Novel Ion Channel Genes in Malaria Parasites.

Ion channels serve many cellular functions including ion homeostasis, volume regulation, signaling, nutrient acquisition, and developmental progression. Although the complex life cycles of malaria parasites necessitate ion and solute flux across membranes, the whole-genome sequencing of the human pathogen Plasmodium falciparum revealed remarkably few orthologs of known ion channel genes. Contrasting with this, biochemical studies have implicated the channel-mediated flux of ions and nutritive solutes across several membranes in infected erythrocytes. Here, I review advances in the cellular and molecular biology of ion channels in malaria parasites. These studies have implicated novel parasite genes in the formation of at least two ion channels, with additional ion channels likely present in various membranes and parasite stages. Computational approaches that rely on homology to known channel genes from higher organisms will not be very helpful in identifying the molecular determinants of these activities. Given their unusual properties, novel molecular and structural features, and essential roles in pathogen survival and development, parasite channels should be promising targets for therapy development.

Whole genome sequencing identifies novel mutations in malaria parasites resistant to artesunate (ATN) and to ATN + mefloquine combination.

Introduction: The global evolution of resistance to Artemisinin-based Combination Therapies (ACTs) by malaria parasites, will severely undermine our ability to control this devastating disease. Methods: Here, we have used whole genome sequencing to characterize the genetic variation in the experimentally evolved Plasmodium chabaudi parasite clone AS-ATNMF1, which is resistant to artesunate + mefloquine. Results and discussion: Five novel single nucleotide polymorphisms (SNPs) were identified, one of which was a previously undescribed E738K mutation in a 26S proteasome subunit that was selected for under artesunate pressure (in AS-ATN) and retained in AS-ATNMF1. The wild type and mutated three-dimensional (3D) structure models and molecular dynamics simulations of the P. falciparum 26S proteasome subunit Rpn2 suggested that the E738K mutation could change the toroidal proteasome/cyclosome domain organization and change the recognition of ubiquitinated proteins. The mutation in the 26S proteasome subunit may therefore contribute to altering oxidation-dependent ubiquitination of the MDR-1 and/or K13 proteins and/or other targets, resulting in changes in protein turnover. In light of the alarming increase in resistance to artemisin derivatives and ACT partner drugs in natural parasite populations, our results shed new light on the biology of resistance and provide information on novel molecular markers of resistance that may be tested (and potentially validated) in the field.

Multi-omics data elucidate parasite-host-microbiota interactions and resistance to Haemonchus contortus in sheep.

BACKGROUND: The integration of molecular data from hosts, parasites, and microbiota can enhance our understanding of the complex biological interactions underlying the resistance of hosts to parasites. Haemonchus contortus, the predominant sheep gastrointestinal parasite species in the tropics, causes significant production and economic losses, which are further compounded by the diminishing efficiency of chemical control owing to anthelmintic resistance. Knowledge of how the host responds to infection and how the parasite, in combination with microbiota, modulates host immunity can guide selection decisions to breed animals with improved parasite resistance. This understanding will help refine management practices and advance the development of new therapeutics for long-term helminth control. METHODS: Eggs per gram (EPG) of feces were obtained from Morada Nova sheep subjected to two artificial infections with H. contortus and used as a proxy to select animals with high resistance or susceptibility for transcriptome sequencing (RNA-seq) of the abomasum and 50 K single-nucleotide genotyping. Additionally, RNA-seq data for H. contortus were generated, and amplicon sequence variants (ASV) were obtained using polymerase chain reaction amplification and sequencing of bacterial and archaeal 16S ribosomal RNA genes from sheep feces and rumen content. RESULTS: The heritability estimate for EPG was 0.12. GAST, GNLY, IL13, MGRN1, FGF14, and RORC genes and transcripts were differentially expressed between resistant and susceptible animals. A genome-wide association study identified regions on chromosomes 2 and 11 that harbor candidate genes for resistance, immune response, body weight, and adaptation. Trans-expression quantitative trait loci were found between significant variants and differentially expressed transcripts. Functional co-expression modules based on sheep genes and ASVs correlated with resistance to H. contortus, showing enrichment in pathways of response to bacteria, immune and inflammatory responses, and hub features of the Christensenellaceae, Bacteroides, and Methanobrevibacter genera; Prevotellaceae family; and Verrucomicrobiota phylum. In H. contortus, some mitochondrial, collagen-, and cuticle-related genes were expressed only in parasites isolated from susceptible sheep. CONCLUSIONS: The present study identified chromosome regions, genes, transcripts, and pathways involved in the elaborate interactions between the sheep host, its gastrointestinal microbiota, and the H. contortus parasite. These findings will assist in the development of animal selection strategies for parasite resistance and interdisciplinary approaches to control H. contortus infection in sheep.

Single-cell transcriptomics of the human parasite Schistosoma mansoni first intra-molluscan stage reveals tentative tegumental and stem-cell regulators.

Schistosomiasis is a major Neglected Tropical Disease, caused by the infection with blood flukes in the genus Schistosoma. To complete the life cycle, the parasite undergoes asexual and sexual reproduction within an intermediate snail host and a definitive mammalian host, respectively. The intra-molluscan phase provides a critical amplification step that ensures a successful transmission. However, the cellular and molecular mechanisms underlying the development of the intra-molluscan stages remain poorly understood. Here, single cell suspensions from S. mansoni mother sporocysts were produced and sequenced using the droplet-based 10X Genomics Chromium platform. Six cell clusters comprising two tegument, muscle, neuron, parenchyma and stem/germinal cell clusters were identified and validated by in situ hybridisation. Gene Ontology term analysis predicted key biological processes for each of the clusters, including three stem/germinal sub-clusters. Furthermore, putative transcription factors predicted for stem/germinal and tegument clusters may play key roles during parasite development and interaction with the intermediate host.

Parasite and genetic diversity of big-scale sand smelt (Atherina boyeri Risso, 1810) populations in their natural and expansion ranges in Ukraine.

The big-scale sand smelt (Atherina boyeri) is an Atlanto-Mediterranean amphidromous fish species found within the Black Sea. Here, we assess differences in the parasite fauna of big-scale sand smelt populations from their natural range in the northwestern Black Sea and from their expansion range in the Lower and Middle River Dnipro. In addition, we undertook a microsatellite analysis to assess the genetic similarity of fish from the different locations. We found that the parasite community of fish in their natural range was wider than that from their expansion range. While the Gulf of Odesa was most distant from all other localities by parasite community composition and the Dnipro Reservoir was characterised by an absence of parasites (newest and most distant expansion locality), only fish from the Danube Delta showed a significant genetic difference. Our results suggest that the parasite community of big-scale sand smelt is primarily influenced by environmental factors, such as habitat type, water salinity and/or prey composition. Both microsatellite analysis and parasite community species composition (e.g. the presence of the marine Telosentis exiguus in the Kakhovka Reservoir and freshwater Raphidascaris sp. in the Gulf of Odesa) confirmed that populations in the River Dnipro reservoirs had, at some time, been connected with native marine populations, thus also confirming the species' amphidromous nature.

Gaps in parasitological research in the molecular era.

We live in the age of molecular biology and '-omics', and molecular methods have opened up unimagined possibilities for biological research, including parasitology. However, too one-sided a focus on new approaches can lead to major gaps as less 'cool' topics are neglected. Selected areas of research are briefly discussed to highlight the gaps caused by the current excessive focus on molecular and '-omics' methods. It is crucial to combine both 'classical' and modern methods without neglecting the complexity of the interactions of parasites with their hosts and the environment (One Health concept), which is even more urgent in today's rapidly changing world. Parasitologists should be more involved in field studies and multidisciplinary assessment of parasites.

In silico prediction of the metabolism of Blastocrithidia nonstop, a trypanosomatid with non-canonical genetic code.

BACKGROUND: Almost all extant organisms use the same, so-called canonical, genetic code with departures from it being very rare. Even more exceptional are the instances when a eukaryote with non-canonical code can be easily cultivated and has its whole genome and transcriptome sequenced. This is the case of Blastocrithidia nonstop, a trypanosomatid flagellate that reassigned all three stop codons to encode amino acids. RESULTS: We in silico predicted the metabolism of B. nonstop and compared it with that of the well-studied human parasites Trypanosoma brucei and Leishmania major. The mapped mitochondrial, glycosomal and cytosolic metabolism contains all typical features of these diverse and important parasites. We also provided experimental validation for some of the predicted observations, concerning, specifically presence of glycosomes, cellular respiration, and assembly of the respiratory complexes. CONCLUSIONS: In an unusual comparison of metabolism between a parasitic protist with a massively altered genetic code and its close relatives that rely on a canonical code we showed that the dramatic differences on the level of nucleic acids do not seem to be reflected in the metabolisms. Moreover, although the genome of B. nonstop is extremely AT-rich, we could not find any alterations of its pyrimidine synthesis pathway when compared to other trypanosomatids. Hence, we conclude that the dramatic alteration of the genetic code of B. nonstop has no significant repercussions on the metabolism of this flagellate.

Revealing the associated microflora hosted by the globally significant parasite Trichostrongylus colubriformis.

Trichostrongylus colubriformis is a parasitic helminth that primarily infects small ruminants, causing substantial economic losses in the livestock industry. Exploring the microbiome of this helminth might provide insights into the potential influence of its microbial community on the parasite's survival. We characterised the intestinal microbiome of T. colubriformis that had been collected from the duodenum of sheep, and compared the helminth microbiome with the duodenal microbiome of its host, aiming to identify contributions from the helminth's environment. At the same time, we explored the isolation of fastidious organisms from the harvested helminth. Primary alpha and beta diversity analyses of bacterial species revealed statistically significant differences between the parasite and the host, in terms of species richness and ecological composition. 16S rRNA differential abundance analysis showed that Mycoplasmoides and Stenotrophomonas were significantly present in T. colubriformis but not in the duodenal microbiome of the sheep. Furthermore, two bacteria, Aeromonas caviae and Aeromonas hydrophila, were isolated from T. colubriformis. Examinations of the genome highlight differences in genome size and profiles of antimicrobial resistance genes. Our results suggest that T. colubriformis carries a specific bacterial community that could be supporting the helminth's long-term survival in the host's digestive system.

Decoding the RNA virome of the tree parasite Armillaria provides new insights into the viral community of soil-borne fungi.

The globally distributed basidiomycete genus Armillaria includes wood decomposers that can act as opportunistic parasites, causing deadly root rot on woody plants. To test whether RNA viruses are involved in this opportunistic behaviour, a large isolate collection of five Armillaria species collected over 40 years in Switzerland from trees, dead wood and soil was analysed. De novo assembly of RNA-Seq data revealed 21 viruses, 14 of which belong to putative new species. Two dsRNA viruses and an unclassified Tymovirales are formally described for the first time for Armillaria. One mitovirus occurred with a high prevalence of 71.1%, while all other viruses were much less prevalent (0.6%-16.9%). About half of all viruses were found only in one fungal species, others occurred in 2-6 fungal species. Co-infections of 2-7 viruses per isolate were not uncommon (34.9%), and most viruses persisted circulating within fungal populations for decades. Some viruses were related to viruses associated with other Armillaria species, supporting the hypothesis that virus transmission can occur between different fungal species. Although no specific correlation between viruses and the fungal trophic strategy was found, this study opens new insights into viral diversity hidden in the soil microbiome.

Emerging roles of the epitranscriptome in parasitic protozoan biology and pathogenesis.

RNA modifications (epitranscriptome) - such as N6-methyladenosine (m6A), 5-methylcytosine (m5C), and pseudouridine (Ψ) - modulate RNA processing, stability, interaction, and translation, thereby playing critical roles in the development, replication, virulence, metabolism, and life cycle adaptations of parasitic protozoa. Here, we summarize potential homologs of the major human RNA modification regulatory factors in parasites, outline current knowledge on how RNA modifications affect parasitic protozoa, highlight the regulation of RNA modifications and their crosstalk, and discuss current progress in exploring RNA modifications as potential drug targets. This review contributes to our understanding of epitranscriptomic regulation of parasitic protozoa biology and pathogenesis and provides new perspectives for the treatment of parasitic diseases.

Genome-wide transcriptome analysis of Echinococcus multilocularis larvae and germinative cell cultures reveals genes involved in parasite stem cell function.

The lethal zoonosis alveolar echinococcosis is caused by tumour-like growth of the metacestode stage of the tapeworm Echinococcus multilocularis within host organs. We previously demonstrated that metacestode proliferation is exclusively driven by somatic stem cells (germinative cells), which are the only mitotically active parasite cells that give rise to all differentiated cell types. The Echinococcus gene repertoire required for germinative cell maintenance and differentiation has not been characterised so far. We herein carried out Illumina sequencing on cDNA from Echinococcus metacestode vesicles, from metacestode tissue depleted of germinative cells, and from Echinococcus primary cell cultures. We identified a set of ~1,180 genes associated with germinative cells, which contained numerous known stem cell markers alongside genes involved in replication, cell cycle regulation, mitosis, meiosis, epigenetic modification, and nucleotide metabolism. Interestingly, we also identified 44 stem cell associated transcription factors that are likely involved in regulating germinative cell differentiation and/or pluripotency. By in situ hybridization and pulse-chase experiments, we also found a new general Echinococcus stem cell marker, EmCIP2Ah, and we provide evidence implying the presence of a slow cycling stem cell sub-population expressing the extracellular matrix factor Emkal1. RNA-Seq analyses on primary cell cultures revealed that metacestode-derived Echinococcus stem cells display an expanded differentiation capability and do not only form differentiated cell types of the metacestode, but also cells expressing genes specific for protoscoleces, adult worms, and oncospheres, including an ortholog of the schistosome praziquantel target, EmTRPMPZQ. Finally, we show that primary cell cultures contain a cell population expressing an ortholog of the tumour necrosis factor α receptor family and that mammalian TNFα accelerates the development of metacestode vesicles from germinative cells. Taken together, our analyses provide a robust and comprehensive characterization of the Echinococcus germinative cell transcriptome, demonstrate expanded differentiation capability of metacestode derived stem cells, and underscore the potential of primary germinative cell cultures to investigate developmental processes of the parasite. These data are relevant for studies into the role of Echinococcus stem cells in parasite development and will facilitate the design of anti-parasitic drugs that specifically act on the parasite germinative cell compartment.

Environmental Detection of Parasites in the Marginalized Paiute Reservations Compared to a Nearby Area.

The amounts of parasite DNA in soil samples from different playgrounds and other public areas can help identify areas of possible microbe transmission and give indications of the possible occurrence of parasite infection in nearby communities. We collected 207 soil samples from parks in Paiute indigenous tribal areas in southwestern Utah and from the higher income city of St. George, Utah, and tested them for 11 parasites that can cause human disease. Molecular tests revealed an elevated odds ratio (OR) of 3.072 (range, 1.114-8.065) for detecting the helminth Trichuris trichiura and an elevated OR of 3.036 (range, 1.101-7.966) for any protozoa (not including Acanthamoeba) in the tribal land playgrounds compared with St. George parks. These findings support previous studies showing that areas in lower socioeconomic communities, especially marginalized communities, tend to have more parasites in the soil, which may lead to higher disease prevalence rates.

New reference genomes to distinguish the sympatric malaria parasites, Plasmodium ovale curtisi and Plasmodium ovale wallikeri.

Despite Plasmodium ovale curtisi (Poc) and wallikeri (Pow) being important human-infecting malaria parasites that are widespread across Africa and Asia, little is known about their genome diversity. Morphologically identical, Poc and Pow are indistinguishable and commonly misidentified. Recent rises in the incidence of Poc/Pow infections have renewed efforts to address fundamental knowledge gaps in their biology, and to develop diagnostic tools to understand their epidemiological dynamics and malaria burden. A major roadblock has been the incompleteness of available reference assemblies (PocGH01, PowCR01; ~ 33.5 Mbp). Here, we applied multiple sequencing platforms and advanced bioinformatics tools to generate new reference genomes, Poc221 (South Sudan; 36.0 Mbp) and Pow222 (Nigeria; 34.3 Mbp), with improved nuclear genome contiguity (> 4.2 Mbp), annotation and completeness (> 99% Plasmodium spp., single copy orthologs). Subsequent sequencing of 6 Poc and 15 Pow isolates from Africa revealed a total of 22,517 and 43,855 high-quality core genome SNPs, respectively. Genome-wide levels of nucleotide diversity were determined to be 2.98 × 10-4 (Poc) and 3.43 × 10-4 (Pow), comparable to estimates for other Plasmodium species. Overall, the new reference genomes provide a robust foundation for dissecting the biology of Poc/Pow, their population structure and evolution, and will contribute to uncovering the recombination barrier separating these species.

Dynamic profiles of lncRNAs reveal a functional natural antisense RNA that regulates the development of Schistosoma japonicum.

Schistosomes are flatworm parasites that undergo a complex life cycle involving two hosts. The regulation of the parasite's developmental processes relies on both coding RNAs and non-coding RNAs. However, the roles of non-coding RNAs, including long non-coding RNAs (lncRNAs) in schistosomes remain largely unexplored. Here we conduct advanced RNA sequencing on male and female S. japonicum during their pairing and reproductive development, resulting in the identification of nearly 8,000 lncRNAs. This extensive dataset enables us to construct a comprehensive co-expression network of lncRNAs and mRNAs, shedding light on their interactions during the crucial reproductive stages within the mammalian host. Importantly, we have also revealed a specific lncRNA, LNC3385, which appears to play a critical role in the survival and reproduction of the parasite. These findings not only enhance our understanding of the dynamic nature of lncRNAs during the reproductive phase of schistosomes but also highlight LNC3385 as a potential therapeutic target for combating schistosomiasis.

Genome-wide association studies of parasite resistance, productivity and immunology traits in Scottish Blackface sheep.

Gastrointestinal parasitism represents a global problem for grazing ruminants, which can be addressed sustainably by breeding animals to be more resistant against infection by parasites. The aim of this study was to assess the genetic architecture underlying traits associated with gastrointestinal parasite resistance, immunological profile and production in meat sheep, and identify and characterise candidate genes affecting these traits. Data on gastrointestinal parasite infection (faecal egg counts for Strongyles (FECS) and Nematodirus (FECN) and faecal oocyst counts for Coccidia, along with faecal soiling scores (DAG), characterised by the accumulation of faeces around the perineum) and production (live weight (LWT)) were gathered from a flock Scottish Blackface lambs at three and four months of age. Data on the immune profile were also collected from a subset of these lambs at two and five months of age. Immune traits included the production of Interferon-γ (IFN-γ), Interleukin (IL)-4 and IL-10 following stimulation of whole blood with pokeweed mitogen (PWM) or antigen from the gastric parasite Teladorsagia circumcincta (T-ci), and serum levels of T. circumcincta-specific immunoglobulin A (IgA). Animals were genotyped with genome-wide DNA arrays, and a total of 1 766 animals and 45 827 Single Nucleotide Polymorphisms (SNPs) were retained following quality control and imputation. Genome-wide association studies were performed for 24 traits. The effects of individual markers with significant effects were estimated, and the genotypic effect solutions were used to estimate additive and dominance effects, and the proportion of additive genetic variance attributed to each SNP locus. A total of 15 SNPs were associated at least at a suggestive level with FECS, FECN, DAG, IgA, PWM-induced IFN-γ and IL-4, and T-ci-induced IL-10. This study uncovered 52 genes closely related to immune function in proximity to these SNPs. A number of genes encoding C-type lectins and killer cell lectin-like family members were close to a SNP associated with FECN, while several genes encoding IL-1 cytokine family members were found to be associated with IgA. Potential candidate genes belonging to or in close proximity with the Major Histocompatibility Complex (MHC) were revealed, including Homeostatic Iron Regulator and butyrophilin coding genes associated with IFN-γ(PWM), and IL-17 coding genes associated with IgA. Due to the importance of the MHC in the control of immune responses, these genes may play an important role in resistance to parasitic infections. Our results reveal a largely complex and polygenic genetic profile of the studied traits in this Scottish Blackface sheep population.

Prevalence of intestinal parasites and comparison of detection techniques for soil-transmitted helminths among newly arrived expatriate labors in Jeddah, Saudi Arabia.

Background: Diversity in clinical signs and symptoms are associated with soil transmitted diseases (STD), which are spread to humans by intestinal worms and transmitted in a variety of ways. There is a need for the present study, which aimed to investigate the prevalence of intestinal parasites and to compare between the common detection techniques for soil-transmitted helminths (STHs) among newly arrived expatriate labors in Jeddah, Saudi Arabia. Methods: A total of 188 stool samples were analyzed by macroscopic examination, and microscopic examination using direct iodine smear and the formal ether sedimentation technique. Trichrome and modified Kinyoun's stains were used to confirm the morphology of any detected protozoa stages and oocyst of Cryptosporidium, respectively. A chromatographic immunoassay kit was used for Entamoeba histolytica, Giardia lamblia and Cryptosporidium. In addition, real-time PCR was employed only to identify various STHs. Results: Out of 188, several types of parasites were detected in 35 samples (18.62%), of which some with multiple infections. Nine samples (4.79%) were positive for Entamoeba coli, seven samples (3.72%) for Trichuris trichiura, six samples (3.19%) for Necator americanus, four samples (2.13%) for Strongyloides stercoralis, four samples (2.13%) for Ascaris lumbricoides, four samples (2.13%) for E. histolytica, three samples (1.60%) for Blastocystis hominis and two samples (1.06%) for Ancylostoma duodenale. In comparison between laboratory techniques for STHs, real-time PCR was able to detect the DNA of 19 samples (10.1%) followed by Ritchie sedimentation technique (18, 9.6%), and direct smear (7, 3.7%) (p > 0.05). Conclusion: The high rate of newly arrived foreign workers infected with intestinal parasites could lead to a risk to society. Continuous and regular surveys are needed to deal with the occurrence of intestinal parasitic infections including STHs. To improve the identification of these infections, we recommend a supporting infrastructure for the application of concentration methods and molecular assays.

Transcriptomic profiling of different developmental stages reveals parasitic strategies of Wohlfahrtia magnifica, a myiasis-causing flesh fly.

BACKGROUND: Wohlfahrtia magnifica is an obligatory parasite that causes myiasis in several warm-blooded vertebrates. Adult females deposit the first-stage larvae directly onto wounds or natural body orifices (e.g., genitalia) of the host, from where they quickly colonize the host tissue and feed on it for development. The infestation of W. magnifica can lead to health issues, welfare concerns, and substantial economic losses. To date, little is known about the molecular mechanisms of the W. magnifica-causing myiasis. RESULTS: In this study, we collected parasitic-stage larvae of W. magnifica from wounds of naturally infested Bactrian camels, as well as pupae and adult flies reared in vitro from the wound-collected larvae, for investigating the gene expression profiles of the different developmental stages of W. magnifica, with a particular focus on examining gene families closely related to the parasitism of the wound-collected larvae. As key proteins related to the parasite-host interaction, 2049 excretory/secretory (ES) proteins were identified in W. magnifica through the integration of multiple bioinformatics approaches. Functional analysis indicates that these ES proteins are primarily involved in cuticle development, peptidase activity, immune response, and metabolic processes. The global investigation of gene expression at different developmental stages using pairwise comparisons and weighted correlation network analysis (WGCNA) showed that the upregulated genes during second-stage larvae were related to cuticle development, peptidase activity, and RNA transcription and translation; during third-stage larvae to peptidase inhibitor activity and nutrient reservoir activity; during pupae to cell and tissue morphogenesis and cell and tissue development; and during adult flies to signal perception, many of them involved in light perception, and adult behavior, e.g., feeding, mating, and locomotion. Specifically, the expression level analysis of the likely parasitism-related genes in parasitic wound-collected larvae revealed a significant upregulation of 88 peptidase genes (including 47 serine peptidase genes), 110 cuticle protein genes, and 21 heat shock protein (hsp) genes. Interestingly, the expression of 2 antimicrobial peptide (AMP) genes, including 1 defensin and 1 diptericin, was also upregulated in the parasitic larvae. CONCLUSIONS: We identified ES proteins in W. magnifica and investigated their functional distribution. In addition, gene expression profiles at different developmental stages of W. magnifica were examined. Specifically, we focused on gene families closely related to parasitism of wound-collected larvae. These findings shed light on the molecular mechanisms underlying the life cycle of the myiasis-causing fly, especially during the parasitic larval stages, and provide guidance for the development of control measures against W. magnifica.

Evaluating evidence for co-geography in the Anopheles-Plasmodium host-parasite system.

The often tight association between parasites and their hosts means that under certain scenarios, the evolutionary histories of the two species can become closely coupled both through time and across space. Using spatial genetic inference, we identify a potential signal of common dispersal patterns in the Anopheles gambiae and Plasmodium falciparum host-parasite system as seen through a between-species correlation of the differences between geographic sampling location and geographic location predicted from the genome. This correlation may be due to coupled dispersal dynamics between host and parasite but may also reflect statistical artifacts due to uneven spatial distribution of sampling locations. Using continuous-space population genetics simulations, we investigate the degree to which uneven distribution of sampling locations leads to bias in prediction of spatial location from genetic data and implement methods to counter this effect. We demonstrate that while algorithmic bias presents a problem in inference from spatio-genetic data, the correlation structure between A. gambiae and P. falciparum predictions cannot be attributed to spatial bias alone and is thus likely a genetic signal of co-dispersal in a host-parasite system.

VESPA: an optimized protocol for accurate metabarcoding-based characterization of vertebrate eukaryotic endosymbiont and parasite assemblages.

Protocols for characterizing taxonomic assemblages by deep sequencing of short DNA barcode regions (metabarcoding) have revolutionized our understanding of microbial communities and are standardized for bacteria, archaea, and fungi. Unfortunately, comparable methods for host-associated eukaryotes have lagged due to technical challenges. Despite 54 published studies, issues remain with primer complementarity, off-target amplification, and lack of external validation. Here, we present VESPA (Vertebrate Eukaryotic endoSymbiont and Parasite Analysis) primers and optimized metabarcoding protocol for host-associated eukaryotic community analysis. Using in silico prediction, panel PCR, engineered mock community standards, and clinical samples, we demonstrate VESPA to be more effective at resolving host-associated eukaryotic assemblages than previously published methods and to minimize off-target amplification. When applied to human and non-human primate samples, VESPA enables reconstruction of host-associated eukaryotic endosymbiont communities more accurately and at finer taxonomic resolution than microscopy. VESPA has the potential to advance basic and translational science on vertebrate eukaryotic endosymbiont communities, similar to achievements made for bacterial, archaeal, and fungal microbiomes.

Whole-genome CRISPR screens to understand Apicomplexan-host interactions.

Apicomplexan parasites are aetiological agents of numerous diseases in humans and livestock. Functional genomics studies in these parasites enable the identification of biological mechanisms and protein functions that can be targeted for therapeutic intervention. Recent improvements in forward genetics and whole-genome screens utilising CRISPR/Cas technology have revolutionised the functional analysis of genes during Apicomplexan infection of host cells. Here, we highlight key discoveries from CRISPR/Cas9 screens in Apicomplexa or their infected host cells and discuss remaining challenges to maximise this technology that may help answer fundamental questions about parasite-host interactions.