activity of NOS, using transgenic technology, in order to induce apoptosis in most, or all, of the ookinetes before they can traverse the midgut epithelium. We would not anticipate creating undue fitness affects by this strategy because the presence of extra NO, caused by feeding a NO donor with the blood meal, did not affect mosquito longevity, at least at the concentrations of nitrite generated in our experiments (which, in the case of 1 mM SNP is estimated to be approximately 10 mM and thus roughly equivalent to the concentration produced by SNAP that significantly reduced prevalence and intensity of infection). In addition, it may be possible to circumvent some of the loss of reproductive fitness that occurs when mosquito immune responses are up-regulated due to infection if all parasites die very early post- infection and are unable to invade the mosquito midgut [83,84]. Enhancing the natural apoptotic death of ookinetes in the mosquito midgut lumen could be an important transmission blocking strategy. Experimental evidence available to date does not supply clues as the evolutionary history or significance of the existence of an apoptosis phenotype in malaria ookinetes. We can speculate that it represents an adaptive, density control strategy that may limit the adverse effects of high infection intensities in the vector and benefit other members of the clone [41]. This could operate via the triggering of mosquito NOS activity in a density dependent way. Equally, density dependent triggers occurring in the mammalian host could be operative in the blood bolus in the midgut lumen. Alternatively, our view of apoptosis in protists may be coloured by knowledge of its functions in metazoans and we may be observing an unavoidable consequence of ookinetes being under oxidative stress or containing defective biochemical pathways and having passed the point of rescue. The latter implying that apoptosis was a mechanism that evolved to be used altruistically in multicellular organisms. Materials and Methods Products were purchased from Sigma-Aldrich Company Ltd. (Dorset, England, UK), unless stated otherwise. Ethics statement All animals were handled in strict accordance with good animal practice as defined by the UK Animal (Scientific Procedures) Act 1986, and all animal work was approved by the UK Home Office, license number PPL 40/2997, and was also approved by the University of Keele Animal Care and Ethical Review Committee. Development of ookinetes in vitro Plasmodium berghei ANKA 2.34 was maintained in Charles River- Derived (CD1) mice by serial passage for a maximum of 8 passages. Mice were pre-treated by interperitoneal injection of phenylhydrazine two days prior to infection and thick and thin blood smears checked for the presence of infected red blood cells and exflagellating gametocytes from day 3 post infection. Blood was collected by cardiac puncture into a heparinized syringe when parasitaemia reached 5–15% and gametocytaemia was approxi- mately 1–2%. Blood was immediately placed in T25 culture flasks and incubated for 16–19 h at 19uC in RPMI 1604 medium, supplemented with 0.4 mM hypozanthine, 24 mM sodium bicarbonate, 10,000 U penicillin and 10 mg streptomycin/l and 10% foetal calf serum (ookinete medium), as described previously [85], to allow the development of ookinetes. In the majority of experiments, ookinetes were separated from blood cells using MidiMacs (Miltenyi Biotec, Germany) magnetic columns [86], pelleted and resuspended in RPMI-1640 medium or Schneider’s medium, before being subjected to different assays. For studies of Apoptosis Triggers in Malaria loss of mitochondrial membrane potential, red blood cells were instead lysed by incubation on ice with 170 mM NH4Cl. Ookinetes were pelleted by centrifugation at 800 g for 10 min and ghost red blood cells removed with the supernatant. Removal of white blood cells from infected blood To determine the effect of removal of white blood cells (WBCs) from infected blood prior to incubation, blood was collected from infected mice and one half passed through a CF11 column (Whatman, England) [38] to remove WBCs. Infected blood, with or without WBCs was incubated in ookinete culture medium for 16 h at 19uC, and then ookinetes were collected and further incubated in Schneider’s insect medium for 0.25, 3 and 6 h before examining for markers of apoptosis. Assays for markers of apoptosis Morphological markers for apoptosis were detected using slight adaptations of techniques applicable for metazoans, as described in [18]. Briefly, ookinetes exhibiting nuclear chromatin conden- sation were detected using acridine orange at a working concentration of 2.5 mg/ml. The nuclei of healthy ookinetes remained unstained whereas nuclei with condensed chromatin stained green and nuclear fragmentation was occasionally visible. Activated caspases-like molecules that have been associated with PCD in P. berghei ookinetes were detected using the CaspaTag fluorescent caspase activity kit (Chemicon International, USA) as described in [16,18]. This contains the pancaspase inhibitor carboxy- fluorescein -Val-Ala-Asp- fluoromethyl ketone (FAM.VAD.fmk). PI was used to exclude cells with compromised membranes. Translocation of phosphatidylserine to the outer surface of the cell membrane was detected with annexin-V FITC apoptosis detection kit according to manufacturer’s instructions but incubation took place at 19uC. Ookinetes with compromised membranes were detected using PI. Ookinetes labeled with a green band around the plasma membrane and no PI staining were considered positive whereas those with both annexin and PI labeling were discounted because annexin could have penetrated the cell and stained PS that had not translocated. Thus late apoptosis could not be distinguished from death by non-apoptotic means (see [18] for a discussion of annexin staining and signs of early or late apoptosis). Loss of mitochondrial trans-membrane potential was detected using a MitoProbeTM JC-I assay kit (Molecular probes, UK) according to manufacturer’s instructions except that incubation with JC-1 was carried out at 19uC [18]. This kit included the mitochondrial membrane potential disrupter, CCCP, which was used to create loss of MOMP in ookinetes. In appropriate experiments, one group of ookinetes was incubated with CCCP for 30 min prior to examination for MOMP loss. Fluorochrome markers of apoptosis were detected using a Leica DM IRB inverted fluorescence microscope under oil immersion (x1000) and recorded on Leica FW 4000 image software (Leica Microsystems, Germany). Viability assays Cells with compromised membranes were detected by staining with either 0.1% erythrosin-B, which stained cells pink (visible under white light), or 250 mM PI. The effect of nitric oxide on ookinetes in vitro In order to determine whether exposure to NO induces the display of markers of apoptosis, ookinetes were incubated with the NO donor SNP following elution from a magnetic column. SNP PLoS ONE | www.plosone.org 12 September 2010 | Volume 5 | Issue 9 | e12634