Apoptosis Triggers in Malaria P,0.001), time post-infection, (F1,12 = 63.379, P,0.001), and treatments by time interaction (F2,12 = 7.05796, P,0.01). Specif- ically, the addition of L-NAME to the blood meal caused a significant reduction in the proportion of ookinetes exhibiting condensed chromatin (P,0.001) compared to those exposed to control treatments at 18 h and 20 h post-blood feeding. Additionally, significantly more L-NAME treated ookinetes were undergoing apoptosis at 20 h than 18 h post-infection (P,0.001) but this increase was not detected in the control treatments, suggesting that inhibition of NOS activity had delayed the induction of apoptosis (Figure 3A). In the anticipation that activation of cysteine proteases may occur upstream of chromatin condensation we monitored the effect of inhibition of NOS activity earlier, at 15 post blood- feeding. Approximately 50% of the 200 ookinetes examined stained positive for caspase-like activity in untreated and D- NAME treated mosquitoes by 15 h post-feeding and a further 20% were permeable to propidium iodide (PI) (which may have obscured staining for caspase-like activity) and thus classed as dead (Figure 3B). An overall significant change in these proportions had occurred by 20 h post-infection (F5,24 = 573.2 P,0.001 (caspase) F5,24 = 47.8 P,0.001(compromised membranes)) (Figure 3C) with Figure 3. The effect of a nitric oxide synthase inhibitor on the expression of apoptosis markers by ookinetes in vivo. An. stephensi were fed on blood containing P. berghei gametocytes (black bars) with the addition of L-NAME (grey bars) or the inactive isomer D-NAME (white bars). A: The proportion of ookinetes with condensed nuclear chromatin at 18 h and 20 h post-infection. B, C:The proportion of ookinetes expressing caspase-like activity or with compromised membranes (CM) 15 h and 20 h post-infection respectively. Error bars represent standard error of the mean (SEM). Bars with different letters are significantly different. doi:10.1371/journal.pone.0012634.g003 PLoS ONE | www.plosone.org 5 September 2010 | Volume 5 | Issue 9 | e12634