the proportion of caspase positive ookinetes with intact mem- branes declining, those with compromised membranes increasing and lack of visible staining for caspase-like activity increasing. Intermediate values were recorded for each parameter at 18 h (data not shown). The addition of L-NAME to a blood meal significantly decreased the proportion of ookinetes with activated caspase-like molecules in their cytoplasm at 15 h (P,0.001) whereas D-NAME had no effect (P.0.1) but by 20 h there was no significant difference between NOS-inhibitor treated ookinetes and the control (P.1.0) or D-NAME (P.0.99). However, significantly fewer L-NAME treated ookinetes had compromised plasma membranes (P,0.02) at this time (Figure 3C). These data again suggest that inhibition of NOS activity gave some initial protection against apoptosis. Further experiments demonstrated that the addition of the NOS inhibitor L-NAME to the blood meal significantly increased the number of P. berghei oocysts developing on the midgut of An. stephensi 12 days post-infection compared with those in the D- NAME treated groups and untreated mosquitoes (see Table 2). No differences in mosquito survivorship were observed across the treatments (data not shown). The effect of an NO donor on mosquito longevity and ookinete development in vivo An assessment was made of the feasibility of enhancing the natural production of NO by the mosquito as a means of killing ookinetes in vivo without affecting mosquito fitness. The effect on mosquito longevity of administration of the NO donor SNP with an infective blood meal was assessed. Neither 100 mM nor 1 mM SNP caused a significant change in the probability of survival compared with untreated mosquitoes (Wilcoxon analyses: 100 mM, P = 0.9 and 1 mM (P = 0.98) (Figure 4A and 4B), demonstrating that one blood meal containing elevated levels of NO did not affect longevity. Additional NO in the blood meal did, however, have an effect on the prevalence of mosquito infections which fell significantly (P,0.01) when mosquitoes fed on an infective blood meal containing 100 mM SNAP (Figure 5A) and mean numbers of ookinetes found in the midgut lumen dropped from 9696120 to 207662 (P,0.001) (See Figure 5B). The effect of reactive oxygen species on ookinetes The effect of L-DOPA on ookinetes was both dose and time dependent. Thus, compared to controls, the increase in ookinetes expressing chromatin condensation after 1, 2 and 4 h exposure to 10 mM L-DOPA was not significantly different (P = 0.9, P = 0.7 and P = 0.9, respectively), but concentrations of 100 and 500 mM L-DOPA applied for 1 h did significantly increase this apoptosis marker (P,0.05). However, at higher concentrations and longer Treatment Control D-NAME L-NAME 48/50 46/50 49/50 Apoptosis Triggers in Malaria incubation times the proportion of parasites with condensed chromatin was no greater than that of untreated samples (Figure 6A). Instead, the proportion of ookinetes with compro- mised membranes rose steadily with time and concentration of L- DOPA (Figure 6B). It should be noted that the increase in apoptotic ookinetes seen when treated with 500 mM L-DOPA for 4 h was not significant (Figure 6B). Another reactive oxygen species generator, H2O2, caused a significant loss of membrane integrity within 1 h of exposure to 100 mM, 500 mM or 1 mM concentrations (P,0.001) and a larger proportion of the population had compromised membranes when incubated with 100 mM, or 500 mM H2O2 for 4 h. However, no increase in the proportion of ookinetes displaying nuclear chromatin condensation occurred following 1 h or 4 h incubation with these concentrations of H2O2 (Figure 7A,B). Discussion We previously reported, that a proportion of P. berghei ookinetes that develop in vitro and in vivo express markers of apoptosis without the addition of known inducers of apoptosis. [16]. The findings reported herein strongly support our hypothesis that NO and/or RNS act as inducers of apoptosis in P. berghei ookinetes in vitro and that this induction is dose and time dependent. We show that incubation with the NO donor SNP significantly increased the proportion of newly formed ookinetes exhibiting nuclear chroma- tin condensation, caspase-like activity and PS translocation when present at concentration that our experiments suggest would produce between 5–10 mM nitrite after 1 h. SNAP also induced caspase-like activity within 1 h at doses that generated low levels of nitrite (,10 mM nitrite after 1 h). In contrast, higher concentra- tions and longer incubation periods were used to study the effect of the NO donor SNOG but the proportion of ookinetes displaying condensed chromatin did not significantly increase after 3 h exposure and a significant increase was only observed after 6 h incubation (expected nitrite production of ,50 mM). It is possible that induction of apoptosis occurs within a narrow range of concentrations of NO or RNS and exposure to high levels of these compounds may cause death by other means such as necrosis. However, this does not explain why we observed this significant increase in chromatin condensation following 6 h incubation. It will be necessary to use several apoptosis markers to confirm that we were definitely observing apoptosis rather than necrosis when ookinetes are exposed to high levels of NO. It would appear that L-DOPA-induction of the expression of apoptotic markers in ookinetes is highly sensitive to concentration and time of exposure. Very low concentrations of L-DOPA did not generate enough ROS to have any effect, whereas exposure to 1 mM or more resulted in loss of membrane integrity without markers of apoptosis being displayed. Although none of the concentrations of H2O2 used in this study induced apoptosis, it is 44.60 (1.62) 40.37 (1.54) 85.91 (5.81) P-value NS NS 0.001* Table 2. The effect of nitric oxide synthase activity on the number of oocysts developing in the midgut of An. stephensi mosquitoes. Mosquitoes surviving after day 12 post-infection {Oocysts/gut (± SEM) {Oocysts developing on the midgut were counted on day 12 post-infection. *Significantly different from the control, untreated group. doi:10.1371/journal.pone.0012634.t002 PLoS ONE | www.plosone.org 6 September 2010 | Volume 5 | Issue 9 | e12634