Revisiting the determinants of malaria transmission.

Malaria parasites have coevolved with humans over thousands of years, mirroring their migration out of Africa. They persist to this day, despite continuous elimination efforts worldwide. These parasites can adapt to changing environments during infection of human and mosquito, and when expanding the geographical range by switching vector species. Recent studies in the human malaria parasite, Plasmodium falciparum, identified determinants governing the plasticity of sexual conversion rates, sex ratio, and vector competence. Here we summarize the latest literature revealing environmental, epigenetic, and genetic determinants of malaria transmission.

Single-cell transcriptomics reveals expression profiles of Trypanosoma brucei sexual stages.

Early diverging lineages such as trypanosomes can provide clues to the evolution of sexual reproduction in eukaryotes. In Trypanosoma brucei, the pathogen that causes Human African Trypanosomiasis, sexual reproduction occurs in the salivary glands of the insect host, but analysis of the molecular signatures that define these sexual forms is complicated because they mingle with more numerous, mitotically-dividing developmental stages. We used single-cell RNA-sequencing (scRNAseq) to profile 388 individual trypanosomes from midgut, proventriculus, and salivary glands of infected tsetse flies allowing us to identify tissue-specific cell types. Further investigation of salivary gland parasite transcriptomes revealed fine-scale changes in gene expression over a developmental progression from putative sexual forms through metacyclics expressing variant surface glycoprotein genes. The cluster of cells potentially containing sexual forms was characterized by high level transcription of the gamete fusion protein HAP2, together with an array of surface proteins and several genes of unknown function. We linked these expression patterns to distinct morphological forms using immunofluorescence assays and reporter gene expression to demonstrate that the kinetoplastid-conserved gene Tb927.10.12080 is exclusively expressed at high levels by meiotic intermediates and gametes. Further experiments are required to establish whether this protein, currently of unknown function, plays a role in gamete formation and/or fusion.

A single-cell atlas of Plasmodium falciparum transmission through the mosquito.

Malaria parasites have a complex life cycle featuring diverse developmental strategies, each uniquely adapted to navigate specific host environments. Here we use single-cell transcriptomics to illuminate gene usage across the transmission cycle of the most virulent agent of human malaria - Plasmodium falciparum. We reveal developmental trajectories associated with the colonization of the mosquito midgut and salivary glands and elucidate the transcriptional signatures of each transmissible stage. Additionally, we identify both conserved and non-conserved gene usage between human and rodent parasites, which point to both essential mechanisms in malaria transmission and species-specific adaptations potentially linked to host tropism. Together, the data presented here, which are made freely available via an interactive website, provide a fine-grained atlas that enables intensive investigation of the P. falciparum transcriptional journey. As well as providing insights into gene function across the transmission cycle, the atlas opens the door for identification of drug and vaccine targets to stop malaria transmission and thereby prevent disease.

Using scRNA-seq to Identify Transcriptional Variation in the Malaria Parasite Ookinete Stage.

The crossing of the mosquito midgut epithelium by the malaria parasite motile ookinete form represents the most extreme population bottleneck in the parasite life cycle and is a prime target for transmission blocking strategies. However, we have little understanding of the clonal variation that exists in a population of ookinetes in the vector, partially because the parasites are difficult to access and are found in low numbers. Within a vector, variation may result as a response to specific environmental cues or may exist independent of those cues as a potential bet-hedging strategy. Here we use single-cell RNA-seq to profile transcriptional variation in Plasmodium berghei ookinetes across different vector species, and between and within individual midguts. We then compare our results to low-input transcriptomes from individual Anopheles coluzzii midguts infected with the human malaria parasite Plasmodium falciparum. Although the vast majority of transcriptional changes in ookinetes are driven by development, we have identified candidate genes that may be responding to environmental cues or are clonally variant within a population. Our results illustrate the value of single-cell and low-input technologies in understanding clonal variation of parasite populations.

Transmission of Artemisinin-Resistant Malaria Parasites to Mosquitoes under Antimalarial Drug Pressure.

Resistance to artemisinin-based combination therapy (ACT) in the Plasmodium falciparum parasite is threatening to reverse recent gains in reducing global deaths from malaria. While resistance manifests as delayed parasite clearance in patients, the phenotype can only spread geographically via the sexual stages and mosquito transmission. In addition to their asexual killing properties, artemisinin and its derivatives sterilize sexual male gametocytes. Whether resistant parasites overcome this sterilizing effect has not, however, been fully tested. Here, we analyzed P. falciparum clinical isolates from the Greater Mekong Subregion, each demonstrating delayed clinical clearance and known resistance-associated polymorphisms in the Kelch13 (PfK13var) gene. As well as demonstrating reduced asexual sensitivity to drug, certain PfK13var isolates demonstrated a marked reduction in sensitivity to artemisinin in an in vitro male gamete formation assay. Importantly, this same reduction in sensitivity was observed when the most resistant isolate was tested directly in mosquito feeds. These results indicate that, under artemisinin drug pressure, while sensitive parasites are blocked, resistant parasites continue transmission. This selective advantage for resistance transmission could favor acquisition of additional host-specificity or polymorphisms affecting partner drug sensitivity in mixed infections. Favored resistance transmission under ACT coverage could have profound implications for the spread of multidrug-resistant malaria beyond Southeast Asia.

Uptake of Plasmodium falciparum Gametocytes During Mosquito Bloodmeal by Direct and Membrane Feeding.

Plasmodium falciparum remains one of the leading causes of child mortality, and nearly half of the world's population is at risk of contracting malaria. While pathogenesis results from replication of asexual forms in human red blood cells, it is the sexually differentiated forms, gametocytes, which are responsible for the spread of the disease. For transmission to succeed, both mature male and female gametocytes must be taken up by a female Anopheles mosquito during its blood meal for subsequent differentiation into gametes and mating inside the mosquito gut. Observed circulating numbers of gametocytes in the human host are often surprisingly low. A pre-fertilization behavior, such as skin sequestration, has been hypothesized to explain the efficiency of human-to-mosquito transmission but has not been sufficiently tested due to a lack of appropriate tools. In this study, we describe the optimization of a qPCR tool that enables the relative quantification of gametocytes within very small input samples. Such a tool allows for the quantification of gametocytes in different compartments of the host and the vector that could potentially unravel mechanisms that enable highly efficient malaria transmission. We demonstrate the use of our gametocyte quantification method in mosquito blood meals from both direct skin feeding on Plasmodium gametocyte carriers and standard membrane feeding assay. Relative gametocyte abundance was not different between mosquitoes fed through a membrane or directly on the skin suggesting that there is no systematic enrichment of gametocytes picked up in the skin.

The Malaria Cell Atlas: Single parasite transcriptomes across the complete Plasmodium life cycle.

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.

Rapid seasonal evolution in innate immunity of wild Drosophila melanogaster.

Understanding the rate of evolutionary change and the genetic architecture that facilitates rapid adaptation is a current challenge in evolutionary biology. Comparative studies show that genes with immune function are among the most rapidly evolving genes across a range of taxa. Here, we use immune defence in natural populations of Drosophila melanogaster to understand the rate of evolution in natural populations and the genetics underlying rapid change. We probed the immune system using the natural pathogens Enterococcus faecalis and Providencia rettgeri to measure post-infection survival and bacterial load of wild D. melanogaster populations collected across seasonal time along a latitudinal transect along eastern North America (Massachusetts, Pennsylvania and Virginia). There are pronounced and repeatable changes in the immune response over the approximately 10 generations between spring and autumn collections, with a significant but less distinct difference observed among geographical locations. Genes with known immune function are not enriched among alleles that cycle with seasonal time, but the immune function of a subset of seasonally cycling alleles in immune genes was tested using reconstructed outbred populations. We find that flies containing seasonal alleles in Thioester-containing protein 3 (Tep3) have different functional responses to infection and that epistatic interactions among seasonal Tep3 and Drosomycin-like 6 (Dro6) alleles underlie the immune phenotypes observed in natural populations. This rapid, cyclic response to seasonal environmental pressure broadens our understanding of the complex ecological and genetic interactions determining the evolution of immune defence in natural populations.

The genetic architecture of defence as resistance to and tolerance of bacterial infection in Drosophila melanogaster.

Defence against pathogenic infection can take two forms: resistance and tolerance. Resistance is the ability of the host to limit a pathogen burden, whereas tolerance is the ability to limit the negative consequences of infection at a given level of infection intensity. Evolutionarily, a tolerance strategy that is independent of resistance could allow the host to avoid mounting a costly immune response and, theoretically, to avoid a co-evolutionary arms race between pathogen virulence and host resistance. Biomedically, understanding the mechanisms of tolerance and how they relate to resistance could potentially yield treatment strategies that focus on health improvement instead of pathogen elimination. To understand the impact of tolerance on host defence and identify genetic variants that determine host tolerance, we defined genetic variation in tolerance as the residual deviation from a binomial regression of fitness under infection against infection intensity. We then performed a genomewide association study to map the genetic basis of variation in resistance to and tolerance of infection by the bacterium Providencia rettgeri. We found a positive genetic correlation between resistance and tolerance, and we demonstrated that the level of resistance is highly predictive of tolerance. We identified 30 loci that predict tolerance, many of which are in genes involved in the regulation of immunity and metabolism. We used RNAi to confirm that a subset of mapped genes have a role in defence, including putative wound repair genes grainy head and debris buster. Our results indicate that tolerance is not an independent strategy from resistance, but that defence arises from a collection of physiological processes intertwined with canonical immunity and resistance.

Convergent Balancing Selection on an Antimicrobial Peptide in Drosophila.

Genes of the immune system often evolve rapidly and adaptively, presumably driven by antagonistic interactions with pathogens [1-4]. Those genes encoding secreted antimicrobial peptides (AMPs), however, have failed to exhibit conventional signatures of strong adaptive evolution, especially in arthropods (e.g., [5, 6]) and often segregate for null alleles and gene deletions [3, 4, 7, 8]. Furthermore, quantitative genetic studies have failed to associate naturally occurring polymorphism in AMP genes with variation in resistance to infection [9-11]. Both the lack of signatures of positive selection in AMPs and lack of association between genotype and immune phenotypes have yielded an interpretation that AMP genes evolve under relaxed evolutionary constraint, with enough functional redundancy that variation in, or even loss of, any particular peptide would have little effect on overall resistance [12, 13]. In stark contrast to the current paradigm, we identified a naturally occurring amino acid polymorphism in the AMP Diptericin that is highly predictive of resistance to bacterial infection in Drosophila melanogaster [13]. The identical amino acid polymorphism arose in parallel in the sister species D. simulans, by independent mutation with equivalent phenotypic effect. Convergent substitutions at the same amino acid residue have evolved at least five times across the Drosophila genus. We hypothesize that the alternative alleles are maintained by balancing selection through context-dependent or fluctuating selection. This pattern of evolution appears to be common in AMPs but is invisible to conventional screens for adaptive evolution that are predicated on elevated rates of amino acid divergence.

Genotype and diet shape resistance and tolerance across distinct phases of bacterial infection.

BACKGROUND: Host defense against pathogenic infection is composed of resistance and tolerance. Resistance is the ability of the host to limit a pathogen burden, whereas tolerance is the ability to limit the deleterious effects of a given pathogen burden. This distinction recognizes that the fittest host does not necessarily have the most aggressive immune system, suggesting that host-pathogen co-evolution involves more than an escalating arms race between pathogen virulence factors and host antimicrobial activity. How a host balances resistance and tolerance and how this balance influences the evolution of host defense remains unanswered. In order to determine how genotype-by-diet interactions and evolutionary costs of each strategy may constrain the evolution of host defense, we measured survival, fecundity, and pathogen burden over five days in ten genotypes of Drosophila melanogaster reared on two diets and infected with the Gram-negative bacterial pathogen Providencia rettgeri. RESULTS: We demonstrated two distinct phases of infection: an acute phase that consists of high mortality, low fecundity, and high pathogen loads, and a chronic phase where there was a substantial but stable pathogen load and mortality and fecundity returned to uninfected levels. We demonstrated genetic variation for resistance in both phases of infection, but found genetic variation for tolerance only in the acute phase. We found genotype-by-diet interactions for tolerance, especially in the acute phase, but genotype-by-diet interaction did not significantly shape resistance. We found a diet-dependent positive relationship between resistance and tolerance and a weak evolutionary cost of resistance, but did not detect any costs of tolerance. CONCLUSIONS: Existing models of tolerance and resistance are overly simplistic. Multi-phase infections such as that studied here are rarely considered, but we show important differences in determination and evolutionary constraints on tolerance and resistance over the two phases of infection. Our observation of genetic variation for tolerance is inconsistent with simple models that predict evolutionary fixation of tolerance alleles, and instead indicate that genetic variation for resistance and tolerance is likely to be maintained by non-independence between resistance and tolerance, condition-dependent evolutionary costs, and environmental heterogeneity.