Table 1. The fold-increase in the percentage of apoptotic ookinetes treated with SNP, relative to untreated ookinetes. Concentration Fold-increase Apoptotic marker Condensed chromatin Activated caspase-like molecules PS translocation after 1 h PS translocation after 4 h 10 mM 100 mM 10 mM 100 mM 500 mM 100 mM 500 mM 1 mM 100 mM 500 mM 1 mM 1.06 2.08** 1.72 3.86** 3.72** 2.17 2.18 2.33 1.88 2.11 3.85* Apoptotic ookinetes were identified using the following markers; condensed chromatin, activated caspase-like molecules or phosphatidyl (PS) translocation to the outer cell membrane. Significant difference from controls, *P,0.05 and ** P,0.001. doi:10.1371/journal.pone.0012634.t001 required for the proportion of annexin positive ookinetes (that did not have compromised membranes) to be significantly higher than untreated parasites (Figure 1D and Table 1). The yield of parasites available for this latter series of experiments was lower than the previous ones and thus the number of ookinetes examined was smaller. Ookinetes that had been collected from gametocytaemic-blood following 18 h of culture were used to determine whether the execution pathway of NO-induced apoptosis involved the mito- chondrion. Ookinetes were incubated for 2 h with 100 mM SNP and then examined for loss of MOMP using a JC-1 assay kit. Approximately 30% of untreated ookinetes exhibited loss of MOMP and a similar proportion to this contained nuclei with condensed chromatin (Figure 2). Further ookinetes were, instead, incubated with carbonyl cyanide 3-chlorophenylhydrazone (CCCP), which causes loss of MOMP, and compared with untreated samples and SNP treated samples. All the ookinetes in samples treated with Apoptosis Triggers in Malaria CCCP had lost MOMP (Kruskal-Wallis p = 0.008), however SNP treatment did not induce further loss of MOMP. Overall, both SNP and CCCP treatments caused a significant increase in the proportion of ookinetes displaying condensed chromatin (F2.9 = 18.93; p = 0.001), and each group differed significantly from the untreated parasites (Figure 2). These data suggest expression of this apoptosis marker in ookinetes may occur via a pathway involving loss of mitochondrial membrane potential (D ym), but that NO-induced apoptosis does not proceed in this way. In order to confirm that the induction of apoptosis seen following incubation with SNP was due to the NO produced by this donor some supplementary investigations were performed using two other donors to deliver NO to ookinetes in culture. It took 500 mM S-Nitroso-N-acetyl-DL-penicillamine (SNAP) to induced a significant increase in the proportion of ookinetes displaying caspase-like activity after 1 h incubation (P,0.01). However S-nitroglutathione (SNOG) only induced a significant increase in the proportion with condensed chromatin at a concentration of 2 mM after incubation for 6 h (P,0.001) (Supplementary Figure S2A and B). Measurement of the nitrite produced by the three donors when dissolved in solutions of supplemented RPMI revealed large differences in the speed and amount of release, with SNAP producing more nitrite that the other two donors during short incubation times whereas SNOG produced much more after 24 h (Supplementary Figure S3). Some of the nitrite produced by these donors may have been oxidized to nitrate during these incubation periods but this would not be detected by the Griess reaction use in this study. The effect of a nitric oxide synthase inhibitor on apoptotic ookinetes in vivo In addition to WBCs in the blood meal, the mosquito midgut epithelium is a source of NO that could induce apoptosis in developing ookinetes. To test this hypothesis, mosquitoes were fed a NOS inhibitor, Nw-nitro-L-arginine methyl ester (L-NAME), the inactive isomer, Nw-nitro-D-arginine methyl ester (D-NAME), or infective blood meal alone. Analysis of the number of ookinetes in the midgut lumen that were exhibiting condensed chromatin in the presence of a NOS inhibitor or the inactive isomer indicat- ed significant differences due to treatments, (F2,12 = 123.86, Figure 2. Loss of mitochondrial membrane potential and the induction of apoptosis in P. berghei ookinetes. Ookinetes were maintained in RPMI alone (controls) or incubated with 100 mM SNP for 2 h or with CCCP for 30 min prior to examination for loss of mitochondrial membrane potential (MOMP) (black bars) or the presence of nuclear chromatin condensation (grey bars). Error bars represent standard error of the mean (SEM), n = 4 with 50–100 ookinetes examined in each experiment. Bars with different letters are significantly different. doi:10.1371/journal.pone.0012634.g002 PLoS ONE | www.plosone.org 4 September 2010 | Volume 5 | Issue 9 | e12634