Malaria parasites adopt a remarkable variety of morphological life stages as they transition through multiple mammalian host and mosquito vector environments. We profiled the single-cell transcriptomes of thousands of individual parasites, deriving the first high-resolution transcriptional atlas of the entire Plasmodium berghei life cycle. We then used our atlas to precisely define developmental stages of single cells from three different human malaria parasite species, including parasites isolated directly from infected individuals. The Malaria Cell Atlas provides both a comprehensive view of gene usage in a eukaryotic parasite and an open-access reference dataset for the study of malaria parasites.

In order to demonstrate souporcell on an external and widely used benchmark dataset, we downloaded the three overlapping mixtures from the demuxlet paper 6. Sample A contains a mixture of four donors' PBMCs, Sample B contains a mixture of four different donors' PBMCs, and Sample C contains a mixture of all 8 donors' PBMCs. We synthetically combined this data into a single dataset and clustered with souporcell. Supp Fig 4a shows that the resulting clusters either contain cells from Sample A or Sample B, but not both as is expected from this experimental setup. We also show that the first cluster of the doublet assignments are also largely consistent with this experimental design (Supp Fig 4b). Supp Figure 4. Demuxlet data Deconvolution of overlapping mixtures To enable identification of which cluster is which individual using the overlapping mixture experimental design outlined in Table 1, we provide a tool shared_samples.py which takes as input two souporcell output directories and the number of samples which are shared. It compares the sum of squared differences of the allele fraction of confident (>95% confident genotype call in all clusters) shared variant calls between clusters in the two experiments and outputs the best matches for the number of shared samples. We tested this using multiple different runs of souporcell on the synthetic mixture of 5 HipSci cell lines with 6% doublets and 5% ambient RNA and gave both as input to the shared_samples.py tool and it correctly assigned the clusters in one run to the clusters in the second experiment which corresponded to the same samples. We also ran souporcell on the three demuxlet datasets separately and ran the shared_samples.py tool on Sample A vs Sample C and Sample B vs Sample C and it confidently identified the non-overlapping clusters in Sample C which correspond to A and B. Demonstration on 21 donor sample We demonstrate that souporcell is capable of demultiplexing many donors by creating a synthetic mixture of 21 different individuals, which given the current recommendations from 10x on cells per run would be a high-end number of donors to multiplex. To generate this 21-donor mix, we used the 5 HipSci samples described in Fig 2 and added to them 16 PBMC samples obtained from the Human Cell Atlas Census of Immune Cells 25. From each dataset we randomly selected 1000 cells with at least 4000 UMIs and simulated 10% doublets and 2.5% ambient RNA by altering the cell barcodes, as described above. We clustered these

Single-cell RNA-sequencing is revolutionising our understanding of seemingly homogeneous cell populations but has not yet been widely applied to single-celled organisms. Transcriptional variation in unicellular malaria parasites from the Plasmodium genus is associated with critical phenotypes including red blood cell invasion and immune evasion, yet transcriptional variation at an individual parasite level has not been examined in depth. Here, we describe the adaptation of a single-cell RNA-sequencing (scRNA-seq) protocol to deconvolute transcriptional variation for more than 500 individual parasites of both rodent and human malaria comprising asexual and sexual life-cycle stages. We uncover previously hidden discrete transcriptional signatures during the pathogenic part of the life cycle, suggesting that expression over development is not as continuous as commonly thought. In transmission stages, we find novel, sex-specific roles for differential expression of contingency gene families that are usually associated with immune evasion and pathogenesis.

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SummaryThe malaria life cycle relies on the successful transfer of the parasite between its human and mosquito hosts. We identified a Plasmodium berghei secreted protein (PBANKA_131270) that plays distinct roles in both the mammal-to-mosquito and the mosquito-tomammal transitions. This protein, here named gamete egress and sporozoite traversal (GEST), plays an important role in the egress of male and female gametes from the vertebrate red blood cell. Interestingly, GEST is also required following the bite of the infected mosquito, for sporozoite progression through the skin. We found PbGEST to be secreted shortly after activation of the intraerythrocytic gametocyte, and during sporozoite migration. These findings indicate that a single malaria protein may have pleiotropic roles in different parasites stages mediating transmission between its insect and mammalian hosts.

The human malaria parasite Plasmodium falciparum is responsible for the majority of malaria-related deaths. Tools allowing the study of the basic biology of P. falciparum throughout the life cycle are critical to the development of new strategies to target the parasite within both human and mosquito hosts. We here present 3D7HT-GFP, a strain of P. falciparum constitutively expressing the Green Fluorescent Protein (GFP) throughout the life cycle, which has retained its capacity to complete sporogonic development. The GFP expressing cassette was inserted in the Pf47 locus. Using this transgenic strain, parasite tracking and population dynamics studies in mosquito stages and exo-erythrocytic schizogony is greatly facilitated. The development of 3D7HT-GFP will permit a deeper understanding of the biology of parasite-host vector interactions, and facilitate the development of high-throughput malaria transmission assays and thus aid development of new intervention strategies against both parasite and mosquito.

The protozoan Plasmodium falciparum has a complex life cycle in which asexual multiplication in the vertebrate host alternates with an obligate sexual reproduction in the anopheline mosquito. Apart from the apparent recombination advantages conferred by sex, P. falciparum has evolved a remarkable biology and adaptive phenotypes to insure its transmission despite the dangers of sex. This review mainly focuses on the current knowledge on commitment to sexual development, gametocytogenesis and the evolutionary significance of various aspects of gametocyte biology. It goes further than pure biology to look at the strategies used to improve successful transmission. Although gametocytes are inevitable stages for transmission and provide a potential target to fight malaria, they have received less attention than the pathogenic asexual stages. There is a need for research on gametocytes, which are a fascinating stage, responsible to a large extent for the success of P. falciparum.

BackgroundGametogenesis and fertilization play crucial roles in malaria transmission. While male gametes are thought to be amongst the simplest eukaryotic cells and are proven targets of transmission blocking immunity, little is known about their molecular organization. For example, the pathway of energy metabolism that power motility, a feature that facilitates gamete encounter and fertilization, is unknown.MethodsPlasmodium berghei microgametes were purified and analysed by whole-cell proteomic analysis for the first time. Data are available via ProteomeXchange with identifier PXD001163.Results615 proteins were recovered, they included all male gamete proteins described thus far. Amongst them were the 11 enzymes of the glycolytic pathway. The hexose transporter was localized to the gamete plasma membrane and it was shown that microgamete motility can be suppressed effectively by inhibitors of this transporter and of the glycolytic pathway.ConclusionsThis study describes the first whole-cell proteomic analysis of the malaria male gamete. It identifies glycolysis as the likely exclusive source of energy for flagellar beat, and provides new insights in original features of Plasmodium flagellar organization.Electronic supplementary materialThe online version of this article (doi:10.1186/1475-2875-13-315) contains supplementary material, which is available to authorized users.