Mutant/mutation
The mutant lacks expression of Pb115

Protein (function)
PBANKA_0931000 (Pb115) is among a group of genes with the following features: conserved in Plasmodium genomes; sexual-stage expression; and containing the sequence for a putative signal peptide or at least one transmembrane domain and with a predicted size of 115 kDa. As shown in the predicted protein features in PlasmoDB, the predicted protein contains a membrane lipoprotein lipid attachment site at the N-terminus and a major facilitator superfamily (MFS) general substrate transporter domain (P value 1.05e-14) near the C terminus. The encoded protein contains 12 transmembrane domains, six within the MFS domain

Phenotype
Normal number of gametocytes and gametes were formed. The Δpb115 parasites showed significant defects in the interactions between the male and female gametes, and as a result, very few zygotes were formed in ookinete cultures.
During our in vitro culture of ookinetes, the zygote numbers formed at 2 h and ookinete numbers at 24 h in the two Δpb115 clones suffered similar levels of reduction (∼95%) compared to the WT parasites.
The Δpb115 parasites showed significant defects in the interactions between the male and female gametes, and as a result, very few zygotes were formed in ookinete cultures. Male gametes from both WT and Δpb115 parasites exhibited similar motility and interaction with the RBCs, as evidenced by the similar numbers of exflagellation centers formed. In WT parasites, male–female attachments (for >3 s) were readily observed; on average 22 attachments were seen in five experiments. By contrast, the number of gametes forming male–female attachments in the Δpb115 parasite lines was drastically reduced (five observed in eight experiments) (P < 0.01), indicating that Δpb115 gametes were defective in recognition and attachment.
Mosquitoes fed on Δpb115-infected mice showed 74% reduction in the prevalence of infection and 96.5% reduction in oocyst density compared to those fed on wild-type P. berghei-infected mice

Additional information
Cross fertilization with the male-defective Δpbs48/45 line and the female-defective Δpfs47 line further indicated that the fertilization defects of the Δpb115 lines were present in both male and female gametes.
We evaluated the transmission-blocking potential of Pb115 by immunization of mice with a recombinant Pb115 fragment. In vivo mosquito feeding assay showed Pb115 immunization conferred modest, but significant transmission reducing activity with 44% reduction in infection prevalence and 39% reduction in oocyst density.

Analysis of a mutant expressing a C-terminal HA-tagged version of Pb115 (RMgm-4687) showed the following and using antibodies against Pb115and HA (IFA and Western analyses):
Pb115 was expressed in both asexual stages (schizonts) and sexual stages (gametocytes, gametes, and ookinetes), and was localized on the plasma membrane of gametes and ookinetes.

In both schizonts and gametocytes, Pb115 fluorescence dispersed throughout the cytoplasm, sometimes with a speckled appearance. In female gametes, the Pb115 signal was found to be associated with the PM only, whereas in male gametes, it was associated with both the flagella and the residual body. Consistent with the Western analysis showing primary association of Pb115 with the PM in ookinetes, IFA also showed association of Pb115 with the PM of ookinetes with the signals partially being co-localized with those of PSOP25. To differentiate internal from external membrane localization, IFA was performed without membrane permeabilization. In this case, schizonts and gametocytes were not labeled, indicating that the Pb115 protein was not localized outside of the membrane of the iRBC. During gamete development, the female gametes showed a similar localization pattern regardless of membrane permeabilization status, demonstrating external membrane localization of the Pb115 on female gametes. Similarly, in male gametes, the fluorescent signals were detected on both residual bodies and flagella-like male gametes. Again, Pb115 was clearly localized on the PM of ookinetes. IFA with developmental stages of the Pb115-HA line using anti-HA mAb showed highly similar patterns of Pb115 localization.

Other mutants
a mutant expressing a C-terminal HA-tagged version of Pb115 (RMgm-4687)