Apoptosis Triggers in Malaria Figure 4. The effect of SNP on the survivorship of P. berghei-infected mosquitoes. Four groups of 38–45 mosquitoes were fed on P. berghei-infected mouse blood (solid black lines, 2 groups), or P. berghei- infected mouse blood with the addition of 100 mM SNP (A) or 1 mM SNP (B) (dotted red lines). Mosquito deaths were recorded daily. doi:10.1371/journal.pone.0012634.g004 possible that lower concentration would have done so. RNS and ROS have been identified as inducers of apoptosis in a variety of protozoans and their effect has been reported to be concentration dependent (see reviews [10–12]). For example, H2O2 at low concentrations (500 mM) caused Tritrichomonas foetus to undergo apoptosis-like death whereas at concentration of 8 mM necrotic death was observed [50]. It is worth noting the large variation in the proportion of apoptotic ookinetes observed at the beginning of different experiments. This could be an unavoidable consequence of having to use different mice as the source of infectious blood. We speculate that parameters such as intensity of asexual infection, number of gametocytes present or parameters such as NO or cytokine levels in the mouse plasma may be responsible for these differences. The steady rise in the proportion of apoptotic ookinetes over time may be due to an increase in the level of RNS in the culture medium, or differences in susceptibility of individual parasites to NO/RNS (possibly associated with differences in ookinete age as development is asynchronous). It is known that, in vitro, late erythrocytic stages of P. falciparum are more vulnerable to the effects of NO produced by SNAP than the early (ring) stages and this effect is dose dependent [33]. In this and our previous studies, we note that approximately 20% of ookinetes remain viable even when exposed to inducers of apoptosis for long periods. Herrer- Ortiz and colleagues [28] also found that 28% of P. berghei ookinetes survive the oxidative stress generated by the combined action of L-DOPA and SNP. They suggested that this was due to the presence of adaptive, protective, molecules produced by ookinetes to fight mosquito defenses such as antioxidants. Even though these parasites originated from a single clone some individuals may be more vulnerable than others. The demonstration that NO or ROS will induce apoptosis-like death of ookinetes in vitro did not, however, demonstrate that NO- or ROS-induced apoptosis has a role to play in the life history of the sporogonic stages of the malaria parasite. To support this proposal it was necessary to identify a potential source of NO, RNS or ROS in the mosquito midgut lumen and to demonstrate that redox PLoS ONE | www.plosone.org 7 September 2010 | Volume 5 | Issue 9 | e12634