Mutant/mutation
The mutant lacks expression of PF16 (PBANKA_091740; PF11_0318)
Note that the PF16 gene is distinct from Pfs16, another gametocyte specific gene identified in P. falciparum (PFD0310w).

Protein (function)
All eukaryotic flagella contain an axoneme, a structure consisting of a central apparatus (a pair of microtubules named C1 and C2) encircled by nine doublet microtubules. PF16 is an important Armadillo (ARM)-repeat protein of the central apparatus of eukaryotic flagella. ARM-repeats consist of a ~42-amino acid structurally conserved repeating motif named after the Drosophila segment polarity gene Armadillo (mammalian homologue, b-catenin). Proteins containing ARM-repeats have diverse roles in eukaryotes, including cell signalling, cytoskeletal organisation and regulation of gene expression. Inactivation of the Chlamydomonas PF16 gene led to loss of the central pair of microtubules and abnormal flagellar function, showing that PF16 is required for flagellar motility and stability of the central apparatus C1 microtubule. Disruption of SPAG6, the mouse PF16 orthologue, results in male infertility, due to loss of sperm motility.RNAi of PF16 in the flagellated protozoan Trypanosoma brucei demonstrates a conserved role in flagellum-dependent motility.

Phenotype
The phenotype analyses indicate that PF16 plays a crucial role maintaining the correct microtubule structure in the central apparatus of the axoneme of the male gamete (see also 'Additional information'). Mutant male gametes show abnormal flagellar movement and reduced fertility, but lack of PF16 expression does not lead to complete sterility.

Additional information
PF16 is expressed in male gametocytes and male gametes as shown by analysis of a mutant parasite that expresses a GFP-tagged form of PF16 (see mutant RMgm-589).

Male gametes showed reduced motility and fertility, with 50% of male gametes being immotile. The flagellar beat pattern of mutant male gametes differed from wild type; it was characterised by a significantly lower beat frequency and beat amplitude together with a significantly reduced speed.

The formation of male gametes was analysed by electron microscopy. The development of the axonemes of Plasmodium male gametes is unusual, as they do not arise from a classical centriole/basal body with nine concentric triple microtubules. Instead, there is an electron dense structure in which nine single microtubules have been identified. This can result a slightly uncoordinated assembly process where the majority of axonemes consist of a central pair of microtubules (central apparatus) encircled by 9 doublets of microtubules with associated dynein arms. However, some axonemes lack the central pair (9+0) or the peripheral doublets forming an ‘S’ shape rather than a circle. Similar structures were observed in the  mutant, however a significant number of the axonemes have one missing central tubule (9+1). The mature microgamete consists of an elongated, undulating axoneme with a flattened electron-dense nucleus in the central region, which follows the contours of the axoneme, all enclosed by a unit membrane. In  wild type parasites, the vast majority (96%) displayed the typical 9+2 arrangement with only rare examples of 9+0 (2%) or 9+1 (2%). However, the mutant showed a marked increase in the number of  microgametes with atypical axonemes. Only 12% had the normal 9+2 structure while 21% had 9+0 and 67% had a 9+1 structure. Therefore, the loss of PbPF16 results in increased numbers of abnormal microgametes with the majority lacking one central microtubule (9+1) and a significant number lacking both central microtubules (9+0).

Other mutants
RMgm-589: a P. berghei mutant expressing a GFP-tagged form of PF16