Cell suicide in protists can be triggered by various extra and intracellular events including nutrient deprivation, heat shock, drug treatment and the presence of nitric oxide (NO), reactive nitrogen species (RNS) and reactive oxygen species (ROS) (see for example reviews [11–13]). Many investigations of parasitic protists are conducted in vitro and apoptosis is induced experimentally by one or other of these triggers. However, protists have been shown to exhibit markers of apoptosis in vivo, without experimental manipulation, and apoptosis has been suggested to play an important role in the life history of some unicellular parasite populations. For example, in trypanosomatids it is thought to act as a mechanism to control population numbers and eliminate particular developmental stages [11,20–23]. Extrinsic inducers of apoptosis in parasitic protozoans must therefore occur in vivo and would be expected to be found in the mosquito midgut lumen following an infective blood meal. The mosquito midgut lumen is an environment that becomes increasing hostile as the blood meal is digested; in part this is due to the presence of NO, RNS and ROS. Peterson and colleagues [24] suggest that haemoglobin, present in the blood meal, catalyses the synthesis of NO metabolites in a reducing environment. An additional source of NO comes from the mosquito as nitric oxide synthase (NOS), present in midgut epithelial cells, catalyses the production of NO during the oxidative deamination of L-arginine to L-citrulline. Inducible NOS expression is upregulated in mosquito midgut epithelial cells in response to malaria infection [25,26] and induction of NOS is proportional to the intensity of infection [27]. Herrera-Oritz and colleagues [28] also reported that NO is produced in midguts of Anopheles pseudopunctipennis cultured with P. berghei. In addition, NOS has been detected within midgut cells that have been invaded by ookinetes [29,30]. NO, or its reactive derivatives, play a part in the immunological reaction of host defence against numerous pathogens [31]. They have been identified as important cytotoxic effector molecules in a variety of parasitic infections of vertebrates, including schistoso- miasis, leishmaniasis and malaria infection [32]. NO has been shown to have a cytotoxic or cytostatic effect on erythrocyte stages of Plasmodium, depending on concentration [33–35] and Rockett and colleagues found that S-nitrosothiols displayed a far greater toxicity to P. falciparum than nitrate which, in turn, was more effective than nitrite [36]. The availability of NO for Plasmodium killing in the vertebrate circulatory system has been questioned [37], nevertheless, it has indisputable effects on malaria transmis- sion [38]: killing gametocytes [39] and ookinetes [40] and nitrate concentration increases in the gut of Plasmodium-infected mosquitoes [41]. In the mosquito, inhibition of NOS activity has been shown to increase the number of oocysts developing on the midgut [26]. Herrera-Ortiz et al [28] found that ookinetes incubated with the NO donor, sodium nitroprusside (SNP) or the ROS generator, L- DOPA, significantly decreased viability and that a combination of both donors was synergistic. Similarly, L-DOPA dependent superoxide anion (O2-), produced in the haemolymph and midgut of Anopheles albimanus, is toxic to ookinetes [40]. L-DOPA, also caused loss of P. berghei gametocyte infectivity [42]. In addition to RNS, ROS are also generated within the midgut cells of An. stephensi during ookinete invasion [43]. We hypothesized that these toxic molecules may be naturally occurring extrinsic triggers of apoptosis in P. berghei ookinetes. NO has been shown to create nitrosative stress which can promote apoptosis by the activation of mitochondrial apoptotic pathways [44] and mitochondrially derived ROS have also been associated with the initiation phase of apoptosis, acting as mediators for different signal transduction pathways. A role for Apoptosis Triggers in Malaria ROS in ancient redox –sensitive pathways leading to apoptosis- like PCD has also been proposed and both RNS and ROS have been shown to act as triggers for apoptosis in Toxoplasma gondii [45] and kinetoplastid parasites of the genera Trypanosoma and Leishmania [46–49]. The purpose of this study was to investigate the hypothesis that NO and ROS cause the death of P. berghei ookinetes by an apoptosis-like (hereafter referred to as apoptosis) process. Investi- gations were performed in vitro and ookinetes examined for several markers of apoptosis following exposure to donors of NO and ROS. Experiments were then designed to determine whether natural sources of NO in the blood meal and mosquito midgut could also induce the display of markers of apoptosis in vivo. The results demonstrated that NO, or its derivatives, do trigger apoptosis in ookinetes in vitro and support the hypothesis that NOS activity is linked to the induction of apoptosis in P. berghei ookinetes whist they are in the mosquito midgut lumen. The role of ROS in inducing apoptosis is less marked, although we do demonstrate that it is toxic to ookinetes in vitro and causes the display of markers of apoptosis in a dose and time dependent manner. Results The effect of white blood cells on the induction of apoptosis White blood cells (WBCs) contain NOS and thus represent a source of NO in the blood. In order to determine whether the removal of these cells from gametocytaemic blood prior to culture affected the proportion of ookinetes that exhibited signs of chromatin condensation, a comparison was made between blood with and without WBCs. Exposure to WBCs caused a small, but overall significant, increase in the proportion of ookinetes with acridine orange staining (depicting condensed chromatin) when maintained in Schneider’s medium for 0.25 h, 3 h and 6 h respectively (,5.5, 4 and 1% increase; F1,35 = 4.76; P,0.05) (see supplementary Figure S1). The remainder of ookinete incubations in this study was performed in the presence of WBCs. The effect of NO donors on ookinetes in vitro Newly formed ookinetes were exposed to 2 mM sodium nitroprusside (SNP) for 0.25 h, 1 h or 4 h and the percentage of ookinetes displaying condensed chromatin was detected using acridine orange staining. An overall significant increase in acridine orange-positive ookinetes occurred (F1,53 = 40.29; P,0.001) and pair wise comparisons showed that this increase was significant after 1 h (,25% increase P,0.001) and 4 h (,20%, P,0.01) incubations (see Figure 1A). Ookinetes were also cultured for 24 h but by this time the proportion of parasites with condensed chromatin in the control group had risen to over 70% and no significant differences were detected between control and treated groups (data not shown). NO thus accelerated the induction of apoptosis in a large proportion of the ookinete population. An investigation into the effect of different concentrations of SNP showed that the effect was dose dependent; although 10 mM SNP did not induce additional ookinetes to undergo chromatin condensation, 100 mM SNP caused a significant increase of 30% (P,0.001) and 500 mM SNP caused an increase of 32% (P,0.001) (Figure 1B). Further experiments incubating ookinetes with 1 mM, 2 mM and 4 mM SNP for 1 h and 4 h all showed significant increases in ookinetes displaying this marker for apoptosis compared with untreated groups at the same time points. However, in no case were all ookinetes induced to undergo apoptosis; between 20 and 30% never displayed chromatin PLoS ONE | www.plosone.org 2 September 2010 | Volume 5 | Issue 9 | e12634