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. Author manuscript; available in PMC: 2019 Nov 27.
Published in final edited form as: Nat Microbiol. 2019 Sep 2;4(12):2226–2236. doi: 10.1038/s41564-019-0539-x

Figure 2: Exclusive molecular markers for C. parvum sexual stages.

Figure 2:

C. parvum were engineered to express Cowp1-mNeon (a & b, Scale Bar = 1 μm) and Cowp1-HA (Supplementary Figure. 3) out of the native locus or cowp1 promoter driven tdTomato out of the ectopic TK locus (c, Scale Bar = 1 μm). Note mNeon labeling of the wall in oocyst purified from infected mice and punctate labeling in female gametes observed in infected HCT-8 cells (Scale Bar= 1 μm). Labeling becomes apparent 42 h into culture and is never observed in asexual meronts or male gametes (b & d). The Cowp1 promoter alone is sufficient to confer female-specific expression to a reporter protein (c-d). Anti-H3K9Ac was used to label the nuclei of females as they stain poorly with DAPI. (e) Male gametes show a characteristic array of microtubules around the nucleus upon staining with an anti-tubulin antibody (e, Scale Bar = 0.5 μm). (d) For time courses cultures were infected with the indicated transgenic strains and triplicate coverslips were fixed and processed for IFA. Parasite stages were scored for HA staining, the mean percentage ± SD of HA positive stages among all parasites is shown for three independent biological replicates. When parasites were engineered to express HAP2-HA out of the native locus antibody staining revealed exclusive labeling of male gamonts (g, Scale Bar = 1 μm) and free gametes (f, Scale Bar = 1 μm). HAP2 labels a single pole per mature gamete. This staining becomes apparent at 42 hours of culture (d, blue). All microscopy experiments shown in this figure were performed three independent times.