Development of pyrom1(-) EEF is significantly reduced within the first 24 hours of hepatic development.

Development of sporozoites within Hepa 1–6 cells was analyzed at 6 hours, 12 hours, and 24 hours post invasion. (A) A double staining assay was carried out to determine the number of intracellular parasites undergoing development inside Hepa1–6 cells at 6 hours post infection The % intracellular parasites was determined by counting at least 30 fields per well. Values shown are the mean % intracellular parasites from duplicate counts ± standard deviation. The values were not statistically significant (P-value 0.17) using unpaired t test analysis. Parasite development at 12 hours (B) and 24 hours (C) post infection was assayed by staining with α-pyCSP and developing parasites per field were counted (at least 30 fields were counted per well). The numbers represented are the mean values of developing parasites/15 fields ± standard deviation. At 12 hours development there is a 45% reduction in pyrom1(-) parasite development (P-value 0.008 using one-way ANOVA analysis) and at 24 hours there is at least a 60% reduction in pyrom1(-) parasite development (P-value 0.001 using one-way ANOVA analysis) D) Images of developing liver stage parasites at 6 hours and 12 hours stained with CSP show that despite reduced numbers, R1KO parasites are capable of undergoing the initial differentiation, transformation, and sphericalization during early hepatic development. (E) Development of pyrom1(-) parasites that survive the initial 24 hours go on to develop normally. Immunofluorescence of exo-erythrocytic forms (EEF) at 40 hours post invasion of Hepa1–6 cells was performed using anti-pyCSP to visualize developing parasites. Pictures of at least 50 EEF/well (duplicate) were taken. Area of EEF was measured using Image J software. Area was determined as an arbitrary value from images taken with the same magnification (40×). Bar graphs represent the mean area ± SEM.