Enhanced Genomic and Transcriptomic Resources for Trichinella pseudospiralis and T. spiralis to Underpin the Discovery of Molecular Differences between Stages and Species.

Nematodes of the genus Trichinella are important pathogens of humans and animals. This study aimed to enhance the genomic and transcriptomic resources for T. pseudospiralis (non-encapsulated phenotype) and T. spiralis (encapsulated phenotype) and to explore transcriptional profiles. First, we improved the assemblies of the genomes of T. pseudospiralis (code ISS13) and T. spiralis (code ISS534), achieving genome sizes of 56.6 Mb (320 scaffolds, and an N50 of 1.02 Mb) and 63.5 Mb (568 scaffolds, and an N50 value of 0.44 Mb), respectively. Then, for each species, we produced RNA sequence data for three key developmental stages (first-stage muscle larvae [L1s], adults, and newborn larvae [NBLs]; three replicates for each stage), analysed differential transcription between stages, and explored enriched pathways and processes between species. Stage-specific upregulation was linked to cellular processes, metabolism, and host-parasite interactions, and pathway enrichment analysis showed distinctive biological processes and cellular localisations between species. Indeed, the secreted molecules calmodulin, calreticulin, and calsyntenin-with possible roles in modulating host immune responses and facilitating parasite survival-were unique to T. pseudospiralis and not detected in T. spiralis. These insights into the molecular mechanisms of Trichinella-host interactions might offer possible avenues for developing new interventions against trichinellosis.

Trematode Genomics and Proteomics.

Trematode infections stand out as one of the frequently overlooked tropical diseases, despite their wide global prevalence and remarkable capacity to parasitize diverse host species and tissues. Furthermore, these parasites hold significant socio-economic, medical, veterinary and agricultural implications. Over the past decades, substantial strides have been taken to bridge the information gap concerning various "omic" tools, such as proteomics and genomics, in this field. In this edition of the book, we highlight recent progress in genomics and proteomics concerning trematodes with a particular focus on the advances made in the past 5 years. Additionally, we present insights into cutting-edge technologies employed in studying trematode biology and shed light on the available resources for exploring the molecular facets of this particular group of parasitic helminths.

Chromosome-level genome assembly of the cereal cyst nematode Heterodera flipjevi.

As an economically important plant parasitic nematode (PPN), Heterodera filipjevi causes great damage on wheat, and now it was widely recorded in many countries. While multiple genomes of PPNs have been published, high-quality genome assembly and annotation on H. filipjevi have yet to be performed. This study presents a chromosome-scale genome assembly and annotation for H. filipjevi, utilizing a combination of Illumina short-read, PacBio long-read, and Hi-C sequencing technologies. The genome consists of 9 pseudo-chromosomes that contain 134.19 Mb of sequence, with a scaffold N50 length of 11.88 Mb. In total, 10,036 genes were annotated, representing 75.20% of the total predicted protein-coding genes. Our study provides the first chromosome-scale genome for H. filipjevi, which is also the inaugural high-quality genome of cereal cyst nematodes (CCNs). It provides a valuable genomic resource for further biological research and pest management of cereal cyst nematodes disease.

Spirocerca lupi draft genome, vaccine and anthelmintic targets.

Spirocerca lupi is a parasitic nematode affecting predominantly domestic dogs. It causes spirocercosis, a disease that is often fatal. The assembled draft genome of S. lupi consists of 13,627 predicted protein-coding genes and is approximately 150 Mb in length. Several known anthelmintic gene targets such as for ß-Tubulin, glutamate, and GABA receptors as well as known vaccine gene targets such as cysteine protease inhibitor and cytokines were identified in S. lupi by comparing orthologs of C. elegans anthelmintic gene targets as well as orthologs to known vaccine candidates. New anthelmintic targets were predicted through an inclusion-exclusion strategy and new vaccine targets were predicted through an immunoinformatics approach. New anthelminthic targets include DNA-directed RNA polymerases, chitin synthase, polymerases, and other enzymes. New vaccine targets include cuticle collagens. These gene targets provide a starting platform for new drug identification and vaccine design.

Phased chromosome-scale genome assembly of an asexual, allopolyploid root-knot nematode reveals complex subgenomic structure.

We present the chromosome-scale genome assembly of the allopolyploid root-knot nematode Meloidogyne javanica. We show that the M. javanica genome is predominantly allotetraploid, comprising two subgenomes, A and B, that most likely originated from hybridisation of two ancestral parental species. The assembly was annotated using full-length non-chimeric transcripts, comparison to reference databases, and ab initio prediction techniques, and the subgenomes were phased using ancestral k-mer spectral analysis. Subgenome B appears to show fission of chromosomal contigs, and while there is substantial synteny between subgenomes, we also identified regions lacking synteny that may have diverged in the ancestral genomes prior to or following hybridisation. This annotated and phased genome assembly forms a significant resource for understanding the origins and genetics of these globally important plant pathogens.

Genome-wide RNA interference of the nhr gene family in barber's pole worm identified members crucial for larval viability in vitro.

Nuclear hormone receptors (NHRs) are emerging target candidates against nematode infection and resistance. However, there is a lack of comprehensive information on NHR-coding genes in parasitic nematodes. In this study, we curated the nhr gene family for 60 major parasitic nematodes from humans and animals. Compared with the free-living model organism Caenorhabditis elegans, a remarkable contraction of the nhr family was revealed in parasitic species, with genetic diversification and conservation unveiled among nematode Clades I (10-13), III (16-42), IV (33-35) and V (25-64). Using an in vitro biosystem, we demonstrated that 40 nhr genes in a blood-feeding nematode Haemonchus contortus (clade V; barber's pole worm) were responsive to host serum and one nhr gene (i.e., nhr-64) was consistently stimulated by anthelmintics (i.e., ivermectin, thiabendazole and levamisole); Using a high-throughput RNA interference platform, we knocked down 43 nhr genes of H. contortus and identified at least two genes that are required for the viability (i.e., nhr-105) and development (i.e., nhr-17) of the infective larvae of this parasitic nematode in vitro. Harnessing this preliminary functional atlas of nhr genes for H. contortus will prime the biological studies of this gene family in nematode genetics, infection, and anthelmintic metabolism within host animals, as well as the promising discovery of novel intervention targets.

WormBase 2024: status and transitioning to Alliance infrastructure.

WormBase has been the major repository and knowledgebase of information about the genome and genetics of Caenorhabditis elegans and other nematodes of experimental interest for over 2 decades. We have 3 goals: to keep current with the fast-paced C. elegans research, to provide better integration with other resources, and to be sustainable. Here, we discuss the current state of WormBase as well as progress and plans for moving core WormBase infrastructure to the Alliance of Genome Resources (the Alliance). As an Alliance member, WormBase will continue to interact with the C. elegans community, develop new features as needed, and curate key information from the literature and large-scale projects.

The impact of differential transposition activities of autonomous and nonautonomous hAT transposable elements on genome architecture and gene expression in Caenorhabditis inopinata.

Transposable elements are DNA sequences capable of moving within genomes and significantly influence genomic evolution. The nematode Caenorhabditis inopinata exhibits a much higher transposable element copy number than its sister species, Caenorhabditis elegans. In this study, we identified a novel autonomous transposable element belonging to the hAT superfamily from a spontaneous transposable element-insertion mutant in C. inopinata and named this transposon Ci-hAT1. Further bioinformatic analyses uncovered 3 additional autonomous hAT elements-Ci-hAT2, Ci-hAT3, and Ci-hAT4-along with over 1,000 copies of 2 nonautonomous miniature inverted-repeat transposable elements, mCi-hAT1 and mCi-hAT4, likely derived from Ci-hAT1 and Ci-hAT4 through internal deletion. We tracked at least 3 sequential transpositions of Ci-hAT1 over several years. However, the transposition rates of the other 3 autonomous hAT elements were lower, suggesting varying activity levels. Notably, the distribution patterns of the 2 miniature inverted-repeat transposable element families differed significantly: mCi-hAT1 was primarily located in the chromosome arms, a pattern observed in the transposable elements of other Caenorhabditis species, whereas mCi-hAT4 was more evenly distributed across chromosomes. Additionally, interspecific transcriptome analysis indicated that C. inopinata genes with upstream or intronic these miniature inverted-repeat transposable element insertions tend to be more highly expressed than their orthologous genes in C. elegans. These findings highlight the significant role of de-silenced transposable elements in driving the evolution of genomes and transcriptomes, leading to species-specific genetic diversity.

Hookworm genomics: dusk or dawn?

Hookworms are parasites, closely related to the model nematode Caenorhabditis elegans, that are a major economic and health burden worldwide. Primarily three hookworm species (Necator americanus, Ancylostoma duodenale, and Ancylostoma ceylanicum) infect humans. Another 100 hookworm species from 19 genera infect primates, ruminants, and carnivores. Genetic data exist for only seven of these species. Genome sequences are available from only four of these species in two genera, leaving 96 others (particularly those parasitizing wildlife) without any genomic data. The most recent hookworm genomes were published 5 years ago, leaving the field in a dusk. However, assembling genomes from single hookworms may bring a new dawn. Here we summarize advances, challenges, and opportunities for studying these neglected but important parasitic nematodes.

Recombination in bdelloid rotifer genomes: asexuality, transfer and stress.

Bdelloid rotifers constitute a class of microscopic animals living in freshwater habitats worldwide. Several strange features of bdelloids have drawn attention: their ability to tolerate desiccation and other stresses, a lack of reported males across the clade despite centuries of study, and unusually high numbers of horizontally acquired, non-metazoan genes. Genome sequencing is transforming our understanding of their lifestyle and its consequences, while in turn providing wider insights about recombination and genome organisation in animals. Many questions remain, not least how to reconcile apparent genomic signatures of sex with the continued absence of reported males, why bdelloids have so many horizontally acquired genes, and how their remarkable ability to survive stress interacts with recombination and other genomic processes.

Genome structure and population genomics of the canine heartworm Dirofilaria immitis.

The heartworm, Dirofilaria immitis, is a filarial parasitic nematode responsible for significant morbidity and mortality in wild and domesticated canids. Resistance to macrocyclic lactone drug prevention represents a significant threat to parasite control and has prompted investigations to understand the genetic determinants of resistance. This study aimed to improve the genomic resources of D. immitis to enable a more precise understanding of how genetic variation is distributed within and between parasite populations worldwide, which will inform the likelihood and rate by which parasites, and in turn, resistant alleles, might spread. We have guided the scaffolding of a recently published genome assembly for D. immitis (ICBAS_JMDir_1.0) using the chromosomal-scale reference genomes of Brugia malayi and Onchocerca volvulus, resulting in an 89.5 Mb assembly composed of four autosomal- and one sex-linked chromosomal-scale scaffolds representing 99.7% of the genome. Publicly available and new whole-genome sequencing data from 32 D. immitis samples from Australia, Italy and the USA were assessed using principal component analysis, nucleotide diversity (Pi) and absolute genetic divergence (Dxy) to characterise the global genetic structure and measure within- and between-population diversity. These population genetic analyses revealed broad-scale genetic structure among globally diverse samples and differences in genetic diversity between populations; however, fine-scale subpopulation analysis was limited and biased by differences between sample types. Finally, we mapped single nucleotide polymorphisms previously associated with macrocyclic lactone resistance in the new genome assembly, revealing the physical linkage of high-priority variants on chromosome 3, and determined their frequency in the studied populations. This new chromosomal assembly for D. immitis now allows for a more precise investigation of selection on genome-wide genetic variation and will enhance our understanding of parasite transmission and the spread of genetic variants responsible for resistance to treatment.

Unraveling the Unusual Subgenomic Organization in the Neopolyploid Free-Living Flatworm Macrostomum lignano.

Whole genome duplication (WGD) is an evolutionary event resulting in a redundancy of genetic material. Different mechanisms of WGD, allo- or autopolyploidization, lead to distinct evolutionary trajectories of newly formed polyploids. Genome studies on such species are important for understanding the early stages of genome evolution. However, assembling neopolyploid is a challenging task due to the presence of 2 homologous (or homeologous) chromosome sets and therefore the existence of the extended paralogous regions in its genome. Post-WGD evolution of polyploids includes cytogenetic diploidization leading to the formation of species, whose polyploid origin might be hidden by disomic inheritance. Earlier we uncovered the hidden polyploid origin of the free-living flatworms of the genus Macrostomum (Macrostomum lignano, M. janickei, and M. mirumnovem). Cytogenetic diploidization in these species is accompanied by intensive chromosomal rearrangements including chromosomes fusions. In this study, we unravel the M. lignano genome organization through generation and sequencing of 2 sublines of the commonly used inbred line of M. lignano (called DV1) differing only in a copy number of the largest chromosome (MLI1). Using nontrivial assembly free comparative analysis of their genomes, we deciphered DNA sequences belonging to MLI1 and validated them by sequencing the pool of microdissected MLI1. Here we presented the uncommon mechanism of genome rediplodization of M. lignano, which consists of (i) presence of 3 subgenomes, which emerged via formation of large fused chromosomes and its variants, and (ii) sustaining their heterozygosity through inter- and intrachromosomal rearrangements.

Thousands of Pristionchus pacificus orphan genes were integrated into developmental networks that respond to diverse environmental microbiota.

Adaptation of organisms to environmental change may be facilitated by the creation of new genes. New genes without homologs in other lineages are known as taxonomically-restricted orphan genes and may result from divergence or de novo formation. Previously, we have extensively characterized the evolution and origin of such orphan genes in the nematode model organism Pristionchus pacificus. Here, we employ large-scale transcriptomics to establish potential functional associations and to measure the degree of transcriptional plasticity among orphan genes. Specifically, we analyzed 24 RNA-seq samples from adult P. pacificus worms raised on 24 different monoxenic bacterial cultures. Based on coexpression analysis, we identified 28 large modules that harbor 3,727 diplogastrid-specific orphan genes and that respond dynamically to different bacteria. These coexpression modules have distinct regulatory architecture and also exhibit differential expression patterns across development suggesting a link between bacterial response networks and development. Phylostratigraphy revealed a considerably high number of family- and even species-specific orphan genes in certain coexpression modules. This suggests that new genes are not attached randomly to existing cellular networks and that integration can happen very fast. Integrative analysis of protein domains, gene expression and ortholog data facilitated the assignments of biological labels for 22 coexpression modules with one of the largest, fast-evolving module being associated with spermatogenesis. In summary, this work presents the first functional annotation for thousands of P. pacificus orphan genes and reveals insights into their integration into environmentally responsive gene networks.

Expression of Hairpin-Enriched Mitochondrial DNA in Two Hairworm Species (Nematomorpha).

Nematomorpha (hairworms) is a phylum of parasitic ecdysozoans, best known for infecting arthropods and guiding their hosts toward water, where the parasite can complete its life cycle. Over 350 species of nematomorphs have been described, yet molecular data for the group remain scarce. The few available mitochondrial genomes of nematomorphs are enriched with long inverted repeats, which are embedded in the coding sequences of their genes-a remarkably unusual feature exclusive to this phylum. Here, we obtain and annotate the repeats in the mitochondrial genome of another nematomorph species-Parachordodes pustulosus. Using genomic and transcriptomic libraries, we investigate the impact of inverted repeats on the read coverage of the mitochondrial genome. Pronounced drops in the read coverage coincide with regions containing long inverted repeats, denoting the 'blind spots' of short-fragment sequencing libraries. Phylogenetic inference with the novel data reveals multiple disagreements between the traditional system of Nematomorpha and molecular data, rendering several genera paraphyletic, including Parachordodes.

The first high-quality chromosome-level genome of the Sipuncula Sipunculus nudus using HiFi and Hi-C data.

Sipuncula is a class of exocoelomic unsegmented animals whose evolutionary relationships are unresolved. The peanut worm Sipunculus nudus is a globally distributed, economically important species belonging to the class Sipuncula. Herein, we present the first high-quality chromosome-level assembly of S. nudus based on HiFi reads and high-resolution chromosome conformation capture (Hi-C) data. The assembled genome was 1,427 Mb, with a contig N50 length of 29.46 Mb and scaffold N50 length of 80.87 Mb. Approximately 97.91% of the genome sequence was anchored to 17 chromosomes. A BUSCO assessment showed that 97.7% of the expectedly conserved genes were present in the genome assembly. The genome was composed of 47.91% repetitive sequences, and 28,749 protein-coding genes were predicted. A phylogenetic tree demonstrated that Sipuncula belongs to Annelida and diverged from the common ancestor of Polychaeta. The high-quality chromosome-level genome of S. nudus will serve as a valuable reference for studies of the genetic diversity and evolution of Lophotrochozoa.

Improving helminth genome resources in the post-genomic era.

Rapid advancement in high-throughput sequencing and analytical approaches has seen a steady increase in the generation of genomic resources for helminth parasites. Now, helminth genomes and their annotations are a cornerstone of numerous efforts to compare genetic and transcriptomic variation, from single cells to populations of globally distributed parasites, to genome modifications to understand gene function. Our understanding of helminths is increasingly reliant on these genomic resources, which are primarily static once published and vary widely in quality and completeness between species. This article seeks to highlight the cause and effect of this variation and argues for the continued improvement of these genomic resources - even after their publication - which is necessary to provide a more accurate and complete understanding of the biology of these important pathogens.

Approaches for CRISPR/Cas9 Genome Editing in C. elegans.

The clustered, regularly interspaced, short, palindromic repeat (CRISPR)-associated (CAS) nuclease Cas9 has been used in many organisms to generate specific mutations and transgene insertions. Here we describe our most up-to-date protocols using the S. pyogenes Cas9 in C. elegans that provides a convenient and effective approach for making heritable changes to the worm genome. We present several considerations when deciding which strategy best suits the needs of the experiment.

First Insights into the Repertoire of Secretory Lectins in Rotifers.

Due to their high biodiversity and adaptation to a mutable and challenging environment, aquatic lophotrochozoan animals are regarded as a virtually unlimited source of bioactive molecules. Among these, lectins, i.e., proteins with remarkable carbohydrate-recognition properties involved in immunity, reproduction, self/nonself recognition and several other biological processes, are particularly attractive targets for biotechnological research. To date, lectin research in the Lophotrochozoa has been restricted to the most widespread phyla, which are the usual targets of comparative immunology studies, such as Mollusca and Annelida. Here we provide the first overview of the repertoire of the secretory lectin-like molecules encoded by the genomes of six target rotifer species: Brachionus calyciflorus, Brachionus plicatilis, Proales similis (class Monogononta), Adineta ricciae, Didymodactylos carnosus and Rotaria sordida (class Bdelloidea). Overall, while rotifer secretory lectins display a high molecular diversity and belong to nine different structural classes, their total number is significantly lower than for other groups of lophotrochozoans, with no evidence of lineage-specific expansion events. Considering the high evolutionary divergence between rotifers and the other major sister phyla, their widespread distribution in aquatic environments and the ease of their collection and rearing in laboratory conditions, these organisms may represent interesting targets for glycobiological studies, which may allow the identification of novel carbohydrate-binding proteins with peculiar biological properties.

Population differentiation and epidemic tracking of Bursaphelenchus xylophilus in China based on chromosome-level assembly and whole-genome sequencing data.

BACKGROUND: Bursaphelenchus xylophilus, the pinewood nematode, kills millions of pine trees worldwide every year, and causes enormous economic and ecological losses. Despite extensive research on population variation, there is little understanding of the population-wide variation spectrum in China. RESULTS: We sequenced an inbred B. xylophilus strain using Pacbio+Illumina+Bionano+Hi-C and generated a chromosome-level assembly (AH1) with six chromosomes of 77.1 Mb (chromosome N50: 12 Mb). The AH1 assembly shows very high continuity and completeness, and contains novel genes with potentially important functions compared with previous assemblies. Subsequently, we sequenced 181 strains from China and the USA and found ~7.8 million single nucleotide polymorphisms (SNPs). Analysis shows that the B. xylophilus population in China can be divided into geographically bounded subpopulations with severe cross-infection and potential migrations. In addition, distribution of B. xylophilus is dominated by temperature zones while geographically associated SNPs are mainly located on adaptation related GPCR gene families, suggesting the nematode has been evolving to adapt to different temperatures. A machine-learning based epidemic tracking method has been established to predict their geographical origins, which can be applied to any other species. CONCLUSION: Our study provides the community with the first high-quality chromosome-level assembly which includes a comprehensive catalogue of genetic variations. It provides insights into population structure and effective tracking method for this invasive species, which facilitates future studies to address a variety of applied, genomic and evolutionary questions in B. xylophilus as well as related species.

Morphological and genomic characterisation of the Schistosoma hybrid infecting humans in Europe reveals admixture between Schistosoma haematobium and Schistosoma bovis.

Schistosomes cause schistosomiasis, the world's second most important parasitic disease after malaria in terms of public health and social-economic impacts. A peculiar feature of these dioecious parasites is their ability to produce viable and fertile hybrid offspring. Originally only present in the tropics, schistosomiasis is now also endemic in southern Europe. Based on the analysis of two genetic markers the European schistosomes had previously been identified as hybrids between the livestock- and the human-infective species Schistosoma bovis and Schistosoma haematobium, respectively. Here, using PacBio long-read sequencing technology we performed genome assembly improvement and annotation of S. bovis, one of the parental species for which no satisfactory genome assembly was available. We then describe the whole genome introgression levels of the hybrid schistosomes, their morphometric parameters (eggs and adult worms) and their compatibility with two European snail strains used as vectors (Bulinus truncatus and Planorbarius metidjensis). Schistosome-snail compatibility is a key parameter for the parasites life cycle progression, and thus the capability of the parasite to establish in a given area. Our results show that this Schistosoma hybrid is strongly introgressed genetically, composed of 77% S. haematobium and 23% S. bovis origin. This genomic admixture suggests an ancient hybridization event and subsequent backcrosses with the human-specific species, S. haematobium, before its introduction in Corsica. We also show that egg morphology (commonly used as a species diagnostic) does not allow for accurate hybrid identification while genetic tests do.