shortly after feeding with NO donors to detect the affects in vivo. We did however demonstrate that addition of the NOS-activity inhibitor, L-NAME, to the infective blood meal significantly decreased the proportion of ookinetes expressing two markers of apoptosis and increased the number of parasites that transformed into oocysts in vivo. This is strong supportive evidence that NO/ RNS generated within the mosquito are inducing ookinete death by apoptosis within the midgut lumen, although we acknowledge that NOS activity in WBSs in the blood bolus would also have been inhibited. Sources of reactive oxygen species Peterson et al. [24] suggest that haem and haematin, released within the mosquito midgut during haemoglobin digestion, could react with iron and oxygen in the blood bolus to form ROS. These toxic metabolites are also produced by the mosquito. Lanz- N – is produced Mendoza and colleagues [40] found that O 2 naturally in the haemolymph and midgut of An. albimanus and is toxic to ookinetes. Additionally, Kumar and colleagues [43] reported P. berghei-induced expression of peroxidase genes in the midgut of An. stephensi, one of which could produce superoxide anion, lead to the accumulation of high levels of hydrogen peroxide and Molina-Cruz et al [58] have shown that midgut catalase expression is suppressed during midgut traversal and suggest that this leads to high local levels of hydrogen peroxide that contribute to limiting Plasmodium infection. The death of ookinetes in vitro following combined treatments with SNP and L- DOPA was attributed to the toxicity of peroxynitrite which was formed from the combination of NO produced from SNP and NO2– produced from L-DOPA [28]. In vivo, ookinetes are developing in a complex redox-active environment (see [24] for a full discussion) and they are thus likely to be exposed to considerable oxidative stress, to which they are highly vulnerable. Possible pathways of induction of apoptosis Following initiation of apoptosis in mammalian cells, two pathways lead to its execution; the intrinsic or mitochondrial dependent pathway and the extrinsic pathway; with considerable cross-talk existing between the two [59]. The former can be initiated by the opening of the inner membrane permeability transition pore or the permeabilization of the outer mitochondrial membrane and release of inter membrane space molecules such as cytochrome c, and Ca+. The latter mechanism is mediated by apoptosis regulators, notably in the Bcl-2 family [60,61]. A mitochondrial dependent pathway had been observed in several parasitic protozoans For example, in T. cruzi complement activation leads to an influx of Ca+, followed by MOMP loss, increase in oxygen radical production, a decrease in the mitochondrial cytochrome c content and initiation of apoptosis [46]. Although members of the Bcl2 family have not been identified in protozoans, homologues of the mitochondrial nuclease, endonuclease G (EndoG), have been described in yeast, T. brucei, L. donovani and L. infantum [62–64]. Additionally a recently described caspase-independent pathway, induced by the presence of a death stimulus, the drug edelfosine, involves the translocation of EndoG straight from the mitochondria of L. infantum to the nucleus. Here it is thought to be involved in DNA degradation [63]. Our findings lend support to the hypothesis that a mitochon- drial dependent apoptotic pathway exists in Plasmodium [15,18], but suggest that this is not likely to be the only signaling pathway. Examination of MOMP in newly developed ookinetes revealed that a similar proportion exhibited a loss of D ym as contained pycnotic nuclei. Treatment to induce MOMP loss was 100% Apoptosis Triggers in Malaria successful and also caused a significant increase in the number of parasites with condensed chromatin but, as with other experiments in this study, ,20% did not exhibit this marker of apoptosis during the period under investigation. This adds more weight to our suggestion that a small proportion of ookinetes may be protected from apoptosis. In contrast to the effect of CCCP incubation, NO did not cause loss of MOMP, giving rise to the conclusion that nitrosative stress-induced apoptosis must operate via an alternative pathway. The induction of apoptosis via nitrosative stress may be attributed to protein nitrosylation [65]. In particular, cysteine proteases could be NO targets via modification of the Cys catalytic residues by S-nitrosylation [66], thus it is possible that NO is activating an apoptosis pathway in the malaria parasite via protein nitrosylation. Extrinsic pathways leading to apoptosis may involve the proteolytic activation of a family of clan CD cysteine proteases, the caspases [67]. These appear to be confined to metazoans, and may form a secondary executive pathway that evolved after the symbiosis that led to the formation of the eukaryotic cell type. Furthermore, they are not found in all metazoan taxa and it is becoming increasing clear that apoptotic cell death does not always involve these cysteine proteases [68]. Despite the reported involvement of caspase-like molecules in PCD in a variety of single celled organisms (listed in [10]), no caspase homologues had so far been identified in any of their genomes [69]. However, other clan CD cysteine proteases, the metacaspases, have been described in several protozoan [70]. They appear to play a role in some apoptosis pathways in yeast (reviewed in [71]) and may be involved in PCD in Trypanoso- matids, Cryptosporidium parvum and Tetrahymena thermophila [72–75]. Although metacaspases have been described in P. falciparum [15] and P. berghei [76] there is, as yet, no evidence that this is the caspase-like molecule that is involved in apoptosis in P. berghei [76] (Arambage and Hurd, unpublished) and clan CA cysteine protease have recently been implicated in programmed cell death in P. falciparum [77]. Indeed, the role that metacaspases play in apoptosis in plants, fungi and protozoans is now being debated; they may have additional or alternative functions [78–80] and the identity of the caspase-like molecule(s) associated with apoptosis in P. berghei and many other protozoans still remains to be determined. Conclusions The majority of ookinetes that form in the mosquito midgut do not succeed in transforming into viable oocysts. Parasite-killing mechanisms such as lysis and melanization operate once the ookinete has traversed the midgut epithelium and attention to date has largely focused on mosquito immune responses that operate at this time. Unless specific markers for apoptosis are used, the majority of ookinetes present in the midgut lumen appear, by light microscopy, to be viable. We suggest that they are not; many of them are already dying by apoptosis and will never invade the midgut. Furthermore NO, RNS and ROS may play a significant role as refractory mechanisms in the redox-active midgut lumen and provide the first line of defence against P. berghei. It is still necessary to determine whether apoptotic death of ookinetes is also an important mechanism for limiting natural mosquito-infections of human parasites such as P. falciparum. We have previously shown that P. falciparum ookinetes do undergo apoptosis in the midgut of An. stephensi in the laboratory [18]. However, the importance of NOS activity in inducing apoptosis, and limiting these infections in the field, is still to be determined and conflicting evidence exists as to the response of NOS to natural Plasmodium infections in An. gambiae [81,82]. If it does prove to be important, we suggest that it may be feasible to enhance the PLoS ONE | www.plosone.org 11 September 2010 | Volume 5 | Issue 9 | e12634