Apoptosis Triggers in Malaria Figure 5. The effect of SNAP on the prevalence and intensity of P. berghei infection in An. gambiae. Mosquitoes were fed on gametocytaemic mouse blood alone or on blood containing 100 mM SNAP and midguts examined 16–18 h post-infection. A: Prevalence of ookinete infections (mean of 5 experiments). B: Intensity of infection (the number of ookinetes recovered from each midgut; infected blood, n = 60, infected blood with SNAP, n = 55. doi:10.1371/journal.pone.0012634.g005 generated reactions initiate PCD pathways in developing zygotes and ookinetes in vivo. Both the blood meal and mosquito tissue have been shown to be sources of these apoptosis triggers. Sources of nitric oxide Plasmodium gametocytes are imbibed with a blood meal that, in addition to infected and uninfected erythrocytes, contains white blood cells, platelets and serum. Malaria infection elevates the level of cytokines such as TNF-a. IL-1 and IFN-c that are known inducers of iNOS expression in mouse macrophages [51]. This induction acts synergistically with haemozoin in mouse [52], but not human, macrophages [53]. WBCs thus represent an endogenous source of NO. Cao et al. [38] reported that infected mouse serum, obtained 5 days after P. yoelii infection, blocks gametocyte infectivity to the mosquito via nitric oxide production and inactivation of gametocyte activity has been associated with the presence of WBCs. Furthermore, WBCs have been shown to remain active in the blood bolus for several hours and thus could contribute to killing mechanisms that operate in the mosquito midgut [39,54]. It is likely that they are also active in gametocytaemic blood that is cultured to produce ookinetes in vitro. Previously, our data had shown that removal of WBCs from in vitro culture caused significant (13%), reduction in the number of apoptotic ookinetes (Al-Olayan and Hurd, unpublished). Here we confirm this finding. In both cases however the difference that the presence of WBCs made, to the proportion of ookinetes displaying markers of apoptosis was small. Therefore we note that the presence of WBCs is unlikely to account for induction of apoptosis in all the ookinetes in our untreated samples in vitro as this ranged from approximately 15 to 40% within 18 h of the culture of gametocytaemic blood, and rose steadily to a plateau that could be as high as 80% after 24 h. It is possible that the production of NO from WBCs might be low when cultured at 19uC (optimal developmental temperature for P. berghei ookinetes) and the NO they produce could play a more important role within mosquitoes, or in cultures kept at the higher temperatures conducive to the development of ookinetes of other species of malaria. However, within our system, we suggest there may be additional sources of NO, or other external triggers, that induce a large proportion of P. berghei ookinetes to undergo apoptosis. PLoS ONE | www.plosone.org 8 September 2010 | Volume 5 | Issue 9 | e12634