Mutant/mutation
The mutant  expresses a C-terminal 3xHA-tagged version of AQP2 and expresses GFP under control of the constitutive eef1a promoter.

Protein (function)
Plasmodium AQP1 is a well-characterized plasma membrane aquaglyceroporin that conducts water, glycerol, and ammonia at high rates. It is expressed throughout parasite asexual blood stages (ABS), sporozoites and infected hepatocytes, and provides parasites access to serum glycerol for membrane lipid synthesis. Whilst the essentiality of AQP1 to ABS is disputed, it is established that the protein is important for progression of hepatic stage infection. A second putative aquaporin, AQP2, has been identified in Plasmodium genomes and reported to be phylogenetically associated more closely to T. gondii AQP1, rather than Plasmodium AQP1. 

From the paper: 
'Phylogenetic analysis along Alphafold structure predictions revealed that AQP2 is distinct from AQP1, other aquaglyceroporins and orthodox aquaporins with regards to both its overall architecture and pore selectivity filter. AQP2 orthologs are found in all plasmodia with available genome sequences. They encode large aquaporin-like proteins of 603 amino acids in P. falciparum (PfAQP2, PF3D7_0810400) and 595 amino acids in P. berghei (PbAQP2, PBANKA_1427100). Multiple sequence alignment identified regions of high conservation mostly associated with predicted a-helixes. One region is substantially divergent between species, both in terms of sequence identity and length: amino acids 110-228 in PfAQP2 and 110-215 in PbAQP2. Insertions in this region cause AQP2s of human parasites of the sub-genus Plasmodium (P. malariae, P. vivax and P. knowlesi) to be considerably longer than other AQP2s. Protein structure predictions made with Alphafold Monomer v2.0 showed that PfAQP2 and PbAQP2, like all aquaporins, have six full transmembrane helices and two half helices, connected by five loops. The variable region corresponds to extracellular loop A that lies between helixes 1 and 2. Loop A is highly charged, with roughly 40% of amino acids carrying either a positive or negative charge. Intracellular loop D is longer than extracellular loop C, the inverse of loop lengths of Plasmodium AQP1. The characteristic aquaporin pore forming NPA motifs are present in all AQP2s, in half-helices 1 and 2 that are part of loops B and E, respectively. While the first NPA motif is present as a canonical NPA. Finally, in all AQP2s, the canonical arginine residue that determines the selectivity filter, invariably found directly adjacent to the second NPA motif, is replaced with phenylalanine (R>F).'

Phenotype
Analysis of a mutant lacking expression of AQP2 (RMgm-5354) showed the following: 
'Normal asexual blood stage development, gametocyte, ookinete and oocyst production. However, sporozoite formation inside oocysts is severely affected. Both Δpbaqp2 oocyst and salivary gland sporozoites were severely reduced.
DAPI staining of Δpbaqp2 oocysts at day 15 after feeding revealed that nuclei were large and disorganized unlike wild type oocysts that had highly organized nuclei, characteristic of sporozoites that had budded off from the central sporoblastoid body.
Almost total absence of Δpbaqp2 sporozoites in the mosquito salivary glands. No transmission to mice by mosquito bite'.

Western blot analysis of aqp2::3xha parasite protein extracts using an antibody against the HA epitope showed the expected ca. 73 kDa protein predominantly in gametocytes – already prior to gametogenesis activation and, at a lower abundance, in mature ookinetes.
In indirect immunofluorescence assays of aqp2::3xha parasites, AQP2::3xHA was seen to specifically localize to vesicle-like structures in the cytoplasm of gametocytes, ookinetes and midgut sporozoites. The number and appearance of these structures varied from 1-2 large spots and several smaller ones in gametocytes, 2-3 and occasionally 1-4 spots in ookinetes, and 2-3 spots in sporozoites. In ookinetes, this pattern resembles that of crystalloid, an elusive organelle exclusive to ookinetes and  young oocysts. Although these spots were often seen adjacent to the hemozoin crystals, the waste product of hemoglobin metabolism and hallmark of the ABS food vacuole and the crystalloid, they never fully colocalized with the hemozoin or located amongst the hemozoin crystals where the crystalloid is found. Indeed, the diameter of these structures in ookinetes (0.2-0.7 μm) was smaller than that of crystalloids (0.3-1.15 μm) as determined by IFAs of the recently identified crystalloid proteins CRYSP and CRONE. The diameter of these structures was more uniform in all sporozoites examined (0.25-0.4 μm).

Additional information

Other mutants