Apoptosis Triggers in Malaria Figure 1. The effect of nitric oxide donors on the induction of apoptosis in P. berghei ookinetes. Ookinetes were incubated in RPMI with or without the addition of SNP at different concentrations or for different time periods and then examined for the presence of different markers of apoptosis. A:The effect of 2 mM SNP on the proportion of ookinetes expressing a marker of apoptosis; data are means of 3 experiments (n = 3) with 300 ookinetes examined in each experiment. B,C: The effect of different concentrations of SNP following 1 h incubation; n = 3, with 100–150 ookinetes examined per experiment). D: The effect of different concentrations of SNP on ookinetes following 4 h incubation; n = 3, with 25–50 ookinetes examined in each experiment. Apoptosis markers examined were condensed nuclear chromatin (A, B), activated caspase-like molecules (C), and PS translocation without loss of membrane integrity (D). Error bars represent standard error of the mean (SEM). Bars with different letters are significantly different. White bars represent controls and patterned bars represent treated ookinetes. doi:10.1371/journal.pone.0012634.g001 condensation, even after 24 h incubation with SNP (data not shown). Two additional markers for apoptosis-like death, the presence of activated caspase-like molecules and the translocation of PS to the outer membrane were monitored after SNP treatment. As the percentage of untreated ookinetes with apoptosis markers varied greatly between experiments, fold increases in the expression of each marker were calculated to facilitate comparison between experiments (Table 1). A similar pattern to chromatin condensa- tion was seen with an overall significant increase in the percentage of ookinetes expressing active caspase-like molecules in SNP- treated ookinetes compared to that of untreated ookinetes (F3,23 = 9.6; P,0.001). This was caused by incubation with 100 mM and 500 mM SNP but not with the lower concentration of 10 mM SNP (see Figure 1C). Although the proportion of untreated ookinetes displaying caspase-like activity was much smaller than those displaying acridine orange staining in the previous experiment the proportion of apoptotic ookinetes in the untreated group was lower in these experiments and a similar fold increase in this marker occurred upon treatment (see Table 1). Examination of ookinetes for PS translocation revealed that incubation with SNP also caused a significant increase in this marker of apoptosis (F3,19 = 8.93; P,0.01) but that higher concentrations (1 mM) and a longer incubation period (4 h) were PLoS ONE | www.plosone.org 3 September 2010 | Volume 5 | Issue 9 | e12634