is essential for gametogenesis of both sexes.

a Predicted GEP1 protein structure with 14 TM domains (green bar) and cytoplasmic N-termini and C-termini. b IFA analysis of GEP1 expression in asexual blood stages (ABS), gametocytes, ookinetes, oocysts, and sporozoites of the 6HA::gep1 parasite using anti-HA antibody. Hoechst 33342 (Blue) is used for nuclear acid stain for all images in this figure. c Western blot analysis of GEP1 in ABS and gametocytes of the 6HA::gep1 parasite. BiP as loading control. d IFA analysis of GEP1 in the 4Myc::gep1 parasite using anti-Myc antibody. e mScarlet fluorescence protein expression driven by the endogenous gep1 promoter in ABS and gametocytes of the ∆gep1mScarlet parasite. f Co-staining of GEP1 and α-Tubulin (male gametocyte specific) in the non-activated (NAG) 6HA::gep1 gametocytes. x/y in the figure is the number of cell displaying signal/the number of cell tested. g and h, P28 expression during in vitro gametocyte to ookinete differentiation. P28 expression is detected in female gametes, fertilized zygotes, and ookinetes in IFA (g) and western blot (h). mpa: minute post activation; hpa, hour post activation. i Day 7 midgut oocyst counts from mosquitoes infected with parasites, including 17XNL, ∆gep1, ∆nek4, or ∆map2 parasite alone, as well as mixtures of ∆gep1/∆nek4, ∆gep1/∆map2, or ∆map2/∆nek4 parasites. ∆nek4 and ∆map2 are female and male gamete-defect parasites, respectively. x/y on the top is the number of mosquito containing oocyst/the number of mosquito dissected; Mosquito infection prevalence is shown above. Scale bar = 5 μm for all images in this figure. Experiments were independently repeated three times in b, c, d, e, f, g, and two times in i. Two-tailed unpaired Student’s t test in i.