Apoptosis Triggers in Malaria Figure 7. The effect of H2O2 on the induction of apoptosis of ookinetes in P. berghei ookinetes in vitro. The effect of incubation with different concentrations of H2O2 for 1 h (A) or 4 h (B). Apoptotic ookinetes displaying chromatin condensation were detected with acridine orange (white bars); ookinetes with compromised membranes were detected with propidium iodide (grey bars). Bars represent means of 3 experiments and at least 25 ookinetes were counted per sample. Error bars represent SEM and bars with different letters are significantly different. doi:10.1371/journal.pone.0012634.g007 at 24–36 h, after ookinete invasion of the midgut [41]. Interestingly, P. falciparum glycosylphosphatidylinositols (GPIs), that would be released into the blood meal by both sexual and asexual stages of the parasite, have been shown to induce AsNOS expression in vivo and in vitro, with signaling likely to be mediated by an insulin signal transduction-like pathway [56]. The malaria pigment, haemazoin, has also been reported to induce NOS gene expression at 24 h post-feeding, thus producing complimentary activity to GPIs [57]. An additional signal, ingested by the mosquito and activated in the midgut, is mammalian latent transforming growth factor (TFG-b1). Acting as a cytokine, it is a potent regulator of AsNOS expression with low doses resulting in sustained induction of anti-parasitic AsNOS [41]. Finally, a TFG-b homologue, As60A, is inducibly expressed in the mosquito in a parasite density-dependent manner. This also induces NO production and acts to control parasite development [27]. Thus many factors contribute to the toxic, apoptosis-inducing environ- ment in which ookinetes are developing in vivo. Early activity of NOS has been shown to limit parasite development in vivo and supplementation of the blood meal with the NOS substrate, L-arginine, significantly reduces the number of oocyst on the midgut of P. berghei-infected An. stephensi. Further- more, feeding the NOS inhibitor, L-NAME, increases parasite burden whilst D-NAME, the inactive isomer, has no effect [26] and Cao and co-workers [38] reported a similar effect when mosquitoes were fed on mice that had previously fed on L-NAME or D-NAME. These data do not, however, tell us whether NO/ RNS are acting by inducing apoptosis in the parasites. In this study, feeding mosquitoes blood meals mixed with an NO donor inhibited the development of ookinetes to such a marked degree that hardly any were available to be examined for markers of apoptosis thus the experiments reported here were not able to demonstrate that the death of ookinetes induced by the addition of a NO donor to the blood meal was by apoptosis. We estimate that the concentration of nitrite produced in the midgut by SNAP (the donor used in this experiment) would have been approximately 10 mM. As gametocytes are highly susceptible to the toxic effects of NO and Al-Olayan and co-workers [16] reported signs of apoptosis in the early zygotic stages, midguts containing sporogonic stages clearly need to be examined very PLoS ONE | www.plosone.org 10 September 2010 | Volume 5 | Issue 9 | e12634