Naturally Occurring Triggers that Induce Apoptosis-Like Programmed Cell Death in Plasmodium berghei Ookinetes Medhat Ali1,2, Ebtesam M. Al-Olayan1,3, Steven Lewis1, Holly Matthews1, Hilary Hurd1* 1 School of Life Sciences, Keele University, Keele, United Kingdom, 2 Department of Zoology, Ain Shams University, Cairo, Egypt, 3 Department of Zoology, King Saud University, Riyadh, Saudi Arabia Abstract Several protozoan parasites have been shown to undergo a form of programmed cell death that exhibits morphological features associated with metazoan apoptosis. These include the rodent malaria parasite, Plasmodium berghei. Malaria zygotes develop in the mosquito midgut lumen, forming motile ookinetes. Up to 50% of these exhibit phenotypic markers of apoptosis; as do those grown in culture. We hypothesised that naturally occurring signals induce many ookinetes to undergo apoptosis before midgut traversal. To determine whether nitric oxide and reactive oxygen species act as such triggers, ookinetes were cultured with donors of these molecules. Exposure to the nitric oxide donor SNP induced a significant increase in ookinetes with condensed nuclear chromatin, activated caspase-like molecules and translocation of phosphatidylserine that was dose and time related. Results from an assay that detects the potential-dependent accumulation of aggregates of JC-1 in mitochondria suggested that nitric oxide does not operate via loss of mitochondrial membrane potential. L-DOPA (reactive oxygen species donor) also caused apoptosis in a dose and time dependent manner. Removal of white blood cells significantly decreased ookinetes exhibiting a marker of apoptosis in vitro. Inhibition of the activity of nitric oxide synthase in the mosquito midgut epithelium using L-NAME significantly decreased the proportion of apoptotic ookinetes and increased the number of oocysts that developed. Introduction of a nitric oxide donor into the blood meal had no effect on mosquito longevity but did reduce prevalence and intensity of infection. Thus, nitric oxide and reactive oxygen species are triggers of apoptosis in Plasmodium ookinetes. They occur naturally in the mosquito midgut lumen, sourced from infected blood and mosquito tissue. Up regulation of mosquito nitric oxide synthase activity has potential as a transmission blocking strategy. Citation: Ali M, Al-Olayan EM, Lewis S, Matthews H, Hurd H (2010) Naturally Occurring Triggers that Induce Apoptosis-Like Programmed Cell Death in Plasmodium berghei Ookinetes. PLoS ONE 5(9): e12634. doi:10.1371/journal.pone.0012634 Editor: Gordon Langsley, INSERM U1016, Institut Cochin, France Received March 30, 2010; Accepted August 10, 2010; Published September 9, 2010 Copyright: 2010 Ali et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funding for insectary facilities was provided by The Wellcome Trust, (069162/Z/02/Z), www.wellcome.ac.uk. EMA and MA received scholarships from the Saudi Arabian Government and the Egyptian Government, respectively. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript Competing Interests: The authors have declared that no competing interests exist. * E-mail: h.hurd@keele.ac.uk Introduction There is a well established body of literature that recognizes programmed cell death (PCD) as a signal dependent, active process of cell suicide that occurs in unicellular eukaryotes as well as metazoans. Its origin predates the evolution of multicellularity and may have arisen during the endosymbiotic incorporation of the mitochondrial precursor into the ancestral eukaryote [1–4]. Galluzzi and colleagues speculate that many of the cell death regulators of metazoan cells may originally have been involved in stress regulation [5]. Three types of PCD have been well characterized in metazoans, including type 1 PCD, or apoptosis [6,7] however, it is now recognized that overlapping pathways leading to different types of PCD death exist, and more complex nomenclature has been suggested [8]. Despite this, new recommendations concerning death-related terminology have recently been published and the used of just the three terms, necrosis, apoptosis and autophagic cell death are recommended [9]. Most of the features that are signatures of apoptosis in metazoans are also present in unicellular eukaryotes, including loss of mitochondrial outer membrane permeability (MOMP), nuclear chromatin condensation, cyto- chrome c release, DNA fragmentation and translocation of phosphatidylserine (PS) to the outer cell membrane (reviewed in [10,11]). Despite this, much of the apoptotic molecular machinery common to multicellular eukaryotes is absent, and different pathways may be leading to the same endpoint [11–13]. Markers of apoptosis have been described in both the vertebrate and vector stages of the malaria parasite [14–17] although, as discussed by Arambage and colleagues [18], conflicting results have emerged from different laboratories, particularly regarding apop- tosis in erythrocytic stages. In the mosquito midgut lumen, motile zygotes or ookinetes are formed around 18 h post-infection and move out of the blood meal bolus before traversing the midgut epithelium to form oocysts beneath the basal lamina. Within the midgut lumen of Anopheles stephensi, over 50% of ookinetes of the rodent malaria, Plasmodium berghei, exhibit markers of apoptosis by 24 h post-infection [16].This cell suicide may help to explain the huge loss of parasites between the ookinete and oocyst stage that has been reported during many Plasmodium/mosquito associations [19]. PLoS ONE | www.plosone.org 1 September 2010 | Volume 5 | Issue 9 | e12634