Dicrocoelium dendriticum (Trematoda) larvae cause their ant hosts to attach themselves to a plant with their mandibles. Infected ants detach a few hours later, then repeat the attach/detach sequence over subsequent days. Recognition of reversibility in altered host behaviours can enhance our understanding of the general phenomenon of host manipulation.

The Xenopus model organism knowledgebase, Xenbase (www.xenbase.org), bridges a wide variety of data types including genomes, anatomy, phenotypes, proteins, diseases and more. The goal of Xenbase is to support Xenopus molecular, cell and developmental biology research, to make these data available to the broader biomedical ecosystem, and accelerate the translation of Xenopus research into knowledge that will improve human health. Connections are made between data through relationships in our core data model and via a series of ontologies that serve as graph-based maps that can be traversed in various dimensions to find connections within our vast corpus of data. Data is input by a team of expert curators applying FAIR data management principles and also via automated pipelines and data processing routines. While our main focus is embryonic development and cell biology, these are often the underlying causes of compromised human health and are therefore invaluable for exploring the medical impacts of DNA sequence variants identified through patient exome or whole genome sequencing. One of the foundational elements in Xenbase with our gene-centric data structure is genomes, and we have recently vastly improved the quality of these core resources for both Xenopus laevis and Xenopus tropicalis. These and an extensive suite of other improvements are described, including updates and upgrades in content types, software and systems.

Motivation Molecular mimicry is used by pathogens to evade the host immune system and manipulate other host cellular processes. It is often mediated by short motifs in non-homologous proteins, whose detection challenges the sensitivity and specificity of existing bioinformatics tools.Results We present mimicDetector, a k-mer-based pipeline for identifying protein-level molecular mimicry between pathogens and their hosts. Applied to 17 globally important pathogens, mimicDetector identified a broad and biologically plausible set of mimicry candidates, including helminth proteins mimicking components of the human complement system and a Leishmania infantum mimic of Reticulon-4, a regulator of immune cell recruitment.Availability and implementation mimicDetector is freely available at https://github.com/kayleerich/mimicDetector/, implemented in Python and Snakemake, and compatible with Unix-based systems.

Structural variation, involving large alterations in chromosome structure, drives genetic diversification and the emergence of new phenotypes. These changes are widespread in natural populations and play an important role in adaptation and speciation. For many species, research has been limited to laboratory adapted strains and experimental evolution, which may not reflect the diversity of structural variants in the wild. Furthermore, technological limitations have proved to be a major barrier to accurate and comprehensive variant calling. In this study, we used PacBio sequencing data from 14 wild Caenorhabditis elegans strains to characterize structural variants in a natural population. With long-reads, we overcame limitations associated with short-read approaches and leveraged population-level data to further refine the accuracy of variant calls. We found that large, rapidly evolving gene families, such as GPCR, F-Box, and C-Type lectin, were prominent among the variants predicted to have phenotypic consequences. These results shed light on the significant role of structural variants in the evolution of Caenorhabditis elegans in its wild habitats and the limitations of treating N2 as the reference wild-type. ### Competing Interest Statement The authors have declared no competing interest.

Genome assemblers are a critical component of genome science, but the choice of assembly software and protocols can be daunting. Here, we investigate genome assembly variation and its implications for gene discovery across three nematode species-Caenorhabditis bovis, Haemonchus contortus, and Heligmosomoides bakeri-highlighting the critical interplay between assembly choice and downstream genomic analysis. Selecting commonly used genome assemblers, we generated multiple assemblies for each species, analyzing their structure, completeness, and effect on gene family analysis. Our findings demonstrate that assembly variations can significantly affect gene family composition, with notable differences in gene families important in anthelmintic discovery and immunomodulation. Despite broadly similar performance using various assembly metrics, comparisons of assemblies with a single species revealed underlying structural rearrangements and inconsistencies in gene content, which would affect downstream analyses. This emphasizes the need for continuous refinement of genome assemblies and their annotations.

Many ant species show dramatic shifts in behaviour when infected with parasites, but the molecular basis of these behavioural changes is not well understood. An example is the wood ant, Formica aserva, which serves as an intermediate host for the lancet liver fluke, Dicrocoelium dendriticum. Infected ants leave their nests during the cool hours of the day, ascend a flower and then attach themselves to a petal with their mandibles. Attached ants remain affixed to an inflorescence overnight, after which they detach, descend the plant and return to their nest. Unless eaten by a grazing mammal (the obligate next host), infected ants repeat this attach-and-detach cycle for the rest of the summer. We used transcriptomics to decipher the potential molecular mechanisms that underlie this reversible behaviour manipulation. Using naturally infected ants, we recreated the manipulation cycle in the laboratory and then evaluated messenger RNA from ant brains at four contrasting phases of manipulation. Among these phases, we found a total of 1349 transcripts differentially expressed between infected and uninfected ant brains. Many of these transcripts are involved in cell signalling pathways, including odorant, gustatory, vision, circadian rhythm and the production of biogenic monoamines and hormones. Metabolism, protein management and DNA repair functions might also play a role at different phases of manipulation. Our combined results are consistent with the idea that the mechanism(s) leading to the attach/detach/repeat sequence of behaviours of fluke-infected ants is multifaceted, involving much more than temperature-dependent contraction/relaxation of the mandibular muscles.

Haem, which binds iron and oxygen, is essential for parasitic nematode growth. Nematodes lack endogenous haem synthesis pathways and acquire haem from their environment or intracellular symbionts. Genes involved in haem degradation and detoxification have been identified in parasitic nematodes who are either specialised blood feeders (haematophages) or reside in the blood stream. Targeting these genes, so limiting parasite growth and promoting parasite death, provides a new therapeutic avenue against blood feeding parasitic nematodes. Heligmosomoides bakeri, a model intestinal parasitic nematode, has not been considered a blood feeder. Adults live in the intestinal lumen and graze on host tissue. However, the earlier larval stages enter the intestinal tissue, where they grow and moult multiple times, an oxygen demanding process. We have shown that, in vivo, likely through nematode-induced damage, many tissue-dwelling H. bakeri are in close vicinity of red blood cells. During this time, infection induces host anemia. Further, tissue-dwelling H. bakeri that have fed on blood have an increased expression of collagen genes compared to those parasites that had not fed on blood. In vitro, this translates to a growth advantage. Our findings suggest blood feeding is more widespread among nematodes than currently described. We argue that biochemical adaptations previously considered to be limited to blood-feeders are found in other nematodes. Moreover, H. bakeri can serve as a model for studying blood-feeding mechanisms and developing anthelminthic strategies to them. ### Competing Interest Statement The authors have declared no competing interest.

Myxozoans are a monophyletic taxon of approximately 2,400 described species of parasites from the phylum Cnidaria. The recent focus on their negative impacts on fisheries, on their evolution from free-living ancestors, and on their emergence into new fish host populations has stressed the critical need for genomic resources for this parasitic group. Here, we describe the genome assembly and annotation of Myxobolus rasmusseni, an emerging parasite of fathead minnows in Alberta, Canada. The assembly is 174.6 Mb in size, 68% of which is made up of repetitive elements, making it one of the most repetitive animal genomes sequenced to date. Through comparisons to other myxozoans, we show that widespread gene loss, a known phenomenon of this group of parasites, is consistent with closely related species. Additionally, we assembled the M. rasmusseni mitochondrial genome, which is nearly twice the size of the typical animal mitochondrial genome yet contains only five of the canonical mitochondrial protein-coding genes and open reading frames not found in other myxozoans. These results add to our understanding of the gene- and genome-level diversity observed in myxozoans.

HpARI is an immunomodulatory protein secreted by the intestinal nematode Heligmosomoides polygyrus bakeri , which binds and blocks IL-33. Here, we find that the H. polygyrus bakeri genome contains 3 HpARI family members, and that these have different effects on IL-33-dependent responses in vitro and in vivo, with HpARI1+2 suppressing, and HpARI3 amplifying these responses. All HpARIs have sub-nanomolar affinity for mouse IL-33, however HpARI3 does not block IL-33-ST2 interactions. Instead, HpARI3 stabilises IL-33, increasing the half-life of the cytokine and amplifying responses to it in vivo. Together these data show that H. polygyrus bakeri secretes a family of HpARI proteins with both overlapping and distinct functions, comprising a complex immunomodulatory arsenal of host-targeted proteins.

Background Structural variant (SV) calling from DNA sequencing data has been challenging due to several factors, including the ambiguity of short-read alignments, multiple complex SVs in the same genomic region, and the lack of “truth” datasets for benchmarking. Additionally, caller choice, parameter settings, and alignment method are known to affect SV calling. However, the impact of FASTQ read order on SV calling has not been explored for long-read data. Results Here, we used PacBio DNA sequencing data from 15 Caenorhabditis elegans strains and four Arabidopsis thaliana ecotypes to evaluate the sensitivity of different SV callers on FASTQ read order. Comparisons of variant call format files generated from the original and permutated FASTQ files demonstrated that the order of input data affected the SVs predicted by each caller. In particular, pbsv was highly sensitive to the order of the input data, especially at the highest depths where over 70% of the SV calls generated from pairs of differently ordered FASTQ files were in disagreement. These demonstrate that read order sensitivity is a complex, multifactorial process, as the differences observed both within and between species varied considerably according to the specific combination of aligner, SV caller, and sequencing depth. In addition to the SV callers being sensitive to the input data order, the SAMtools alignment sorting algorithm was identified as a source of variability following read order randomization. Conclusion The results of this study highlight the sensitivity of SV calling on the order of reads encoded in FASTQ files, which has not been recognized in long-read approaches. These findings have implications for the replication of SV studies and the development of consistent SV calling protocols. Our study suggests that researchers should pay attention to the input order sensitivity of read alignment sorting methods when analyzing long-read sequencing data for SV calling, as mitigating a source of variability could facilitate future replication work. These results also raise important questions surrounding the relationship between SV caller read order sensitivity and tool performance. Therefore, tool developers should also consider input order sensitivity as a potential source of variability during the development and benchmarking of new and improved methods for SV calling.

IntroductionIntestinal roundworms cause chronic debilitating disease in animals, including humans. Traditional experimental models of these types of infection use a large single-dose infection. However, in natural settings, hosts are exposed to parasites on a regular basis and when mice are exposed to frequent, smaller doses of Heligmosomoides polygyrus, the parasites are cleared more quickly. Whether this more effective host response has any negative consequences for the host is not known. ResultsUsing a trickle model of infection, we found that worm clearance was associated with known resistance-related host responses: increased granuloma and tuft cell numbers, increased levels of granuloma IgG and decreased intestinal transit time, as well as higher serum IgE levels. However, we found that the improved worm clearance was also associated with an inflammatory phenotype in and around the granuloma, increased smooth muscle hypertrophy/hyperplasia, and elevated levels of Adamts gene expression. DiscussionTo our knowledge, we are the first to identify the involvement of this protein family of matrix metalloproteinases (MMPs) in host responses to helminth infections. Our results highlight the delicate balance between parasite clearance and host tissue damage, which both contribute to host pathology. When continually exposed to parasitic worms, improved clearance comes at a cost.

Background: The Myxozoa is a group of at least 2,400 endoparasites within the phylum Cnidaria. All myxozoans have greatly reduced in size and morphology compared to free-living members of the phylum. They are best known for causing disease in economically important fish across the world; for example, Myxobolus cerebralis causes Whirling Disease, which can kill 90% of infected juvenile salmonid fish. In 2017, a potentially new myxozoan species was identified in Alberta. Myxobolus sp. causes distinct lesions in fathead minnows, which are ultimately fatal. Here, we sequenced, assembled and analyzed the genome of Myxobolus sp. to understand how the parasite interacts with its fish host and identify potential strategies to counter this emerging threat. Results: At 185 Mb, the Myxobolus sp. genome is the largest myxozoan genome sequenced so far. This large genome size is, in part, due to the high repetitive content; 68% of the genome was interspersed repeats, with the MULE-MuDR transposon covering 18% of the Myxobolus sp. genome. Similar to myxozoan genomes, the Myxobolus sp. genome has lost many genes well conserved in other eukaryotes. However, we also identified multiple expansions in gene families (serine proteases, hexokinases, and FLYWCH-domain containing proteins) which suggests their functional importance in the parasite. The mitochondrial genome of Myxobolus sp. encodes only five of the thirteen protein-coding genes typically found in animals. We found that the mitochondrial gene atp6 was transferred to the nucleus and acquired a mitochondria-targeting signal in Myxobolus sp. Conclusions: Our study provides valuable insights into myxozoan biology and identify promising avenues for future research. We also propose that M. rasmusseni is promising myxozoan model to explore host-parasite interactions in these parasites.

Single-cell atlases aim to collect the gene expression information for every cell type in an organism but can be challenging to perform in non-model organisms. To try to circumvent the problem of having no verified cell type markers in the parasitic nematode Heligmosomoides bakeri to use for an atlas, we attempted to use orthologs of verified markers from the closely related model organism Caenorhabditis elegans . This resulted in a useful comparison between the two worms for each of the cell types recovered in preliminary H. bakeri single-cell RNA-sequencing. For H. bakeri males and females, robustly recovered cell types include the gametes, embryos, and male intestine, while hypodermis, neurons, muscles, and pharyngeal cells were under-represented cell types. The two worms appear to have a similar hypodermis, cuticle, eggshell, and spermatogenesis process. On the other hand, putative cell identities and cell cycle scores suggest the intestine and muscle cells in H. bakeri may still be cycling and dividing, unlike in C. elegans . Additionally, embryogenesis and early development appear to be quite different between the two worms, with only eight out of 94 confirmed paternal contributions to the embryo in C. elegans (with an ortholog) predicted to also be paternal contributions in H. bakeri . Overall, this new dataset allowed me to move beyond the presence or absence of orthologs to include their tissue specificity and expression level similarities and differences when comparing these two worms to better identify biological processes and traits in a parasitic nematode that are modelled well by C. elegans .### Competing Interest StatementThe authors have declared no competing interest.

Heligmosomoides bakeri (often mistaken for Heligmosomoides polygyrus) is a promising model for parasitic nematodes with the key advantage of being amenable to study and manipulation within a controlled laboratory environment. While draft genome sequences are available for this worm, which allow for comparative genomic analyses between nematodes, there is a notable lack of information on its gene expression. We generated biologically replicated RNA-seq datasets from samples taken throughout the parasitic life of H. bakeri. RNA from tissue-dwelling and lumen-dwelling worms, collected under a dissection microscope, was sequenced on an Illumina platform. We find extensive transcriptional sexual dimorphism throughout the fourth larval and adult stages of this parasite and identify alternative splicing, glycosylation, and ubiquitination as particularly important processes for establishing and/or maintaining sex-specific gene expression in this species. We find sex-linked differences in transcription related to aging and oxidative and osmotic stress responses. We observe a starvation-like signature among transcripts whose expression is consistently upregulated in males, which may reflect a higher energy expenditure by male worms. We detect evidence of increased importance for anaerobic respiration among the adult worms, which coincides with the parasite’s migration into the physiologically hypoxic environment of the intestinal lumen. Furthermore, we hypothesize that oxygen concentration may be an important driver of the worms encysting in the intestinal mucosa as larvae, which not only fully exposes the worms to their host’s immune system but also shapes many of the interactions between the host and parasite. We find stage- and sex-specific variation in the expression of immunomodulatory genes and in anthelmintic targets. We examine how different the male and female worms are at the molecular level and describe major developmental events that occur in the worm, which extend our understanding of the interactions between this parasite and its host. In addition to generating new hypotheses for follow-up experiments into the worm’s behavior, physiology, and metabolism, our datasets enable future more in-depth comparisons between nematodes to better define the utility of H. bakeri as a model for parasitic nematodes in general.

IntroductionMolecular mimicry is a strategy used by parasites to evade the host’s immune system and facilitate transmission to a new host. To date, high-throughput examples of molecular mimicry have been limited to comparing protein sequences. However, recent advances in the prediction of tertiary structural models, led by Deepmind’s AlphaFold, enable the comparison of thousands of proteins from parasites and their hosts at the structural level, allowing for the identification of more mimics. Here, we present the first proteome-level search for tertiary structure similarity between proteins from Plasmodium falciparum, a malaria-causing parasite, and humans.MethodsWe assembled a database of experimentally-characterized protein tertiary structures (from the Protein Data Bank) and AlphaFold-generated protein tertiary structures from P. falciparum, human, and 15 negative control species, i.e., species not infected by P. falciparum. We aligned human and control structures to the parasite structures using Foldseek. ResultsWe identified molecular mimicry in three proteins that have been previously proposed as mediators of Plasmodium-human interactions. By extending this approach to all P. falciparum proteins, we identified an additional 41 potential mimics that are supported by additional experimental data. DiscussionOur findings demonstrate a valuable application of AlphaFold-derived tertiary structural models, and we discuss key considerations for its effective use in other host-parasite systems.

Pathogens have evolved sophisticated strategies to manipulate host signaling pathways, including the phenomenon of molecular mimicry, where pathogen-derived biomolecules imitate host biomolecules. In this study, we resurrected, updated, and optimized a sequence-based bioinformatics pipeline to identify potential molecular mimicry candidates between humans and 32 pathogenic species whose proteomes’ 3D structure predictions were available at the start of this study. We observed considerable variation in the number of mimicry candidates across pathogenic species, with pathogenic bacteria exhibiting fewer candidates compared to fungi and protozoans. Further analysis revealed that the candidate mimicry regions were enriched in solvent-accessible regions, highlighting their potential functional relevance. We identified a total of 1,878 mimicked regions in 1,439 human proteins, and clustering analysis indicated diverse target proteins across pathogen species. The human proteins containing mimicked regions revealed significant associations between these proteins and various biological processes, with an emphasis on host extracellular matrix organization and cytoskeletal processes. However, immune-related proteins were underrepresented as targets of mimicry. Our findings provide insights into the broad range of host-pathogen interactions mediated by molecular mimicry and highlight potential targets for further investigation. This comprehensive analysis contributes to our understanding of the complex mechanisms employed by pathogens to subvert host defenses and we provide a resource to assist researchers in the development of novel therapeutic strategies.

The apicomplexan parasite Cyclospora cayetanensis causes foodborne gastrointestinal disease in humans. Here, we report the first hybrid assembly for C. cayetanensis, which uses both Illumina MiSeq and Oxford Nanopore Technologies MinION platforms to generate genomic sequence data. The final genome assembly consists of 44,586,677 bases represented in 313 contigs.

The accurate characterization of structural variation is crucial for our understanding of how large chromosomal alterations affect phenotypic differences and contribute to genome evolution. Whole-genome sequencing is a popular approach for identifying structural variants, but the accuracy of popular tools remains unclear due to the limitations of existing benchmarks. Moreover, the performance of these tools for predicting variants in non-human genomes is less certain, as most tools were developed and benchmarked using data from the human genome. To evaluate the use of long-read data for the validation of short-read structural variant calls, the agreement between predictions from a short-read ensemble learning method and long-read tools were compared using real and simulated data from Caenorhabditis elegans . The results obtained from simulated data indicate that the best performing tool is contingent on the type and size of the variant, as well as the sequencing depth of coverage. These results also highlight the need for reference datasets generated from real data that can be used as ‘ground truth’ in benchmarks.

ABSTRACTGenome-wide methods offer a powerful approach to detect signatures of drug selection in parasite populations in the field. However, their application to parasitic nematodes has been limited because of both a lack of suitable reference genomes and the difficulty of obtaining field populations with sufficiently well-defined drug selection histories. Consequently, there is little information on the genomic signatures of drug selection for parasitic nematodes in the field and on how best to detect them. This study was designed to address these knowledge gaps using field populations of Haemonchus contortus with well-defined and contrasting benzimidazole-selection histories, leveraging a recently completed chromosomal-scale reference genome assembly. We generated a panel of 49,393 ddRADseq markers and used this resource to genotype 20 individual H. contortus adult worms from each of four H. contortus populations: two from closed sheep flocks that had an approximately 20-year history of frequent treatment exclusively with benzimidazole drugs, and two populations with a history of little or no drug treatment. The populations were chosen from the same geographical region to limit population structure in order to maximize the sensitivity of the approach. A clear signature of selection was detected on the left arm of chromosome I centered on the isotype-1 β-tubulin gene in the benzimidazole-selected but not the unselected populations. Two additional, but weaker, signatures of selection were detected; one near the middle of chromosome I and one near the isotype-2 β-tubulin locus on chromosome II. We examined genetic differentiation between populations, and nucleotide diversity and linkage disequilibrium within populations to define these two additional regions as encompassing five genes and a single gene. We also compared the relative power of using pooled versus individual worm sequence data to detect genomic selection signatures and how sensitivity is impacted by sequencing depth, worm number, and population structure.In summary, this study used H. contortus field populations with well-defined drug selection histories to provide the first direct genome-wide evidence for any parasitic nematode that the isotype-1 β-tubulin gene is the quantitatively most important benzimidazole resistance locus. It also identified two additional genomic regions that likely contain benzimidazole-resistance loci of secondary importance. Finally, this study provides an experimental framework to maximize the power of genome-wide approaches to detect signatures of selection driven by anthelmintic drug treatments in field populations of parasitic nematodes.AUTHOR SUMMARYBenzimidazoles are important anthelmintic drugs for human and animal parasitic nematode control with ∼0.5 billion children at risk of infection treated annually worldwide. Drug resistance is common in livestock parasites and a growing concern in humans. Haemonchus contortus is the most important model parasite system used to study anthelmintic resistance and a significant livestock pathogen. It is also one of the few parasitic nematodes with a chromosomal-scale genome assembly. We have undertaken genome-wide scans using a dense RADseq marker panel on worms from natural field populations under differing levels of benzimidazole selection. We show that there is a single predominant genomic signature of selection in H. contortus associated with benzimidazole selection centred on the isotype-1 β-tubulin locus. We also identify two weaker signatures of selection indicative of secondary drug resistance loci. Additionally, we assess the minimum data requirements for parameters including worm number, sequence depth, marker density needed to detect the signatures of selection and compare individual to Poolseq analysis. This work is the first genome-wide study in a parasitic nematode to provide direct evidence of the isotype-1 β-tubulin locus being the single predominant benzimidazole resistance locus and provides an experimental framework for future population genomic studies on anthelmintic resistance.

Cyclospora cayetanensis is an emerging foodborne parasite that causes cyclosporiasis, an enteric disease of humans. Domestically acquired outbreaks have been reported in Canada every spring or summer since 2013. To date, investigations into the potential sources of infection have relied solely on epidemiological data. To supplement the epidemiological data with genetic information, we genotyped 169 Canadian cyclosporiasis cases from stool specimens collected from 2010 to 2021 using an existing eight-marker targeted amplicon deep (TADS) scheme specific to C. cayetanensis as previously described by the US Centers for Disease Control and Prevention (CDC). This is the first study to genotype Canadian Cyclospora cayetanensis isolates, and it focuses on evaluating the genotyping performance and genetic clustering. Genotyping information was successfully collected with at least part of one of the markers in the TADS assay for 97.9% of specimens, and 81.1% of cyclosporiasis cases met the minimum requirements to genetically cluster into 20 groups. The performance of the scheme suggests that examining cyclosporiasis cases genetically will be a valuable tool for supplementing epidemiological outbreak investigations and to minimize further infections. Further research is required to expand the number of discriminatory markers to improve genetic clustering.