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Background: For several decades, the European wildcat (Felis silvestris) has gradually been returning to the forests of Germany, mainly in the central and southwestern regions. To increase the knowledge about this threatened species, the endoparasite status of dead found specimens from Germany was surveyed., Methods: A total of 118 wildcats were examined for endoparasites in peritoneal organs and skeletal muscles. Owing to decomposition or incomplete carcasses, 104 gastrointestinal tracts (stomachs and intestines), 101 livers with gallbladders, 99 urinary bladders, as well as kidneys of 95 and skeletal muscles of 112 specimens were available for examination. All detected parasites were identified morphologically to genus or species level, followed by molecular examinations of one to ten specimens of each parasite species., Results: Overall endoparasite prevalence in peritoneal organs was 99.0% (103/104). Among the 99.0% (103/104) infected gastrointestinal tracts, the most frequent species were Toxocara cati (95.2% [99/104]), Hydatigera kamiyai (84.6% [88/104]), Mesocestoides litteratus (69.2% [72/104]), Strongyloides spp. (58.7% [61/104]), Cylicospirura petrowi (37.5% [39/104]), Ancylostoma tubaeforme (31.7% [33/104]), Capillaria putorii (24.0% [25/104]), and Echinococcus multilocularis (18.3% [19/104]). In 77.8% (77/99) of the urinary bladders, Capillaria plica and/or Capillaria feliscati were detected. Moreover, the liver fluke Metorchis bilis occurred in 2.0% (2/101) of the livers, and roundworm larvae (presumably Toxocara spp.) were detected in 33.0% (37/112) of the muscle samples., Conclusions: These results show a broad spectrum of endoparasite species infecting European wildcats in Germany. It might be assumed that some of the endoparasites could pose a risk to domestic cats (Felis catus) and humans through spillover events, or may be transmitted from domestic cats to the free-ranging population, posing a potential risk to wildcats., Competing Interests: Declarations Ethics approval and consent to participate All organs originated from dead found specimens of F. silvestris collected within the framework of the project Monitoring of dead wildcats in Rhineland-Palatinate (Totfundmonitoring Wildkatze in Rheinland-Pfalz) of the German Federation for the Environment and Nature Conservation, Friends of the Earth Germany, state association of Rhineland-Palatinate [Bund fuer Umwelt und Naturschutz Deutschland (BUND), Landesverband Rheinland-Pfalz]. Collection of the dead found F. silvestris was permitted by the responsible Nature Conservation Agencies [19]. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

In conservation strategies, getting precise and repeatable information on the species' diet and health without relying on invasive or laborious methods is challenging. Here, we developed an efficient and non-invasive workflow for the sequencing and analysis of four taxonomic markers from fecal DNA to characterize the gut microbiota, parasites, and plants and lichens composing the winter diet of caribou (Rangifer tarandus), Canada's most iconic endangered species. Sequencing of the 18S rRNA gene of eukaryotes from seven locations in Manitoba and Saskatchewan, Canada, allowed for the detection of five genera of parasites in caribou feces (including Nematodirella and Parelaphostrongylus) with variable frequency of occurrence depending on sampling location and sex. Our workflow also revealed a rich winter plant and lichen diet in caribou, with respectively 29 and 18 genera identified across all samples through plant and fungal ITS2 sequencing. Relationships between the gut microbiota and both the diet and parasite richness were also identified. Of note, the Central Saskatchewan sampling location was characterized by a clearly distinct gut microbiota which could be linked to an epiphytic lichen-rich diet. Overall, our results showed the potential of this multi-marker DNA metabarcoding workflow as an efficient tool to provide insights into the species biology and ecology., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. His Majesty the King in Right of Canada as represented by the Minister of Natural Resources and Arnaud Droit, Paul Wilson, Gigi Pittoello, Susan Kutz.)

Recent advancements in next-generation sequencing (NGS) technologies have created new opportunities for comprehensive screening of multiple parasite species. In this study, we cloned the 18 S rDNA V9 region of 11 species of intestinal parasites into plasmids. Equal amounts and concentrations of these 11 plasmids were pooled, and amplicon NGS targeting the 18 S rDNA V9 region was performed using the Illumina iSeq 100 platform. A total of 434,849 reads were identified, and all 11 parasite species were detected, although the number of output reads for each parasite varied. The read count ratio, in descending order, was as follows: Clonorchis sinensis, 17.2%; Entamoeba histolytica, 16.7%; Dibothriocephalus latus, 14.4%; Trichuris trichiura, 10.8%; Fasciola hepatica, 8.7%; Necator americanus, 8.5%; Paragonimus westermani, 8.5%; Taenia saginata, 7.1%; Giardia intestinalis, 5.0%; Ascaris lumbricoides, 1.7%; and Enterobius vermicularis, 0.9%. We found that the DNA secondary structures showed a negative association with the number of output reads. Additionally, variations in the amplicon PCR annealing temperature affected the relative abundance of output reads for each parasite. These findings can be applied to improve parasite detection methodologies and ultimately enhance efforts to control and prevent intestinal parasitic infections., (© 2024. The Author(s).)

Among different climatic zones in India, the tropical monsoon region comprises a diverse ecosystem characterized by the endemic nature of several parasites including certain emerging and re-emerging vector-borne pathogens of humans, whereas a systematic investigation of the occurrence of different parasites among domestic goats in this area is not yet explored. The goal of the present study is to explore the parasite diversity focusing on molecular identification of vector-borne hemoparasites and its health impacts on domestic goats reared in the tropical monsoon climate zone of Kerala, India. Among 227 goats presented to the Teaching Veterinary Clinical Complex (TVCC) in the monsoon months of 2023, thirty animals were recruited for the study. The animals were screened for the presence of different hemoparasites (Anaplasma spp., Theileria spp., and Babesia spp.), ectoparasites (ticks, lice, and fleas), and gastrointestinal (GI) parasites (hookworms, threadworms, tapeworms, whipworms, and coccidia). The isolated hemoparasites were further characterized by sequencing and phylogenetic analysis. The correlation studies to elucidate the association between the occurrence of different parasites and clinical manifestations (hyperthermia, pallor of mucous membrane, circulatory failure, respiratory signs, neurological instability, and GI signs), blood picture (anemia, leukopenia, thrombocytopenia), demographics (sex and age), and treatment history (hemoparasitic therapy, ectoparasiticidal application, and prophylactic deworming) were conducted. The co-infection status of these parasites was also evaluated. A substantial portion of the goats in the study group was found to be affected by vector-borne hemoparasitic diseases and their arthropod vectors or GI parasites or both. This can be attributed to the constantly warm and humid climate of the region, which is favorable for the survival and growth of different life cycle stages of these parasites and vectors. A strategic parasitic disease surveillance-cum-control program is the need of the hour for ensuring climate resilience and profitable goat farming in the region., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Vegetable and fruit contamination is recognized as a significant parasite transmission route. This review presents the current state of vegetables ad fruits contamination with food-borne parasitic protozoa worldwide. We consider the methodologies and strategies for detecting parasitic stages developed in the last decade and the contamination data. Asia had the highest number of reports (94 studies), followed by Africa (74 studies). At the country level, with 41 studies, Iran had the most reports among other countries, followed by Nigeria (28 studies). According to the studies included in the current review, 41.22% of vegetables and fruits were contaminated with different species of protozoan parasites. Among different continents, Asia accounted for the highest contamination rate of protozoan parasites (57.12%). Giardia spp. (10%) had the highest contamination rate in vegetables and fruits, followed by Entamoeba coli (8%), E. histolytica/dispar (7%), and Cryptosporidium spp. (6%). This study provides essential data for health authorities to develop food safety programs. The presence of protozoan parasites in fruits and vegetables highlights the critical need for maintaining rigorous food safety measures across the entire production and distribution process, particularly in countries that are major producers and distributors of these food items., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The practice of hybridization is carried out globally in fish farms. Here, we present the first record of the parasitic fauna of hybrids among genus Colossoma and Piaractus in natural environments. We identified a total of 48 hybrids, nine F1 hybrids (nuclear DNA from both species present in the cross) and 38 advanced hybrids (nuclear DNA from one species), both from crosses between Piaractus brachypomus and Piaractus mesopotamicus, and one F1 "tambacu" corresponding to cross between Colossoma macropomum and Piaractus mesopotamicus. This is the first record of Anacanthorus penilabiatus, Anacanthorus toledoensis, Mymarothecium viatorum, Mymarothecium ianwhittington, Haementeria sp., Dadaytrema oxycephala, Rondonia rondoni, and Echinorhynchus gomesi parasitizing hybrids collected in a natural environment. With this, we expand knowledge about the diversity of fish and parasites in the upper Paraná River and warn about the risk that fish escapes can cause in the basin., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The Anthropocene is seeing the human footprint rapidly spreading to all of Earth's ecosystems. The fast-changing biotic and abiotic conditions experienced by all organisms are exerting new and strong selective pressures, and there is a growing list of examples of human-induced evolution in response to anthropogenic impacts. No organism is exempt from these novel selective pressures. Here, we synthesise current knowledge on human-induced evolution in eukaryotic parasites of animals, and present a multidisciplinary framework for its study and monitoring. Parasites generally have short generation times and huge fecundity, features that predispose them for rapid evolution. We begin by reviewing evidence that parasites often have substantial standing genetic variation, and examples of their rapid evolution both under conditions of livestock production and in serial passage experiments. We then present a two-step conceptual overview of the causal chain linking anthropogenic impacts to parasite evolution. First, we review the major anthropogenic factors impacting parasites, and identify the selective pressures they exert on parasites through increased mortality of either infective stages or adult parasites, or through changes in host density, quality or immunity. Second, we discuss what new phenotypic traits are likely to be favoured by the new selective pressures resulting from altered parasite mortality or host changes; we focus mostly on parasite virulence and basic life-history traits, as these most directly influence the transmission success of parasites and the pathology they induce. To illustrate the kinds of evolutionary changes in parasites anticipated in the Anthropocene, we present a few scenarios, either already documented or hypothetical but plausible, involving parasite taxa in livestock, aquaculture and natural systems. Finally, we offer several approaches for investigations and real-time monitoring of rapid, human-induced evolution in parasites, ranging from controlled experiments to the use of state-of-the-art genomic tools. The implications of fast-evolving parasites in the Anthropocene for disease emergence and the dynamics of infections in domestic animals and wildlife are concerning. Broader recognition that it is not only the conditions for parasite transmission that are changing, but the parasites themselves, is needed to meet better the challenges ahead., (© 2024 The Author(s). Biological Reviews published by John Wiley & Sons Ltd on behalf of Cambridge Philosophical Society.)

Parasites are generally associated with lower income countries in tropical and subtropical areas. Still, they are also prevalent in low-income communities in the southern United States. Studies characterizing the epidemiology of parasites in the United States are limited, resulting in little comprehensive understanding of the problem. This study investigated the environmental contamination of parasites in the southern United States by determining each parasite's contamination rate and burden in five low-income communities. A total of 499 soil samples of approximately 50 g were collected from public parks and private residences in Alabama, Louisiana, Mississippi, South Carolina, and Texas. A technique using parasite floatation, filtration, and bead-beating was applied to dirt samples to concentrate and extract parasite DNA from samples and detected via multiparallel quantitative polymerase chain reaction (qPCR). qPCR detected total sample contamination of Blastocystis spp. (19.03%), Toxocara cati (6.01%), Toxocara canis (3.61%), Strongyloides stercoralis (2.00%), Trichuris trichiura (1.80%), Ancylostoma duodenale (1.42%), Giardia intestinalis (1.40%), Cryptosporidium spp. (1.01%), Entamoeba histolytica (0.20%), and Necator americanus (0.20%). The remaining samples had no parasitic contamination. Overall parasite contamination rates varied significantly between communities: western Mississippi (46.88%), southwestern Alabama (39.62%), northeastern Louisiana (27.93%), southwestern South Carolina (27.93%), and south Texas (6.93%) (P <0.0001). T. cati DNA burdens were more significant in communities with higher poverty rates, including northeastern Louisiana (50.57%) and western Mississippi (49.60%) compared with southwestern Alabama (30.05%) and southwestern South Carolina (25.01%) (P = 0.0011). This study demonstrates the environmental contamination of parasites and their relationship with high poverty rates in communities in the southern United States.

As sequencing genomes has become increasingly popular, the need for annotation of the resulting assemblies is growing. Structural and functional annotation is still challenging as it includes finding the correct gene sequences, annotating other elements such as RNA and being able to submit those data to databases to share it with the community. Compared to de novo assembly where contiguous chromosomes are a sign of high quality, it is difficult to visualize and assess the quality of annotation. We developed the Companion web server to allow non-experts to annotate their genome using a reference-based method, enabling them to assess the output before submitting to public databases. In this update paper, we describe how we have included novel methods for gene finding and made the Companion server more efficient for annotation of genomes of up to 1 Gb in size. The reference set was increased to include genomes of interest for human and animal health from the fungi and arthropod kingdoms. We show that Companion outperforms existing comparable tools where closely related references are available., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: The initial step to interpreting putative biological functions from comparative multi-omics studies usually starts from a differential expressed gene list followed by functional enrichment analysis (FEA). However, most FEA packages are designed exclusively for humans and model organisms. Although parasitic protozoan is the most important pathogen in the tropics, no FEA package is available for protozoan functional (ProFun) enrichment analysis. To speed up comparative multi-omics research on parasitic protozoans, we constructed ProFun, a web-based, user-friendly platform for the research community., Methods: ProFun utilizes the Docker container, ShinyProxy, and R Shiny to construct a scalable web service with load-balancing infrastructure. We have integrated a series of visual analytic functions, in-house scripts, and custom-made annotation packages to create three analytical modules for 40 protozoan species: (1) Gene Overlaps; (2) Over-representation Analysis (ORA); (3) Gene Set Enrichment Analysis (GSEA)., Results: We have established ProFun, a web server for functional enrichment analysis of differentially expressed genes. FEA becomes as simple as pasting a list of gene IDs into the textbox of our website. Users can customize enrichment parameters and results with just one click. The intuitive web interface and publication-ready charts enable users to reveal meaningful biological events and pinpoint potential targets for further studies., Conclusion: ProFun is the first web application that enables gene functional enrichment analysis of parasitic protozoans. In addition to supporting FEA analysis, ProFun also allows the comparison of FEA results across complicated experimental designs. ProFun is freely available at http://dalek.cgu.edu.tw:8080/app/profun., (Copyright © 2024. Published by Elsevier B.V.)

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., Competing Interests: Declaration of interests The author declares no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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., (© 2024 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

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., (© 2024. The Author(s).)

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., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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., Competing Interests: Conflicts of interest The author(s) declare no conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Cassiano, Martinelli, Mottin, Neves, Andrade, Ferreira and Cravo.)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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., (© 2024. The Author(s).)

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.

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., (© 2024. The Author(s).)

Plasmodium species adapt a complex lifecycle with multiple phenotypes to survive inside various cell types of humans and mosquitoes. Stage-specific gene expression in the developmental stages of parasites is tightly controlled in Plasmodium species; however, the underlying mechanisms have yet to be explored. Genome organization and gene expression for each stage of the malaria parasite need to be better characterized. Recent studies indicated that epigenetic modifications of histone proteins play a vital role in chromatin plasticity. Like other eukaryotes, Plasmodium species N-terminal tail modifications form a distinct "histone code," which creates the docking sites for histone reader proteins, including gene activator/repressor complexes, to regulate gene expression. The emerging research findings shed light on various unconventional epigenetic changes in histone proteins' core/globular domain regions, which might contribute to the chromatin organization in different developmental stages of the malaria parasite. The malaria parasite lost many transcription factors during evolution, and it is proposed that the nature of local chromatin structure essentially regulates the stage-specific gene expression. This review highlights recent discoveries of unconventional histone globular domain epigenetic modifications and their functions in regulating chromatin structure dynamics in various developmental stages of malaria parasites., (© 2023 Wiley-VCH GmbH.)

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., (© 2024. The Author(s).)

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., (© 2024. The Author(s).)

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.

Small pelagic fishes represent one of the most important food resources off the Northwest coast of Africa. Despite their economic significance, little is known about the infections with flesh invading myxosporean parasites of genus Kudoa (Cnidaria, Myxozoa). Heavy infections in the flesh may be associated with post-mortem myoliquefaction, commonly known as 'soft flesh'. This condition may reduce the quality and marketability of the fish fillet, resulting in both economic losses to the fishing industry and loss of consumer confidence. In this study, we investigated Kudoa-induced 'soft flesh' occurrence in European anchovy Engraulis encrasicolus, European pilchard Sardina pilchardus, and Atlantic chub mackerel Scomber colias caught in 2019 off the Moroccan Atlantic coast. Five hundred specimens of each fish species were examined for 'soft flesh' by texture testing and visual inspection 48 h post-catch. 'Soft flesh' occurred in 0.2 % of the European anchovies, 1.4 % of the European pilchard, and in 4.4 % of the Atlantic chub mackerel. Microscopic examination of muscle samples revealed that 'soft flesh'-affected fish were infected with myxospores of K. thyrsites-like morphotype. Analysis of the kudoid SSU rDNA sequence obtained from European pilchard and the Atlantic chub mackerel identified these as K. thyrsites (100 % identity), whereas analysis of the sequence from European anchovy identified the presence of K. encrasicoli (100 % identity). Even if there are no known human health consequences associated with the ingestion of these Kudoa species, the unsightly appearance of some infected fillets is a food quality issue, that can eventually lead to reduced marketability and value., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

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 (FEC S ) and Nematodirus (FEC N ) 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 FEC S , FEC N , 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 FEC N, 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., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

MicroRNAs (miRNAs) are small noncoding RNAs that oversee gene modulation. They are integral to cellular functions and can migrate between species, leading to cross-kingdom gene suppression. Recent breakthroughs in helminth genome studies have sparked curiosity about helminth RNA regulators and their ability to regulate genes across species. Growing data indicate that helminth miRNAs have a significant impact on the host's immune system. Specific miRNAs from helminth parasites can merge with the host's miRNA system, implying that parasites could exploit their host's regulatory machinery and function. This review highlights the role of cross-kingdom helminth-derived miRNAs in the interplay between host and parasite, exploring potential routes for their uptake, processing, and consequences in host interaction., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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., (© 2024 The Authors. Environmental Microbiology published by Applied Microbiology International and John Wiley & Sons Ltd.)

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Cheng et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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., Competing Interests: The authors declare that they have no competing interests., (© 2024 Al-Refai and Wakid.)

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, EmTRPM PZQ . 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Herz, Zarowiecki, Wessels, Pätzel, Herrmann, Braun, Holroyd, Huckvale, Bergmann, Spiliotis, Koziol, Berriman and Brehm.)

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., (© 2024. The Author(s).)

The bulk of higher order organismal genomes is comprised of transposable element (TE) copies, i.e. genetic parasites. The host-parasite relation is multi-faceted, varying across genomic region (genic versus intergenic), life-cycle stages, tissue-type and of course in health versus pathological state. The reach of functional genomics though, in investigating genotype-to-phenotype relations, has been limited when TEs are involved. The aim of this review is to highlight recent progress made in understanding how TE origin biochemical activity interacts with the central dogma stages of the host genome. Such interaction can also bring about modulation of the immune context and this could have important repercussions in disease state where immunity has a role to play. Thus, the review is to instigate ideas and action points around identifying evolutionary adaptations that the host genome and the genetic parasite have evolved and why they could be relevant., (© The Author(s) 2022. Published by Oxford University Press.)

Importance: Malaria is caused by parasites of the genus Plasmodium , and reached a global disease burden of 247 million cases in 2021. To study drug resistance mutations and parasite population dynamics, whole-genome sequencing of patient blood samples is commonly performed. However, the predominance of human DNA in these samples imposes the need for time-consuming laboratory procedures to enrich Plasmodium DNA. We used the Oxford Nanopore Technologies' adaptive sampling feature to circumvent this problem and enrich Plasmodium reads directly during the sequencing run. We demonstrate that adaptive nanopore sequencing efficiently enriches Plasmodium reads, which simplifies and shortens the timeline from blood collection to parasite sequencing. In addition, we show that the obtained data can be used for monitoring genetic markers, or to generate nearly complete genomes. Finally, owing to its inherent mobility, this technology can be easily applied on-site in endemic areas where patients would benefit the most from genomic surveillance., Competing Interests: The authors declare no conflict of interest.

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., (© 2024. The Author(s).)

Ribosomes from different species can markedly differ in their composition by including dozens of ribosomal proteins that are unique to specific lineages but absent in others. However, it remains unknown how ribosomes acquire new proteins throughout evolution. Here, to help answer this question, we describe the evolution of the ribosomal protein msL1/msL2 that was recently found in ribosomes from the parasitic microorganism clade, microsporidia. We show that this protein has a conserved location in the ribosome but entirely dissimilar structures in different organisms: in each of the analyzed species, msL1/msL2 exhibits an altered secondary structure, an inverted orientation of the N-termini and C-termini on the ribosomal binding surface, and a completely transformed 3D fold. We then show that this fold switching is likely caused by changes in the ribosomal msL1/msL2-binding site, specifically, by variations in rRNA. These observations allow us to infer an evolutionary scenario in which a small, positively charged, de novo-born unfolded protein was first captured by rRNA to become part of the ribosome and subsequently underwent complete fold switching to optimize its binding to its evolving ribosomal binding site. Overall, our work provides a striking example of how a protein can switch its fold in the context of a complex biological assembly, while retaining its specificity for its molecular partner. This finding will help us better understand the origin and evolution of new protein components of complex molecular assemblies-thereby enhancing our ability to engineer biological molecules, identify protein homologs, and peer into the history of life on Earth., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Trypanosoma cruzi is a protozoan parasite causing Chagas disease, with a complex life cycle involving different stages in insect vectors and mammalian hosts. Amastigotes are an intracellular form that replicates in the cytoplasm of host cells, and recent studies suggested that dormant forms may be contributing to parasite persistence, suggesting cellular heterogeneity among amastigotes. We investigated here if a transcriptomic approach could identify some heterogeneity in intracellular amastigotes and identify a dormant population. We used gene expression data derived from bulk RNA-sequencing of T. cruzi infection of human fibroblasts for deconvolution using CDSeq, which allows to simultaneously estimate amastigote cell-type proportions and cell-type-specific expression profiles. Six amastigote subpopulations were identified, confirming intracellular amastigotes heterogeneity, and one population presented characteristics of non-replicative dormant parasites, based on replication markers and TcRAD51 expression. Transcriptomic approaches appear to be powerful to understand T. cruzi cell differentiation and expansion of these studies could provide further insight on the role different cell types in parasite persistence and Chagas disease pathogenesis., Competing Interests: Declaration of competing interest The authors declare to have no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Acanthocephalans of the order Polymorphida mainly parasitic in birds and mammals, are of veterinary, medical and economic importance. However, the evolutionary relationships of its 3 families (Centrorhynchidae, Polymorphidae and Plagiorhynchidae) remain under debate. Additionally, some species of Polymorphida (i.e. Bolbosoma spp. and Corynosoma spp.) are recognized as zoonotic parasites, associated with human acanthocephaliasis, but the mitochondrial genomes for representatives of Bolbosoma and Corynosoma have not been reported so far. In the present study, the complete mitochondrial genomes B. nipponicum and C. villosum (Acanthocephala: Polymorphidae) are reported for the first time, which are 14 296 and 14 241 bp in length, respectively, and both contain 36 genes [including 12 PCGs, 22 tRNA genes and 2 rRNA genes] and 2 non-coding regions ( NCR1 and NCR2 ). The gene arrangement of some tRNAs in the mitogenomes of B. nipponicum and C. villosum differs from that found in all other acanthocephalans, except Polymorphus minutus . Phylogenetic results based on concatenated amino acid (AA) sequences of the 12 protein-coding genes (PCGs) strongly supported that the family Polymorphidae is a sister to the Centrorhynchidae rather than the Plagiorhynchidae, and also confirmed the sister relationship of the genera Bolbosoma and Corynosoma in the Polymorphidae based on the mitogenomic data for the first time. Our present findings further clarified the phylogenetic relationships of the 3 families Plagiorhynchidae, Centrorhynchidae and Polymorphidae, enriched the mitogenome data of the phylum Acanthocephala (especially the order Polymorphida), and provided the resource of genetic data for diagnosing these 2 pathogenic parasites of human acanthocephaliasis.

To date, only a small number of regulatory transcription factors have been predicted from the genome of Plasmodium and Apicomplexan parasites. We previously identified a nuclear factor named Prx regulatory element-binding protein (PREBP) from Plasmodium falciparum. PREBP had been suggested to bind to the cis-element in the promoter of an antioxidant pf1-cys-prx gene, thereby promoting the expression of downstream genes. PREBP has 4 putative K homology (KH) domains, which are known to bind RNA and single-stranded DNA. In this study, to understand the detailed action of PREBP in parasite cells, we first observed that in living parasite cells, PREBP was localized in the nucleus in the trophozoite and schizont stages, in which the expression of the target pf1-cys-prx was enhanced. The interaction of PREBP and the cis-element of pf1-cys-prx in the parasite cells was also confirmed. Further, the activities of PREBP deletion mutants were analyzed, and regions with repeated KH domains in PREBP seemed to be responsible for the recognition of the cis-element. These results led us to hypothesize that Plasmodium and other Apicomplexan parasites might have a transcription factor family with KH domains. Bioinformatic analysis revealed a putative ortholog group including PREBP and several Plasmodium and Apicomplexan factors with KH domains. One of the P. falciparum-derived factors, which were included in the putative ortholog group, was found to be localized at the nucleus in the trophozoite stage, indicating that it might be a novel transcription factor. The discovery of PREBP and putative transcription factors with KH domains suggested that multi-functional proteins with KH domains possibly evolved in the Apicomplexan organisms. They might play key roles in transcriptional regulatory processes that are essential for living organisms and may even represent unique drug targets for malaria therapy., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Komaki-Yasuda, Kano. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background-Aim: Protistan communities have a major contribution to biochemical processes and food webs in coastal ecosystems. However, related studies are scarce and usually limited in specific groups and/or sites. The present study examined the spatial structure of the entire protistan community in seven different gulfs and three different depths in a regional Mediterranean Sea, aiming to define taxa that are important for differences detected in the marine microbial network across the different gulfs studied as well as their trophic interactions., Methods: Protistan community structure analysis was based on the diversity of the V2-V3 hypervariable region of the 18S rRNA gene. Operational taxonomic units (OTUs) were identified using a 97% sequence identity threshold and were characterized based on their taxonomy, trophic role, abundance and niche specialization level. The differentially abundant, between gulfs, OTUs were considered for all depths and interactions amongst them were calculated, with statistic and network analysis., Results: It was shown that Dinophyceae, Bacillariophyta and Syndiniales were the most abundant groups, prevalent in all sites and depths . Gulfs separation was more striking at surface corroborating with changes in environmental factors, while it was less pronounced in higher depths. The study of differentially abundant, between gulfs, OTUs revealed that the strongest biotic interactions in all depths occurred between parasite species (mainly Syndiniales ) and other trophic groups. Most of these species were generalists but not abundant highlighting the importance of rare species in protistan community assemblage., Conclusion: Overall this study revealed the emergence of parasites as important contributors in protistan network regulation regardless of depth., Competing Interests: Konstantinos Kormas is an Academic Editor for PeerJ., (©2023 Meziti et al.)

Haemosporidian parasites that infect birds (Apicomplexa: Haemosporida) are blood parasites that require an invertebrate host (vector) and a vertebrate host for their lifecycle and cause malaria-like diseases. This group of parasites has provided valuable insights into host specificity, virulence, and parasite dispersal. Additionally, they have played a significant role in reshaping our understanding of the evolutionary history of apicomplexans. In order to accurately identify species and to address phylogenetic questions such as the timing of the haemosporidian radiation, the use of a sufficiently large genetic data set is crucial. However, acquiring this genetic data poses significant challenges. In this research, a sensitive nested PCR assay was developed. This assay allows for the easy amplification of complete mitochondrial genomes of haemosporidian parasites in birds, even during the chronic stage of infection. The effectiveness of this new nested PCR assay was evaluated using blood and tissue samples of birds with verified single parasite infections from previous studies. The approach involves amplifying four overlapping fragments of the mitochondrial genome and requires DNA extracts from single-infected samples. This method successfully amplified the complete mitochondrial genomes of 24 distinct haemosporidian parasite lineages found in various bird species. This data is invaluable for conducting phylogenetic analyses and accurately defining species. Furthermore, this study proposes the existence of at least 15 new haemosporidian parasite species based on the genetic information obtained. Data regarding pGRW04, previously categorized as Plasmodium relictum like pSGS1 and pGRW11, indicates that the pGRW04 lineage is actually a separate, hidden Plasmodium species., (© 2023. The Author(s).)

The distribution of avian haemosporidians of the genus Leucocytozoon in the Neotropics remains poorly understood. Recent studies confirmed their presence in the region using molecular techniques alone, but evidence for gametocytes and data on putative competent hosts for Leucocytozoon are still lacking outside highland areas. We combined morphological and molecular data to characterize a new Leucocytozoon species infecting a non-migratory red-legged seriema ( Cariama cristata ), the first report of a competent host for Leucocytozoon in Brazil. Leucocytozoon cariamae n. sp. is distinguished from the Leucocytozoon fringillinarum group by its microgametocytes that are not strongly appressed to the host cell nucleus. The bird studied was coinfected with Haemoproteus pulcher , and we present a Bayesian phylogenetic analysis based on nearly complete mitochondrial genomes of these 2 parasites. Leucocytozoon cariamae n. sp. morphology is consistent with our phylogenetic analysis indicating that it does not share a recent common ancestor with the L . fringillinarum group. Haemoproteus pulcher and Haemoproteus catharti form a monophyletic group with Haemocystidium parasites of Reptilia, supporting the polyphyly of the genus Haemoproteus . We also discussed the hypothesis that H. pulcher and H. catharti may be avian Haemocystidium , highlighting the need to study non-passerine parasites to untangle the systematics of Haemosporida.

Both parasitoids and entomopathogenic fungi are becoming increasingly crucial for managing pest populations. Therefore, it is essential to carefully consider the potential impact of entomopathogenic fungi on parasitoids due to their widespread pathogenicity and the possible overlap between these biological control tools during field applications. However, despite their importance, little research has been conducted on the pathogenicity of entomopathogenic fungi on parasitoids. In our study, we aimed to address this knowledge gap by investigating the interaction between the well-known entomopathogenic fungus Beauveria bassiana , and the pupal endoparasitoid Pteromalus puparum . Our results demonstrated that the presence of B. bassiana significantly affected the survival rates of P. puparum under laboratory conditions. The pathogenicity of B. bassiana on P. puparum was dose- and time-dependent, as determined via through surface spraying or oral ingestion. RNA-Seq analysis revealed that the immune system plays a primary and crucial role in defending against B. bassiana . Notably, several upregulated differentially expressed genes (DEGs) involved in the Toll and IMD pathways, which are key components of the insect immune system, and antimicrobial peptides were rapidly induced during both the early and late stages of infection. In contrast, a majority of genes involved in the activation of prophenoloxidase and antioxidant mechanisms were downregulated. Additionally, we identified downregulated DEGs related to cuticle formation, olfactory mechanisms, and detoxification processes. In summary, our study provides valuable insights into the interactions between P. puparum and B. bassiana , shedding light on the changes in gene expression during fungal infection. These findings have significant implications for the development of more effective and sustainable strategies for pest management in agriculture.

We recently described a targeted amplicon deep sequencing (TADS) strategy that utilizes a nested PCR targeting the 18S rDNA gene of blood-borne parasites. The assay facilitates selective digestion of host DNA by targeting enzyme restriction sites present in vertebrates but absent in parasites. This enriching of parasite-derived amplicon drastically reduces the proportion of host-derived reads during sequencing and results in the sensitive detection of several clinically important blood parasites including Plasmodium spp., Babesia spp., kinetoplastids, and filarial nematodes. Despite these promising results, high costs and the laborious nature of metagenomics sequencing are prohibitive to the routine use of this assay in most laboratories. We describe and evaluate a new metagenomic approach that utilizes a set of primers modified from our original assay that incorporates Illumina barcodes and adapters during the PCR steps. This modification makes amplicons immediately compatible with sequencing on the Illumina MiSeq platform, removing the need for a separate library preparation, which is expensive and time-consuming. We compared this modified assay to our previous nested TADS assay in terms of preparation speed, limit of detection (LOD), and cost. Our modifications reduced assay turnaround times from 7 to 5 days. The cost decreased from approximately $40 per sample to $11 per sample. The modified assay displayed comparable performance in the detection and differentiation of human-infecting Plasmodium spp., Babesia spp., kinetoplastids, and filarial nematodes in clinical samples. The LOD of this modified approach was determined for malaria parasites and remained similar to that previously reported for our earlier assay (0.58 Plasmodium falciparum parasites/µL of blood). These modifications markedly reduced costs and turnaround times, making the assay more amenable to routine diagnostic applications., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Myxobolus lentisuturalis is a myxosporean parasite infecting the musculature both of goldfish (Carassius auratus auratus) and gibel carp (Carassius auratus gibelio). The species was originally described in China from gibel carp that is a common fish for sport fishing in Hungary meanwhile is one of the most popular farmed fish in China due to its high demand. Eighteen gibel carp with distortions were collected from a barrage pond in southern Hungary. All fish had large humps in the dorsolateral region due to infection of the muscle between the head and the dorsal fin. The swollen degenerated tissue was filled with myxozoan spores, which were collected for morphological and molecular studies. By size and morphology, the spores were consistent with morphological description of M. lentisuturalis. Histopathological examination showed that the formation of plasmodia containing myxospores leads to severe destruction of muscle tissue. The 18S ribosomal DNA and 28S ribosomal DNA data of the samples presented matched with previous sequences of M. lentisuturalis in GenBank. Phylogenetic analyses confirmed that our sequences belong to a monophyletic group with them supported by a high bootstrap. This study highlights the occurrence of a highly pathogenic myxozoan, M. lentisuturalis in Hungary as a new geographical location., (© 2023 John Wiley & Sons Ltd.)

Multigene families often play an important role in host-parasite interactions. One of the largest multigene families in Theileria parva, the causative agent of East Coast fever, is the T. parva repeat (Tpr) gene family. The function of the putative Tpr proteins remains unknown. The initial publication of the T. parva reference genome identified 39 Tpr family open reading frames (ORFs) sharing a conserved C-terminal domain. Twenty-eight of these are clustered in a central region of chromosome 3, termed the "Tpr locus", while others are dispersed throughout all four nuclear chromosomes. The Tpr locus contains three of the four assembly gaps remaining in the genome, suggesting the presence of additional, as yet uncharacterized, Tpr gene copies. Here, we describe the use of long-read sequencing to attempt to close the gaps in the reference assembly of T. parva (located among multigene families clusters), characterize the full complement of Tpr family ORFs in the T. parva reference genome, and evaluate their evolutionary relationship with Tpr homologs in other Theileria species. We identify three new Tpr family genes in the T. parva reference genome and show that sequence similarity among paralogs in the Tpr locus is significantly higher than between genes outside the Tpr locus. We also identify sequences homologous to the conserved C-terminal domain in five additional Theileria species. Using these sequences, we show that the evolution of this gene family involves conservation of a few orthologs across species, combined with gene gains/losses, and species-specific expansions., (© 2023. The Author(s).)

Vector-transmitted haemosporidians are among the most common parasites in birds, but our knowledge of the inter-specific patterns of infection rates and the parasite community composition is far from complete because of the unequal distribution of the screening effort across bird families and genera. To assess infection rates and the diversity of haemosporidians from the genera Plasmodium, Haemoproteus, and Leucocytozoon in marsh terns, which represent poorly explored in this regard genus of the family gulls, terns, and skimmers (Laridae), we screened two species: the Whiskered Tern (Chlidonias hybrida) and the Black Tern (Chlidonias niger). We sampled these long-distance migratory birds on breeding grounds: the Whiskered Tern in south-central Poland and north-central Ukraine, and the Black Tern-in north-central Ukraine. We found that birds from both species were infected only sporadically, with prevalence at the population level not exceeding 3.4%. Only parasites from the genera Plasmodium and Leucocytozoon were detected. There was neither an inter-specific difference nor a difference between populations of the Whiskered Tern in infection rates. In total, we registered three lineages-one Plasmodium and two Leucocytozoon-that were previously recorded in other bird species, and two unidentified Plasmodium infections. One of the lineages (Leucocytozoon LARCAC02) represents a specialist parasite with the host range restricted to larids and geographic range restricted to Poland, and two others (Plasmodium SGS1 and Leucocytozoon CIAE02) represent generalist parasites with very broad host and geographic ranges. This study reinforces the existing evidence that terns host parasites from genera Haemoproteus, Plasmodium, and Leucocytozoon only sporadically., (© 2023. The Author(s).)

Apicomplexan parasites are a large and diverse clade of protists responsible for significant diseases of humans and animals. Central to the ability of these parasites to colonize their host and evade immune responses is an expanded repertoire of gene-expression programs that requires the coordinated action of complex transcriptional networks. DNA-binding proteins and chromatin regulators are essential orchestrators of apicomplexan gene expression that often act in concert. Although apicomplexan genomes encode various families of putative DNA-binding proteins, most remain functionally and mechanistically unexplored. This review highlights the versatile role of myeloblastosis (Myb) domain-containing proteins in apicomplexan parasites as transcription factors and chromatin regulators. We explore the diversity of Myb domain structure and use phylogenetic analysis to identify common features across the phylum. This provides a framework to discuss functional heterogeneity and regulation of Myb domain-containing proteins particularly emphasizing their role in parasite differentiation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Aims: Diets and parasites influence the gut bacterial symbionts of bumble bees, but potential interactive effects remain overlooked. The main objective of this study was to assess the isolated and interactive effects of sunflower pollen, its phenolamides, and the widespread trypanosomatid Crithidia sp. on the gut bacterial symbionts of Bombus terrestris males., Methods and Results: Bumble bee males emerged in microcolonies fed on either (i) willow pollen (control), (ii) sunflower pollen, or (iii) willow pollen spiked with phenolamide extracts from sunflower pollen. These microcolonies were infected by Crithidia sp. or were pathogen-free. Using 16S rRNA amplicon sequencing (V3-V4 region), we observed a significant alteration of the beta diversity but not of the alpha diversity in the gut microbial communities of males fed on sunflower pollen compared to males fed on control pollen. Similarly, infection by the gut parasite Crithidia sp. altered the beta diversity but not the alpha diversity in the gut microbial communities of males, irrespective of the diet. By contrast, we did not observe any significant alteration of the beta or alpha diversity in the gut microbial communities of males fed on phenolamide-enriched pollen compared to males fed on control pollen. Changes in the beta diversity indicate significant dissimilarities of the bacterial taxa between the treatment groups, while the lack of difference in alpha diversity demonstrates no significant changes within each treatment group., Conclusions: Bumble bees harbour consistent gut microbiota worldwide, but our results suggest that the gut bacterial communities of bumble bees are somewhat shaped by their diets and gut parasites as well as by the interaction of these two factors. This study confirms that bumble bees are suitable biological surrogates to assess the effect of diet and parasite infections on gut microbial communities., (© The Author(s) 2023. Published by Oxford University Press on behalf of Applied Microbiology International.)

Microturbellarians are abundant and ubiquitous members of marine meiofaunal communities around the world. Because of their small body size, these microscopic animals are rarely considered as hosts for parasitic organisms. Indeed, many protists, both free-living and parasitic ones, equal or surpass meiofaunal animals in size. Despite several anecdotal records of "gregarines", "sporozoans", and "apicomplexans" parasitizing microturbellarians in the literature-some of them dating back to the nineteenth century-these single-celled parasites have never been identified and characterized. More recently, the sequencing of eukaryotic microbiomes in microscopic invertebrates have revealed a hidden diversity of protist parasites infecting microturbellarians and other meiofaunal animals. Here we show that apicomplexans isolated from twelve taxonomically diverse rhabdocoel taxa and one species of proseriate collected in four geographically distinct areas around the Pacific Ocean (Okinawa, Hokkaido, and British Columbia) and the Caribbean Sea (Curaçao) all belong to the apicomplexan genus Rhytidocystis. Based on comprehensive molecular phylogenies of Rhabdocoela and Proseriata inferred from both 18S and 28S rDNA sequences, as well as a molecular phylogeny of Marosporida inferred from 18S rDNA sequences, we determine the phylogenetic positions of the microturbellarian hosts and their parasites. Multiple lines of evidence, including morphological and molecular data, show that at least nine new species of Rhytidocystis infect the microturbellarian hosts collected in this study, more than doubling the number of previously recognized species of Rhytidocystis, all of which infect polychaete hosts. A cophylogenetic analysis examining patterns of phylosymbiosis between hosts and parasites suggests a complex picture of overall incongruence between host and parasite phylogenies, and varying degrees of geographic signals and taxon specificity., (© 2023. The Author(s).)