FIG 2.

Characterization of C. parvum intracellular stages over the first 48 h of infection in HCT-8 cells. (A) Diagram of the C. parvum intracellular life cycle. Single-nucleus trophozoites replicate mitotically to form eight mature type I merozoites that egress and reinvade host cells. After at least two rounds of asexual replication, parasites divide into four mature type II meronts, which differentiate into one of two sexual life stages, macrogamonts or multinucleated microgamonts. (B to H) Immunostaining of parasite stages with rabbit polyclonal anti-C. parvum (Cp, red) and either EdU, a fluorescent thymidine analog that incorporates into replicating DNA (green), or the nuclear stain Hoechst (blue). Life cycle stages were distinguished as follows: trophozoites, with the presence of a single nuclei (B); type I meronts with the presence of two nuclei C), four EdU⁺ nuclei (D), or eight nuclei (E); type II meronts with four EdU⁻ nuclei (F); microgamonts with more than eight nuclei (G); and macrogamonts, with the presence of wall-forming bodies visible by phase contrast (black arrows) (H). Scale bars = 5 μm. (I) Abundance of each life cycle stage as a percentage of total C. parvum at the indicated hours postinfection (hpi). For each time point, EdU was added to the culture 2 h prior to fixation and antibody labeling, and the number of parasites at each stage was counted for 10 fields of view with a 100× oil objective. The number of total parasites counted per time point is shown above bar graph. Troph, trophozoites; Macro, macrogamonts; Micro, microgamonts. (J to L) Gene expression of a predicted transporter protein (cgd2_800) (J), the microgametocyte-specific gene HAP2 (cgd8_2220) (K), and a macrogamont oocyst wall protein 8 (cgd6_200) (L) at specified times postinfection with C. parvum sporozoites. Gene expression profiles are from a single experiment with three replicates per time point. Values are plotted as the means ± SD.