cGMP-PDE (A) and cAMP-PDE (B) activity was measured in membrane fractions from various purified parasite stages in wild type and pdeδ parasites. (C) Total intracellular cGMP concentration was measured in 24 h paramagnetic bead-purified ookinetes. For all datasets the graphs show the mean of three independently purified samples in each case, with error bars indicating standard deviations.

(A) Sagittal section through wild type ookinete. (B). Detail of wild type apical complex. (C) Detail of pdeδ apical complex. (D) Detail of pdeδ subpellicular microtubules in transverse section (detail of F). (E) Detail of pdeδ IMC (detail of F). (F). Sagittal section of whole “round” form pdeδ ookinete. Arrows point to interruptions in the IMC (C, E and F). Scale bars denote 500 nm in all images. Abbreviations: ApC; Apical Complex, Co; Collar, Cr; Crystalloid, Pg; Pigment Vacuole, IMC; Inner Membrane Complex, Mi; Micronemes, MTL; Subpellicular Microtubules in Longitudinal Section, MTT; Subpellicular Microtubules in Traverse Section, Nu; Nucleus, OM; Outer Pellicle Membrane.

(A) Male gamete formation (exflagellation), (B) macrogamete-to-mature ookinete conversion rate (determined as % of “banana”-shaped cells stained with female-derived cell specific antibody) and (C) average oocysts numbers on midguts dissected 10 days post infection were compared. Data were from 3 independent experiments and error bars show standard deviations. Different gliding speeds of wild type (D) and gcβ (E) ookinetes in Matrigel™ is illustrated with representative frames from time lapse videos (Videos S1 and S2). The black arrow marks the apical end of the ookinete and scale bar denotes 10 µm. (F) Speed of individual ookinetes (diamonds) from 24 h cultures, measured over a 10 min period in 3 mutants compared to wild type. The thick black lines denote mean. (G) Impact of Cmpd 1 on gliding of wild type ookinetes. * = significantly different, Student's T-test, p≤0.01.

(A) Male gamete formation (exflagellation), (B) macrogamete-to-mature ookinete conversion rate (determined as % of “banana”-shaped cells stained with female-derived cell specific antibody) and (C) average oocysts numbers on midguts dissected 10 days post infection were compared. Data were from 3 independent experiments and error bars show standard deviations. (D) Giemsa-stained blood films illustrating normal stages of ookinete differentiation and aberrant forms additionally present in pdeδ ookinete cultures. Scale bars indicate 2 µm. (E) Ookinete stages as a percentage of the total number of female derived cells on Giemsa-stained blood films. Slides were prepared from in vitro cultures at the times indicated. Error bars represent standard deviations in three biological replicates. (F) Forward motility and (G) rotation speed of pdeδ and wild type ookinetes as a function of cell length from 15–22 hour in vitro cultures were determined using the Matrigel™ motility assay. Forward speed was averaged over 10 minutes. The rotational component of ookinete motility was determined as (number of turns×circumference at widest point) and averaged over 10 min. Best fit and correlation coefficient r are given for pdeδ ookinetes only. Examples of motility of pdeδ ookinetes with normal, stumpy and round morphology are shown in Videos S3, S4 and S5.

(A) Conversion rate to mature ookinetes was scored for 22 h cultures, to which Cmpd 1 or vehicle had been added from 3 hours of culture. (B) Southern blot analysis comparing genotypes of three pdeδ gcβ double knockout clones (p/g cl. 3–5) obtained from a cross to the parental mutants and wild type. Genomic DNA was digested and probed as for single mutants (Figs. 2 and 5). Effect of pdeδ gcβ double mutant (C), and pdeδ cdpk3 double mutant (F) on conversion to morphologically mature ookinetes in 22-hour cultures. (D) Effect of pdeδ gcβ double mutant on average gliding speed in Matrigel™. (E) Three parasite clones obtained from the pdeδ and cdpk3 genetic cross (p/c) were genotyped by Southern hybridization, along with wild type and parental clones. To examine disruption/deletion of the targeted locus genomic DNA was digested and probed as in Fig. 5 for pdeδ, or as described previously (Siden-Kiamos et al., 2006) for cdpk3. In the left panel a 4.3 kb band is recognised by a 5' probe in EcoRI-digested DNA in wild type and appearance of a larger 5.8 kb fragment is diagnostic of pdeδ deletion. In the right panel a 3 kb and a 0.9 kb fragment recognised in EcoRI/HindIII digested DNA by a cdpk3-specific probe indicate an intact cdpk3 locus only in wild type and pdeδ, and there absence indicates deletion of cdpk3 in all other clones (see Figure S2 G). (G) Effect of the pdeδ cdpk3 double mutant on average gliding speed of individual, morphologically mature ookinetes after 15–18 hour of culture. Statistical analysis was done by two-tailed t-test), p≤0.02 (A, C, D, F and G). For all graphs error bars show standard deviations in 3 biological replicates, * = significantly greater than pdeδ. Φ = significantly less than wild type. Ψ = significantly greater speed than cdpk3 ookinetes.