was added to ookinete medium at different concentrations ranging from 10 mM to 4 mM and ookinetes incubated at 19uC for periods of 15 min to 24 h. Following incubation, ookinetes were checked for signs of condensed chromatin, caspase-like activity, transloca- tion of phosphatidylserine to the outer plasma membrane, loss of mitochondrial membrane potential or loss of plasma membrane integrity. The proportion of ookinetes displaying these markers was compared with those incubated in RPMI or Schneider’s medium alone. Investigations were conducted in a similar manner using two other donors, SNOG and SNAP to demonstrate that induction of apoptosis markers could occur with other NO donors. NO released by NO donors The Griess reaction [87] was used to make a comparison of the production of NO, in the form of nitrite, by SNP, SNAP and SNOG. Donors were dissolved in supplemented RPMI-1640 (without phenol red) at concentrations ranging from 10 mM to 4 mM and incubated at 19uC for various periods up to 24 h. Nitrite production was determined using a standard curve made from serial dilution of sodium nitrite (range 200–3.125 mM). The effect of reactive oxygen species (ROS) on ookinetes in vitro Two sources were used to provide reactive oxygen species in the RPMI incubation medium; 3, 4-dihydroxy-L-phenylalanin (L- DOPA) and hydrogen peroxide (H2O2) at different concentrations ranging from 10 mM to 1 mM. Ookinetes were incubated at 19uC with or without these compounds for 1 h or 4 h and markers for apoptosis-like cell death monitored. The effect of nitric oxide synthase activity on ookinetes in vivo Three to six day-old female An. stephensi (Dubai) were starved overnight before blood feeding. Three groups of females were fed gametocytaemic mouse blood via an artificial membrane feeder (Haemotek Membrane Feeding System). The NOS inhibitor, L- NAME (1 mg/ml) was added to the blood in one feeder, 1 mg/ml of D-NAME, the inactive isomer, to the second feeder and gametocytaemic mouse blood alone was added to the third feeder, as a control. Fully engorged mosquitoes were separated and transferred to cages supplied with 10% glucose solution and maintained at 19–20uC. Midguts from five mosquitoes per group were dissected at 18 h and 20 h post-feeding, the contents stained with an equal volume of acridine orange (25 mg/ml PBS) and immediately observed using fluorescein filters. The percentage of ookinetes with condensed chromatin per midgut was determined in each of 3 experiments and 200 parasites were counted each time. In a further experiment that followed the same protocol, five mosquitoes from each treatment group were dissected at 15, and 20 h post-feeding and 200 ookinetes in the blood meal of each mosquito were examined for caspase-like activity using Caspa- TagTM a fluorescein caspase activity kit (Intergen Company) according to manufacturers instructions, except that incubations were performed at 19–20uC. The proportion of ookinetes that had a compromised membrane was monitored by observing PI penetration. This protocol was repeated with another generation of mosquitoes except that 50 infected mosquitoes were maintained for twelve days post-infection. A record was kept of the number of mosquitoes dying during the experiment. The remaining mosqui- toes from each group were dissected, their guts stained with 0.05% mercurichrome to enhance oocyst visualization and the number of oocysts per midgut counted. Apoptosis Triggers in Malaria The effect of nitric oxide donors on mosquito fitness To determine the effect on mosquito longevity of the presence of additional NO in a mosquito midgut during blood feeding, mosquitoes were fed on P. berghei -infected blood through an artificial membrane feeder. Three to six day-old mosquitoes from the same generation were either fed on blood from a gametocy- taemic mouse containing 100 mM SNP or the infected blood alone. The experimental protocol was repeated with a new generation of mosquitoes, this time with the addition of 1 mM SNP to blood in one of the membrane feeders. Mosquitoes were maintained in standard conditions in the insectary and fed ad libitum on 10% glucose supplemented with 0.05% para-aminoben- zoic acid, and antibiotics (10000 U penicillin and 7 mg strepto- mycin/l) and deaths recorded daily. The effect of nitric oxide donors on Plasmodium development in vivo Mosquitoes were fed via an artificial membrane feeder on blood from a gametocytaemic mouse with or without the addition of SNAP at a final concentration of 100 mM. Mosquitoes were dissected 16–18 h post-blood meal and the number of ookinetes in the midgut lumen, diluted 1:4 with PBS, was counted. This experiment was repeated five times and the prevalence and intensity of ookinete infection in the NO donor-treated and untreated mosquitoes was compared. Statistical analysis Data were analysed using two-way ANOVA with a general linear model for two parameters, or one-way ANOVA for one parameter. Data were tested for normality using Shapiro-Wilks’ W or Anderson-Darling normality tests and, where necessary, percentage data were first transformed using arcsine transforma- tion before being submitted to analysis. Follow-up Tukey’s paired comparison tests were run when significant differences were identified by ANOVA. Following log transformation of data, a t- test was used to compare means of ookinete prevalence and intensity in mosquitoes fed a blood meal with or without a NO donor. Kaplan-Meier analysis was used to analyse mosquito survivorship. An alpha value was set at 0.05 for all analyses. Analyses were performed using a Minitab 13.1 or Statistica’ 99 Edition Package, version 5.5 for Windows (StatSoft Inc., Tulsa: OK, USA). Supporting Information Figure S1 The effect of removal of WBCs from gametocytaemic blood culture on ookinete apoptosis at 0.25, 3 and 6h post collection. Ookinetes showing chromatin condensation were identified by staining with acridine orange. These data represent a mean of three experiments (n = 3) each with two replicates of counts and each count = 50 ookinetes. Error bars represent SEM. Bars with different letters are significantly different. Found at: doi:10.1371/journal.pone.0012634.s001 (0.08 MB TIF) Figure S2 The effect of NO donors on the induction of apoptosis-like death in P. berghei ookinetes. Ookinetes were incubated in RPMI with or without the addition of NO donors at different concentrations or for different time periods and then examined for the presence of markers of apoptosis A: The effect of 2mM SNOG on ookinetes, n = 5 with 100–200 ookinetes examined in each experiment. B: The effect of different concentration of SNAP on ookinetes incubated for 1h; n = 3, 75 ookinetes were examined in each experiment. Apoptosis markers PLoS ONE | www.plosone.org 13 September 2010 | Volume 5 | Issue 9 | e12634