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Parasites of the genus Plasmodium infect a wide range of mammalian hosts including humans, primates, bats and arboreal rodents. A hallmark of Plasmodium spp. is the very narrow host range, indicative of matching parasite-host coevolution. Accordingly, their respective genomes harbour many unique genes and gene families that typically encode proteins involved in host cell recognition and remodelling. Whether and to what extent conserved proteins that are shared across Plasmodium spp. also exert distinct species-specific roles remains largely untested. Here, we present detailed functional profiling of the female gametocyte-specific ATP-binding cassette transporter gABCG2 in the murine parasite Plasmodium berghei and compare our findings with data from the orthologous gene in the human parasite Plasmodium falciparum. We show that P. berghei gABCG2 is female-specific and continues to be expressed in zygotes and ookinetes. In contrast to a distinct localization to Iipid-rich gametocyte-specific spots as observed in P. falciparum, the murine malaria parasite homolog is found at the parasite plasma membrane. Plasmodium berghei lacking gABCG2 displays fast asexual blood-stage replication and increased proportions of female gametocytes, consistent with the corresponding P. falciparum knock-out phenotype. Strikingly, cross-species replacement of gABCG2 in either the murine or the human parasite did not restore normal growth rates. The lack of successful complementation despite high conservation across Plasmodium spp. is an indicator of distinct adaptations and tight parasite-host coevolution. Hence, incompatibility of conserved genes in closely related Plasmodium spp. might be more common than previously anticipated., (Copyright © 2020 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

The most critical bottleneck in the generation of recombinant Plasmodium berghei parasites is the mandatory in vivo cloning step following successful genetic manipulation. This study describes a new technique for rapid selection of recombinant P. berghei parasites. The method is based on flow cytometry to isolate isogenic parasite lines and represents a major advance for the field, in that it will speed the generation of recombinant parasites as well as cut down on animal use significantly. High expression of GFP during blood infection, a prerequisite for robust separation of transgenic lines by flow cytometry, was achieved. Isogenic recombinant parasite populations were isolated even in the presence of a 100-fold excess of wild-type (WT) parasites. Aquaglyceroporin (AQP) loss-of-function mutants and parasites expressing a tagged AQP were generated to validate this approach. aqp(-) parasites grow normally within the WT phenotypic range during blood infection of NMRI mice. Similarly, colonization of the insect vector and establishment of an infection after mosquito transmission were unaffected, indicating that AQP is dispensable for life cycle progression in vivo under physiological conditions, refuting its use as a suitable drug target. Tagged AQP localized to perinuclear structures and not the parasite plasma membrane. We suggest that flow-cytometric isolation of isogenic parasites overcomes the major roadblock towards a genome-scale repository of mutant and transgenic malaria parasite lines., (Copyright © 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)