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.

Deciphering complex genome rearrangements in C. elegans using short-read whole genome sequencing.

Genomic rearrangements cause congenital disorders, cancer, and complex diseases in human. Yet, they are still understudied in rare diseases because their detection is challenging, despite the advent of whole genome sequencing (WGS) technologies. Short-read (srWGS) and long-read WGS approaches are regularly compared, and the latter is commonly recommended in studies focusing on genomic rearrangements. However, srWGS is currently the most economical, accurate, and widely supported technology. In Caenorhabditis elegans (C. elegans), such variants, induced by various mutagenesis processes, have been used for decades to balance large genomic regions by preventing chromosomal crossover events and allowing the maintenance of lethal mutations. Interestingly, those chromosomal rearrangements have rarely been characterized on a molecular level. To evaluate the ability of srWGS to detect various types of complex genomic rearrangements, we sequenced three balancer strains using short-read Illumina technology. As we experimentally validated the breakpoints uncovered by srWGS, we showed that, by combining several types of analyses, srWGS enables the detection of a reciprocal translocation (eT1), a free duplication (sDp3), a large deletion (sC4), and chromoanagenesis events. Thus, applying srWGS to decipher real complex genomic rearrangements in model organisms may help designing efficient bioinformatics pipelines with systematic detection of complex rearrangements in human genomes.

Characterization of the complete mitochondrial genome of the swine kidney worm Stephanurus dentatus (Nematoda: Syngamidae) and phylogenetic implications.

Swine stephanuriasis caused by kidney worm Stephanurus dentatus is a parasitic disease in tropical and subtropical countries, leading to economic losses. Despite its significance as a pathogen, the phylogenetic position and taxonomic status of this nematode remain poorly understood. Mitochondrial (mt) genome sequences are known to provide useful genetic markers for investigations in these areas, but mt genome sequences are lacking for S. dentatus. In the present study, we determined the complete mt genome sequences of S. dentatus with an Illumina platform and compared it with the mt genomes of other closely related species. The circular mt genome was 13,735 bp in size with 36 genes. All genes are transcribed in the same direction and the mt gene arrangement is identified as a GA3 pattern, that is the most common pattern of gene arrangement observed in nematodes to date. Phylogenetic analysis using concatenated amino acid sequences of 12 protein-coding genes supported the hypothesis that S. dentatus was closely related to the family Chabertiidae. Our results provided insights into the phylogenetic relationship of the family Syngamidae within the superfamily Strongyloidea.

Potato cyst nematodes Globodera rostochiensis and G. pallida.

TAXONOMY: Phylum Nematoda; class Chromadorea; order Rhabditida; suborder Tylenchina; infraorder Tylenchomorpha; superfamily Tylenchoidea; family Heteroderidae; subfamily Heteroderinae; Genus Globodera. BIOLOGY: Potato cyst nematodes (PCN) are biotrophic, sedentary endoparasitic nematodes. Invasive (second) stage juveniles (J2) hatch from eggs in response to the presence of host root exudates and subsequently locate and invade the host. The nematodes induce the formation of a large, multinucleate syncytium in host roots, formed by fusion of up to 300 root cell protoplasts. The nematodes rely on this single syncytium for the nutrients required to develop through a further three moults to the adult male or female stage. This extended period of biotrophy-between 4 and 6 weeks in total-is almost unparalleled in plant-pathogen interactions. Females remain at the root while adult males revert to the vermiform body plan of the J2 and leave the root to locate and fertilize the female nematodes. The female body forms a cyst that contains the next generation of eggs. HOST RANGE: The host range of PCN is limited to plants of the Solanaceae family. While the most economically important hosts are potato (Solanum tuberosum), tomato (Solanum lycopersicum), and aubergine (Solanum melongena), over 170 species of Solanaceae are thought to be potential hosts for PCN (Sullivan et al., 2007). DISEASE SYMPTOMS: Symptoms are similar to those associated with nutrient deficiency, such as stunted growth, yellowing of leaves and reduced yields. This absence of specific symptoms reduces awareness of the disease among growers. DISEASE CONTROL: Resistance genes (where available in suitable cultivars), application of nematicides, crop rotation. Great effort is put into reducing the spread of PCN through quarantine measures and use of certified seed stocks. USEFUL WEBSITES: Genomic information for PCN is accessible through WormBase ParaSite.

Molecular evidence of hybridization between pig and human Ascaris indicates an interbred species complex infecting humans.

Human ascariasis is a major neglected tropical disease caused by the nematode Ascaris lumbricoides. We report a 296 megabase (Mb) reference-quality genome comprised of 17,902 protein-coding genes derived from a single, representative Ascaris worm. An additional 68 worms were collected from 60 human hosts in Kenyan villages where pig husbandry is rare. Notably, the majority of these worms (63/68) possessed mitochondrial genomes that clustered closer to the pig parasite Ascaris suum than to A. lumbricoides. Comparative phylogenomic analyses identified over 11 million nuclear-encoded SNPs but just two distinct genetic types that had recombined across the genomes analyzed. The nuclear genomes had extensive heterozygosity, and all samples existed as genetic mosaics with either A. suum-like or A. lumbricoides-like inheritance patterns supporting a highly interbred Ascaris species genetic complex. As no barriers appear to exist for anthroponotic transmission of these 'hybrid' worms, a one-health approach to control the spread of human ascariasis will be necessary.

Genomic and transcriptomic variation defines the chromosome-scale assembly of Haemonchus contortus, a model gastrointestinal worm.

Haemonchus contortus is a globally distributed and economically important gastrointestinal pathogen of small ruminants and has become a key nematode model for studying anthelmintic resistance and other parasite-specific traits among a wider group of parasites including major human pathogens. Here, we report using PacBio long-read and OpGen and 10X Genomics long-molecule methods to generate a highly contiguous 283.4 Mbp chromosome-scale genome assembly including a resolved sex chromosome for the MHco3(ISE).N1 isolate. We show a remarkable pattern of conservation of chromosome content with Caenorhabditis elegans, but almost no conservation of gene order. Short and long-read transcriptome sequencing allowed us to define coordinated transcriptional regulation throughout the parasite's life cycle and refine our understanding of cis- and trans-splicing. Finally, we provide a comprehensive picture of chromosome-wide genetic diversity both within a single isolate and globally. These data provide a high-quality comparison for understanding the evolution and genomics of Caenorhabditis and other nematodes and extend the experimental tractability of this model parasitic nematode in understanding helminth biology, drug discovery and vaccine development, as well as important adaptive traits such as drug resistance.

Long-read RNA sequencing of human and animal filarial parasites improves gene models and discovers operons.

Filarial parasitic nematodes (Filarioidea) cause substantial disease burden to humans and animals around the world. Recently there has been a coordinated global effort to generate, annotate, and curate genomic data from nematode species of medical and veterinary importance. This has resulted in two chromosome-level assemblies (Brugia malayi and Onchocerca volvulus) and 11 additional draft genomes from Filarioidea. These reference assemblies facilitate comparative genomics to explore basic helminth biology and prioritize new drug and vaccine targets. While the continual improvement of genome contiguity and completeness advances these goals, experimental functional annotation of genes is often hindered by poor gene models. Short-read RNA sequencing data and expressed sequence tags, in cooperation with ab initio prediction algorithms, are employed for gene prediction, but these can result in missing clade-specific genes, fragmented models, imperfect mapping of gene ends, and lack of isoform resolution. Long-read RNA sequencing can overcome these drawbacks and greatly improve gene model quality. Here, we present Iso-Seq data for B. malayi and Dirofilaria immitis, etiological agents of lymphatic filariasis and canine heartworm disease, respectively. These data cover approximately half of the known coding genomes and substantially improve gene models by extending untranslated regions, cataloging novel splice junctions from novel isoforms, and correcting mispredicted junctions. Furthermore, we validated computationally predicted operons, manually curated new operons, and merged fragmented gene models. We carried out analyses of poly(A) tails in both species, leading to the identification of non-canonical poly(A) signals. Finally, we prioritized and assessed known and putative anthelmintic targets, correcting or validating gene models for molecular cloning and target-based anthelmintic screening efforts. Overall, these data significantly improve the catalog of gene models for two important parasites, and they demonstrate how long-read RNA sequencing should be prioritized for ongoing improvement of parasitic nematode genome assemblies.

Comparative Genomics Reveals Novel Target Genes towards Specific Control of Plant-Parasitic Nematodes.

Plant-parasitic nematodes cause extensive annual yield losses to worldwide agricultural production. Most cultivated plants have no known resistance against nematodes and the few bearing a resistance gene can be overcome by certain species. Chemical methods that have been deployed to control nematodes have largely been banned from use due to their poor specificity and high toxicity. Hence, there is an urgent need for the development of cleaner and more specific control methods. Recent advances in nematode genomics, including in phytoparasitic species, provide an unprecedented opportunity to identify genes and functions specific to these pests. Using phylogenomics, we compared 61 nematode genomes, including 16 for plant-parasitic species and identified more than 24,000 protein families specific to these parasites. In the genome of Meloidogyne incognita, one of the most devastating plant parasites, we found ca. 10,000 proteins with orthologs restricted only to phytoparasitic species and no further homology in protein databases. Among these phytoparasite-specific proteins, ca. 1000 shared the same properties as known secreted effectors involved in essential parasitic functions. Of these, 68 were novel and showed strong expression during the endophytic phase of the nematode life cycle, based on both RNA-seq and RT-qPCR analyses. Besides effector candidates, transcription-related and neuro-perception functions were enriched in phytoparasite-specific proteins, revealing interesting targets for nematode control methods. This phylogenomics analysis constitutes a unique resource for the further understanding of the genetic basis of nematode adaptation to phytoparasitism and for the development of more efficient control methods.

The tapeworm interactome: inferring confidence scored protein-protein interactions from the proteome of Hymenolepis microstoma.

BACKGROUND: Reference genome and transcriptome assemblies of helminths have reached a level of completion whereby secondary analyses that rely on accurate gene estimation or syntenic relationships can be now conducted with a high level of confidence. Recent public release of the v.3 assembly of the mouse bile-duct tapeworm, Hymenolepis microstoma, provides chromosome-level characterisation of the genome and a stabilised set of protein coding gene models underpinned by bioinformatic and empirical data. However, interactome data have not been produced. Conserved protein-protein interactions in other organisms, termed interologs, can be used to transfer interactions between species, allowing systems-level analysis in non-model organisms. RESULTS: Here, we describe a probabilistic, integrated network of interologs for the H. microstoma proteome, based on conserved protein interactions found in eukaryote model species. Almost a third of the 10,139 gene models in the v.3 assembly could be assigned interaction data and assessment of the resulting network indicates that topologically-important proteins are related to essential cellular pathways, and that the network clusters into biologically meaningful components. Moreover, network parameters are similar to those of single-species interaction networks that we constructed in the same way for S. cerevisiae, C. elegans and H. sapiens, demonstrating that information-rich, system-level analyses can be conducted even on species separated by a large phylogenetic distance from the major model organisms from which most protein interaction evidence is based. Using the interolog network, we then focused on sub-networks of interactions assigned to discrete suites of genes of interest, including signalling components and transcription factors, germline multipotency genes, and genes differentially-expressed between larval and adult worms. Results show not only an expected bias toward highly-conserved proteins, such as components of intracellular signal transduction, but in some cases predicted interactions with transcription factors that aid in identifying their target genes. CONCLUSIONS: With key helminth genomes now complete, systems-level analyses can provide an important predictive framework to guide basic and applied research on helminths and will become increasingly informative as new protein-protein interaction data accumulate.

Genome-wide identification and characterization of eukaryotic protein kinases.

The tropical liver fluke, Fasciola gigantica is a food-borne parasite responsible for the hepatobiliary disease fascioliasis. The recent completion of F. gigantica genome sequencing by our group has provided a platform for the systematic analysis of the parasite genome. Eukaryotic protein kinases (ePKs) are regulators of cellular phosphorylation. In the present study, we used various computational and bioinformatics tools to extensively analyse the ePKs in F. gigantica (FgePKs) genome. A total of 455 ePKs were identified that represent ~2% of the parasite genome. Out of these, 214 ePKs are typical kinases (Ser/Thr- and Tyr-specific ePKs), and 241 were other kinases. Several FgePKs were found to possess unusual domain architectures, which suggests the diverse nature of the proteins that can be exploited for designing novel inhibitors. 115 kinases showed <35% query coverage when compared to human ePKs highlighting significant divergences in their respective kinomes, further providing a platform for novel structure-based drug designing. This study provides a platform that may open new avenues into our understanding of helminth biochemistry and drug discovery.

Toxocara "omics" and the promises it holds for medicine and veterinary medicine.

Toxocariasis is one of the most neglected worldwide zoonoses that is caused by larval nematode parasites of the genus Toxocara, Toxocara canis, and to a lesser extent, Toxocara cati, whose migration mechanism is still largely unknown. Fortunately, some advanced tools have been employed, such as genomics, transcriptomics, and proteomics, to better understand the molecular biology and regulatory mechanisms of Toxocara. Using genomics and transcriptomics, we can identify a large number of genes that participate in the development of Toxocara and the interaction of parasites and their hosts and can predict the functions of unknown genes by comparing them with other relevant species. Using proteomics, we can identify somatic proteins and excretory and secretory (ES) proteins that perform specific biological functions in tissue degradation, pathogen invasion, immune evasion or modulation. These "omics" techniques also can contribute enormously to the development of new drugs, vaccines and diagnostic tools for toxocariasis. In a word, by utilizing "omics", we can better understand the Toxocara and toxocariasis. In this review, we summarized the representative achievements in Toxocara and the interaction between Toxocara spp. and their hosts based on expressed sequence tags (ESTs), microarray gene expression, next-generation sequencing (NGS) technologies and liquid chromatography-tandem mass spectrometry (LC-MS/MS), hoping to better understand the molecular biology of Toxocara, and contribute to new progress in the application areas of new drugs, vaccines and diagnostic tool for toxocariasis in the future.

High prevalence of Ancylostoma caninum infection in black-eared opossums (Didelphis aurita) in an urban environment.

Marsupials of the genus Didelphis, such as black-eared opossums (Didelphis aurita), are common synanthropic animals in urban areas of Brazil. These marsupials are frequently parasitized by numerous helminth species, including ancylostomatid nematodes. This study aimed to report the occurrence of Ancylostoma caninum in black-eared opossums captured in an urban environment of Southeastern Brazil and discuss the potential impact of these findings for public health. From January to June 2019, we collected fecal samples from 49 restrained opossums and evaluated by a simple flotation method; Helminth eggs were observed at different magnifications and identified according to morphological and morphometric features. Genomic DNA was extracted from Ancylostomatidae eggs and screened by duplex PCR for Ancylostoma spp. and Necator americanus using primers that amplify a region of internal transcribed spacer 2 and the 28S ribosomal RNA (ITS2-28S rRNA). Ancylostoma spp. eggs were detected in 65.3% (32/49) of the animals. Sequence analysis revealed 100% homology with A. caninum sequences from GenBank. Our results demonstrate a new host-parasite interaction for A. caninum, suggesting that black-eared opossums may participate in the zoonotic cycle of this parasite in urban areas of Brazil.

Sex chromosome evolution in parasitic nematodes of humans.

Sex determination mechanisms often differ even between related species yet the evolution of sex chromosomes remains poorly understood in all but a few model organisms. Some nematodes such as Caenorhabditis elegans have an XO sex determination system while others, such as the filarial parasite Brugia malayi, have an XY mechanism. We present a complete B. malayi genome assembly and define Nigon elements shared with C. elegans, which we then map to the genomes of other filarial species and more distantly related nematodes. We find a remarkable plasticity in sex chromosome evolution with several distinct cases of neo-X and neo-Y formation, X-added regions, and conversion of autosomes to sex chromosomes from which we propose a model of chromosome evolution across different nematode clades. The phylum Nematoda offers a new and innovative system for gaining a deeper understanding of sex chromosome evolution.

Loss of an H3K9me anchor rescues laminopathy-linked changes in nuclear organization and muscle function in an Emery-Dreifuss muscular dystrophy model.

Mutations in the nuclear structural protein lamin A produce rare, tissue-specific diseases called laminopathies. The introduction of a human Emery-Dreifuss muscular dystrophy (EDMD)-inducing mutation into the C. elegans lamin (LMN-Y59C), recapitulates many muscular dystrophy phenotypes, and correlates with hyper-sequestration of a heterochromatic array at the nuclear periphery in muscle cells. Using muscle-specific emerin Dam-ID in worms, we monitored the effects of the mutation on endogenous chromatin. An increased contact with the nuclear periphery along chromosome arms, and an enhanced release of chromosomal centers, coincided with the disease phenotypes of reduced locomotion and compromised sarcomere integrity. The coupling of the LMN-Y59C mutation with the ablation of CEC-4, a chromodomain protein that anchors H3K9-methylated chromatin at the nuclear envelope (NE), suppressed the muscle-associated disease phenotypes. Deletion of cec-4 also rescued LMN-Y59C-linked alterations in chromatin organization and some changes in transcription. Sequences that changed position in the LMN-Y59C mutant, are enriched for E2F (EFL-2)-binding sites, consistent with previous studies suggesting that altered Rb-E2F interaction with lamin A may contribute to muscle dysfunction. In summary, we were able to counteract the dominant muscle-specific defects provoked by LMNA mutation by the ablation of a lamin-associated H3K9me anchor, suggesting a novel therapeutic pathway for EDMD.

The Genome of Setaria digitata: A Cattle Nematode Closely Related to Human Filarial Parasites.

Here we present the draft genome sequence of Setaria digitata, a parasitic nematode affecting cattle. Due to its similarity to Wuchereria bancrofti, the parasitic nematode that causes lymphatic filariasis in humans, S. digitata has been used as a model organism at the genomic level to find drug targets which can be used for the development of novel drugs and/or vaccines for human filariasis. Setaria digitata causes cerebrospinal nematodiasis in goats, sheep, and horses posing a serious threat to livestock in developing countries. The genome sequence of S. digitata will assist in finding candidate genes to use as drug targets in both S. digitata and W. bancrofti. The assembled draft genome is ∼90 Mb long and contains 8,974 genomic scaffolds with a G+C content of 31.73%.

Genome and Karyotype Reorganization after Whole Genome Duplication in Free-Living Flatworms of the Genus Macrostomum.

The genus Macrostomum represents a diverse group of rhabditophoran flatworms with >200 species occurring around the world. Earlier we uncovered karyotype instability linked to hidden polyploidy in both M. lignano (2n = 8) and its sibling species M. janickei (2n = 10), prompting interest in the karyotype organization of close relatives. In this study, we investigated chromosome organization in two recently described and closely related Macrostomum species, M. mirumnovem and M. cliftonensis, and explored karyotype instability in laboratory lines and cultures of M. lignano (DV1/10, 2n = 10) and M. janickei in more detail. We revealed that three of the four studied species are characterized by karyotype instability, while M. cliftonensis showed a stable 2n = 6 karyotype. Next, we performed comparative cytogenetics of these species using fluorescent in situ hybridization (FISH) with a set of DNA probes (including microdissected DNA probes generated from M. lignano chromosomes, rDNA, and telomeric DNA). To explore the chromosome organization of the unusual 2n = 9 karyotype discovered in M. mirumnovem, we then generated chromosome-specific DNA probes for all chromosomes of this species. Similar to M. lignano and M. janickei, our findings suggest that M. mirumnovem arose via whole genome duplication (WGD) followed by considerable chromosome reshuffling. We discuss possible evolutionary scenarios for the emergence and reorganization of the karyotypes of these Macrostomum species and consider their suitability as promising animal models for studying the mechanisms and regularities of karyotype and genome evolution after a recent WGD.

Within-Population Genome Size Variation is Mediated by Multiple Genomic Elements That Segregate Independently during Meiosis.

Within-species variation in genome size has been documented in many animals and plants. Despite its importance for understanding eukaryotic genome diversity, there is only sparse knowledge about how individual-level processes mediate genome size variation in populations. Here, we study a natural population of the rotifer Brachionus asplanchnoidis whose members differ up to 1.9-fold in diploid genome size, but were still able to interbreed and produce viable offspring. We show that genome size is highly heritable and can be artificially selected up or down, but not below a certain basal diploid genome size for this species. Analyses of segregation patterns in haploid males reveal that large genomic elements (several megabases in size) provide the substrate of genome size variation. These elements, and their segregation patterns, explain the generation of new genome size variants, the short-term evolutionary potential of genome size change in populations, and some seemingly paradoxical patterns, like an increase in genome size variation among highly inbred lines. Our study suggests that a conceptual model involving only two variables, 1) a basal genome size of the population, and 2) a vector containing information on additional elements that may increase genome size in this population (size, number, and meiotic segregation behavior), can effectively address most scenarios of short-term evolutionary change of genome size in a population.

Genome-wide identification of ABC transporters in monogeneans.

ATP-Binding Cassette (ABC) transporters are proteins that actively mediate the transport of a wide variety of molecules, including drugs. Thus, in parasitology, ABC transporters have gained attention as potential targets for therapeutic drugs. Among the parasitic Platyhelminthes, ABC transporters have been identified and classified in a few species of Trematoda and Cestoda but not in Monogenea. Monogeneans are mainly ectoparasites of marine and freshwater fish, although they can also be found on other aquatic organisms. Severe epizootics caused by monogeneans have been reported around the world, mainly in confined and/or overcrowded fish. The purpose of this study was to identify the ABC transporters in four species of monogeneans (Gyrodactylus salaris, Protopolystoma xenopodis, Eudiplozoon nipponicum and Neobenedenia melleni) for which genomic resources are publicly available. For comparative purposes, ABC transporters were also identified in endoparasitic (Schistosoma mansoni and Echinococcus granulosus) and free-living (Macrostomun lignano and Schmidtea mediterranea) platyhelminths. Thirty-two putative ABC transporters were identified in the genome of G. salaris, 40 in the genome of P. xenopodis, 46 in the transcriptome of E. nipponicum and 9 in a rather limited ESTs set available for N. melleni. Of the eight ABC subfamilies (A-H) known in metazoans, subfamily H was the only one not found in any monogenean species. In contrast, ABCC was the best represented subfamily. Phylogenetic analyses showed a few cases of one-to-one orthologous relationships, which agree with results from other metazoan species. We found some monogenean ABC members related to subfamilies B, C and G involved in drug resistance in humans. This information may be useful for future functional studies on ABC transporters in monogeneans.

The genome of a subterrestrial nematode reveals adaptations to heat.

The nematode Halicephalobus mephisto was originally discovered inhabiting a deep terrestrial aquifer 1.3 km underground. H. mephisto can thrive under conditions of abiotic stress including heat and minimal oxygen, where it feeds on a community of both chemolithotrophic and heterotrophic prokaryotes in an unusual ecosystem isolated from the surface biosphere. Here we report the comprehensive genome and transcriptome of this organism, identifying a signature of adaptation: an expanded repertoire of 70 kilodalton heat-shock proteins (Hsp70) and avrRpt2 induced gene 1 (AIG1) proteins. The expanded Hsp70 genes are transcriptionally induced upon growth under heat stress, and we find that positive selection is detectable in several members of this family. We further show that AIG1 may have been acquired by horizontal gene transfer (HGT) from a rhizobial fungus. Over one-third of the genes of H. mephisto are novel, highlighting the divergence of this nematode from other sequenced organisms. This work sheds light on the genomic basis of heat tolerance in a complete subterrestrial eukaryotic genome.