Figure 1.
PF16 gene deletion reduces male gamete flagellar motility and ookinete (zygote) formation.
A. Differentiation of male gametocytes to male gametes (as determined by quantifying exflagellation centres) is similar in both wild type Plasmodium (WT) and two pf16 mutant clones (pf16.3 and pf16.4) suggesting the emergence of male gametes is not affected (P = 0.3699 and P = 0.3789 for clones 3 and 4 respectively). Three independent replicates are plotted (established on 10 independent fields on a slide, with a 40x objective). B. Average number of motile gametes after emergence is reduced in both mutant clones (pf16.3 and pf16.4) compared to wild type (WT) P = 0.0019 for both clones. Three independent replicates of 50 microgametes each were counted. C. Quantification of ookinete conversion (ratio of ookinetes to round cells expressed as a percentage). There was a decrease in the frequency of ookinete formation in both mutant clones when compared to wild type (WT). (P = 0.0017 and P = 0.0045 for clone 3 and 4 respectively). Three independent replicates of 100 events were counted (macrogametes + ookinetes). D, E, F. The male gamete flagellum was analysed for speed (D; P<0.0001 for both clones), beat amplitude (E; P<0.0001 for both clones) and beat frequency (F; P = 0.0180 and P<0.0001 for clones 3 and 4 respectively) in wild type and the two pf16 mutant clones. In all these analyses the values were significantly lower (P<0.001) for both the mutant clones in comparison to wild type. See also Supplementary movie VS1and VS2. 30 male gametes from three independent samples were quantified for each analysis.
Figure 2.
Plasmodium PF16 is expressed in the male gametocyte and gametes.
Immunolocalisation of GFP in PF16-GFP-expressing parasites, both in the male gametocyte (0 min) and during male gamete formation during the exflagellation process (5–15 min). The parasites were co-stained with an anti tubulin antibody (red). Hoechst staining (blue) indicates the presence of DNA. Note the increase in the intensity of GFP localisation with the increase in time, and at the end of exflagellation when motile gametes form (15 min). Scale bar, 5 µm.
Figure 3.
Comparison of the microgametocytes and microgametes of wild type P. berghei and pf16 mutant P. berghei by electron microscopy.
A. Section through a wild type microgametocyte showing the large central nucleus (N) and longitudinal- and cross-sections of axonemes (A) within the cytoplasm. Scale bar, 1 µm. Insert. Enlargement of an axoneme cross-section from a mutant microgametocyte showing the presence of a single central microtubule (9+1). Scale bar, 100 nm. B. Detail of the cytoplasm of a wild type microgamete illustrating the variability of the axoneme structure: some have two central microtubules (arrow), others have no central tubules (arrowhead), some peripheral duplet microtubules form an “S” shaped structure (double arrowheads). Scale bar, 100 nm. C. Longitudinal section through a wild type microgamete showing the undulating axoneme forming the flagellum (F) with the closely adhering electron-dense nucleus (N). Scale bar, 100 nm. D. Cross-section through the central region of a wild type microgamete showing the nucleus and the normal 9+2 organisation of the axoneme. Scale bar, 100 nm. E-G. Cross-sections through microgametes of the PF16 mutant showing the variable axonemal appearance with a few showing the normal 9+2 structure (E), the majority showing 9+1 (F) and a number with a 9+0 appearance (G). Scale bar, 100 nm. H. Quantification of microtubule arrangements in the axonemes of wild type (WT) and pf16 mutant microgametes.
Figure 4.
Comparison of Structure and Sequence analyses of Plasmodium berghei PF16.
A. Top view – cartoon representation of homology models of Plasmodium and Chlamydomonas PF16, coloured by spectrum from N-terminus (blue) to C-terminus (red). Middle view – electrostatic surfaces of the modelled proteins (view rotated 180° around the y axis with respect to top view). The accessible surface area is coloured according to electrostatic potential calculated using APBS from -10kBT/e (red) to + 10kBT/e (blue) [47]. Bottom view – surface representation of Plasmodium PF16 displaying exposed lysine residues. Those shown in green represent lysine residues conserved in Chlamydomonas PF16, with those in orange representing lysine residues found only in Plasmodium PF16. Figures produced using PyMol (The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC). B. Maximum likelihood tree of proteins from 33 different species. Mouse SPAG6 (blue), P. berghei PF16 (red) and Chlamydomonas PF16 (green) are highlighted. The accession number of each species is given in the supplementary material. The tree was made using the WAG model of protein evolution with gamma distributed rates at sites and 1000 bootstrapped replicates implemented in PhyML [48]. Branches with bootstrap support less than 70% are collapsed. Asterisk (*) signifies bootstrap support greater than 90%. The scale bar indicates 0.2 substitutions per site.