Apoptosis Triggers in Malaria Figure 6. The effect of L-DOPA on the induction of apoptosis in P. berghei ookinetes in vitro. A: Ookinetes were incubated in supplemented RPMI containing different concentration of L-DOPA for 1 h (bars with small dots), 2 h (bars with strips) and 4 h (bars with large dots) and the proportion containing condensed nuclear chromatin was calculated. B: Ookinetes were incubated with L-DOPA for 4 h and those with compromised membranes (dead ookinetes) were identified by staining with erythrosin B (grey bars), while ookinetes showing chromatin condensation were identified with acridine orange (white bars). Bars are a mean of 3 experiments (n = 3) with at least 50 ookinetes examined in each experiment. Error bars represent SEM. doi:10.1371/journal.pone.0012634.g006 Within the mosquito midgut, Luckhart and co-workers have identified inflammatory levels of reactive nitrogen intermediates in the blood meal [41] with nitrates and metal nitrosyls elevated following an infective blood meal Nitrogen oxides present in blood meals were measured using a nitric oxide analyzer and normalized against heme and were significantly elevated to ,6 mM/heme mM at 12.5 h post infective blood meal [55]. These authors suggested that, in contrast to the situation in the mammalian host, haemoglobin drives the production of nitric oxide metabolites in the reducing environment of the blood meal [24]. NO can react with haemoglobin forming a variety of complexes including oxygen radicals and nitroxyl that are also implicated in apoptosis induction. It would seem that once infected blood enters the midgut, conditions there give rise to the generation of additional NO within the blood bolus. In addition to NO and RNS imbibed with the blood meal, the mosquito midgut cells are themselves a source of NO. Blood meals containing P. berghei induce up-regulation of NOS expression in the midgut of An. gambiae [25] and An. stephensi [26]. This NOS activity has a biphasic induction in An. stephensi with a peak 6 h post- infection, when parasites are developing in the midgut lumen, and PLoS ONE | www.plosone.org 9 September 2010 | Volume 5 | Issue 9 | e12634