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BackgroundSchistosoma mansoni is a flatworm that causes a neglected tropical disease affecting millions worldwide. Most flatworms are hermaphrodites but schistosomes have genotypically determined male (ZZ) and female (ZW) sexes. Sex is essential for pathology and transmission, however, the molecular determinants of sex remain unknown and is limited by poorly resolved sex chromosomes in previous genome assemblies.ResultsWe assembled the 391.4 Mb S. mansoni genome into individual, single-scaffold chromosomes, including Z and W. Manual curation resulted in a vastly improved gene annotation, resolved gene and repeat arrays, trans-splicing, and almost all UTRs. The sex chromosomes each comprise pseudoautosomal regions and single sex-specific regions. The Z-specific region contains 932 genes, but on W all but 29 of these genes have been lost and the presence of five pseudogenes indicates that degeneration of W is ongoing. Synteny analysis reveals an ancient chromosomal fusion corresponding to the oldest part of Z, where only a single gene—encoding the large subunit of pre-mRNA splicing factor U2AF—has retained an intact copy on W. The sex-specific copies of U2AF have divergent N-termini and show sex-biased gene expression.ConclusionOur assembly with fully resolved chromosomes provides evidence of an evolutionary path taken to create the Z and W sex chromosomes of schistosomes. Sex-linked divergence of the single U2AF gene, which has been present in the sex-specific regions longer than any other extant gene with distinct male and female specific copies and expression, may have been a pivotal step in the evolution of gonorchorism and genotypic sex determination of schistosomes.

Background Chromosome-level assemblies are indispensable for accurate gene prediction, synteny assessment, and understanding higher-order genome architecture. Reference and draft genomes of key helminth species have been published, but little is yet known about the biology of their chromosomes. Here, we present the complete genome of the tapeworm Hymenolepis microstoma, providing a reference quality, end-to-end assembly that represents the first fully assembled genome of a spiralian/lophotrochozoan, revealing new insights into chromosome evolution. Results Long-read sequencing and optical mapping data were added to previous short-read data enabling complete re-assembly into six chromosomes, consistent with karyology. Small genome size (169 Mb) and lack of haploid variation (1 SNP/3.2 Mb) contributed to exceptionally high contiguity with only 85 gaps remaining in regions of low complexity sequence. Resolution of repeat regions reveals novel gene expansions, micro-exon genes, and spliced leader trans-splicing, and illuminates the landscape of transposable elements, explaining observed length differences in sister chromatids. Syntenic comparison with other parasitic flatworms shows conserved ancestral linkage groups indicating that the H. microstoma karyotype evolved through fusion events. Strikingly, the assembly reveals that the chromosomes terminate in centromeric arrays, indicating that these motifs play a role not only in segregation, but also in protecting the linear integrity and full lengths of chromosomes. Conclusions Despite strong conservation of canonical telomeres, our results show that they can be substituted by more complex, species-specific sequences, as represented by centromeres. The assembly provides a robust platform for investigations that require complete genome representation.

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., Stephen Doyle et al. report the chromosome-scale genome assembly and transcriptome sequence data of Haemonchus contortus, a key parasitic nematode model. These data show nearly complete conservation of chromosome content with C. elegans and brings insight into transcriptional regulation and chromosome-wide genetic diversity in this important pathogen.

Hundreds of millions of people are infected with whipworms (Trichuris trichiura), large metazoan parasites that live in the caecum and proximal colon. Whipworms inhabit distinct multi-intracellular epithelial burrows that have been described as syncytial tunnels. However, the interactions between first-stage (L1) larvae and the host epithelia that determine parasite invasion and establishment in the syncytium remain unclear. In vivo experiments investigating these events have been severely hampered by the limited in situ accessibility to intracellular infective larvae at the bottom of the crypts of Lieberkuhn, and the lack of genetic tools such as fluorescent organisms that are readily available for other pathogens but not parasitic nematodes. Moreover, cell lines, which do not mimic the complexity of the intestinal epithelium, have been unsuccessful in supporting infection by whipworm larvae. Here, we show that caecaloids grown in an open crypt-like conformation recapitulate the caecal epithelium. Using this system, we establish in vitro infections with T. muris L1 larvae for the first-time, and provide clear evidence that syncytial tunnels are formed at this early stage. We show that larval whipworms are completely intracellular but woven through multiple cells. Using the caecaloids, we are able to visualise the pathways taken by the larvae as they burrow through the epithelial cells. We also demonstrate that larvae degrade the mucus layers overlaying the epithelium, enabling them to access the cells below. We show that early syncytial tunnels are composed of enterocytes and goblet cells that are alive and actively interacting with the larvae during the first 24 h of the infection. Progression of infection results in damage to host cells and by 72 h post-infection, we show that desmosomes of cells from infected epithelium widen and some host cells appear to become liquified. Collectively, our work unravels processes mediating the intestinal epithelium invasion by whipworms and reveals new specific interactions between the host and the parasite that allow the whipworm to establish on its multi-intracellular niche. Our study demonstrates that caecaloids can be used as a relevant in vitro model to investigate the infection biology of T. muris during the early colonisation of its host.

Graphical abstract, Highlights • Development of new methods to generate, culture and characterise mouse caecaloids is described. • Caecaloids recapitulate the caecal epithelium composition and spatial organisation. • Caecaloids can be used to study host–caecal pathogen interactions in vitro. • Trichuris muris EVs exert novel immunoregulatory effects on intestinal epithelial cells., The caecum, an intestinal appendage in the junction of the small and large intestines, displays a unique epithelium that serves as an exclusive niche for a range of pathogens including whipworms (Trichuris spp.). While protocols to grow organoids from small intestine (enteroids) and colon (colonoids) exist, the conditions to culture organoids from the caecum have yet to be described. Here, we report methods to grow, differentiate and characterise mouse adult stem cell-derived caecal organoids, termed caecaloids. We compare the cellular composition of caecaloids with that of enteroids, identifying differences in intestinal epithelial cell populations that mimic those found in the caecum and small intestine. The remarkable similarity in the intestinal epithelial cell composition and spatial conformation of caecaloids and their tissue of origin enables their use as an in vitro model to study host interactions with important caecal pathogens. Thus, exploiting this system, we investigated the responses of caecal intestinal epithelial cells to extracellular vesicles secreted/excreted by the intracellular helminth Trichuris muris. Our findings reveal novel immunoregulatory effects of whipworm extracellular vesicles on the caecal epithelium, including the downregulation of responses to nucleic acid recognition and type-I interferon signalling.

BackgroundHaemonchus contortusis a globally distributed and economically important gastrointestinal pathogen of small ruminants, and has become the key nematode model for studying anthelmintic resistance and other parasite-specific traits among a wider group of parasites including major human pathogens. Two draft genome assemblies forH. contortuswere reported in 2013, however, both were highly fragmented, incomplete, and differed from one another in important respects. While the introduction of long-read sequencing has significantly increased the rate of production and contiguity ofde novogenome assemblies broadly, achieving high quality genome assemblies for small, genetically diverse, outcrossing eukaryotic organisms such asH. contortusremains a significant challenge.ResultsHere, 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. We show a remarkable pattern of almost complete conservation of chromosome content (synteny) withCaenorhabditis elegans, but almost no conservation of gene order. Long-read transcriptome sequence data has allowed us to define coordinated transcriptional regulation throughout the life cycle of the parasite, and refine our understanding ofcis- andtrans-splicing relative to that observed inC. elegans. Finally, we use this assembly to give a comprehensive picture of chromosome-wide genetic diversity both within a single isolate and globally.ConclusionsTheH. contortusMHco3(ISE).N1 genome assembly presented here represents the most contiguous and resolved nematode assembly outside of theCaenorhabditisgenus to date, together with one of the highest-quality set of predicted gene features. These data provide a high-quality comparison for understanding the evolution and genomics ofCaenorhabditisand other nematodes, and extends the experimental tractability of this model parasitic nematode in understanding pathogen biology, drug discovery and vaccine development, and important adaptive traits such as drug resistance.

WormBase (https://wormbase.org/) is a mature Model Organism Information Resource supporting researchers using the nematode Caenorhabditis elegans as a model system for studies across a broad range of basic biological processes. Toward this mission, WormBase efforts are arranged in three primary facets: curation, user interface and architecture. In this update, we describe progress in each of these three areas. In particular, we discuss the status of literature curation and recently added data, detail new features of the web interface and options for users wishing to conduct data mining workflows, and discuss our efforts to build a robust and scalable architecture by leveraging commercial cloud offerings. We conclude with a description of WormBase's role as a founding member of the nascent Alliance of Genome Resources.

International audience; Peptidoglycan recognition proteins (PGRPs) constitute the primary means of bacterial recognition in insects. Recent work in the model organism Drosophila has revealed the mechanisms by which the complement of PGRPs refine the sensitivity of different tissues to bacterial elicitors, permitting the persistence of commensal bacteria in the gut whilst maintaining vigilance against bacterial infection. Here, we use in vivo knockdowns and in vitro pull-down assays to investigate the role of the three major isoforms of the transmembrane receptor of the Imd pathway, PGRPLC, in basal immunity in the Anopheles coluzzii mosquito midgut. Our results indicate that the mosquito midgut is regionalized in its expression of immune effectors and of PGRPLC1. We show that PGRPLC1 and PGRPLC3 are pulled down with polymeric DAP-type peptidoglycan, while PGRPLC2 and PGRPLC3 co-precipitate in the presence of TCT, a peptidoglycan monomer. These data suggest that, as found in Drosophila, discrimination of polymeric and monomeric PGN by Anopheles PGRPLC participates in the regulation of the Imd pathway.

WormBase ParaSite ( parasite.wormbase.org ) is a comprehensive resource for the genomes of parasitic nematodes and flatworms (helminths). It currently includes genomic data for over 100 helminth species, adding value by way of consistent functional annotation, gene comparative analysis and gene expression analysis. We provide several ways of exploring the data including a choice of genome browsers, genome and gene summary pages, text and sequence searching, a query wizard, bulk downloads, and programmatic interfaces. WormBase ParaSite is released three to six times per year, and is developed in collaboration with WormBase ( www.wormbase.org ) and Ensembl Genomes ( www.ensemblgenomes.org ).

WormBase (http://www.wormbase.org) is an important knowledge resource for biomedical researchers worldwide. To accommodate the ever increasing amount and complexity of research data, WormBase continues to advance its practices on data acquisition, curation and retrieval to most effectively deliver comprehensive knowledge about Caenorhabditis elegans, and genomic information about other nematodes and parasitic flatworms. Recent notable enhancements include user-directed submission of data, such as micropublication; genomic data curation and presentation, including additional genomes and JBrowse, respectively; new query tools, such as SimpleMine, Gene Enrichment Analysis; new data displays, such as the Person Lineage browser and the Summary of Ontology-based Annotations. Anticipating more rapid data growth ahead, WormBase continues the process of migrating to a cutting-edge database technology to achieve better stability, scalability, reproducibility and a faster response time. To better serve the broader research community, WormBase, with five other Model Organism Databases and The Gene Ontology project, have begun to collaborate formally as the Alliance of Genome Resources.

WormBase ParaSite ( parasite.wormbase.org ) is a comprehensive resource for the genomes of parasitic nematodes and flatworms (helminths). It currently includes genomic data for over 100 helminth species, adding value by way of consistent functional annotation, gene comparative analysis and gene expression analysis. We provide several ways of exploring the data including a choice of genome browsers, genome and gene summary pages, text and sequence searching, a query wizard, bulk downloads, and programmatic interfaces. WormBase ParaSite is released three to six times per year, and is developed in collaboration with WormBase ( www.wormbase.org ) and Ensembl Genomes ( www.ensemblgenomes.org ).

The recent explosion of interest in the mosquito gut microbiota has opened up the possibility of several novel interventions for vectorborne diseases. Through introduction of specific bacterial isolates to mosquito hosts, which directly affect the disease agents or induce mosquito resistance to them, or by targeting the microbiota interaction with the mosquito immune system causing dysbiosis and reduction of fitness, there are several potential means to exploiting the microbiota to lower disease transmission. The success of any of these strategies depends on a detailed, system-based understanding of the microbiota and its relationship with the mosquito host. On the one hand, the contribution of the microbiota to mosquito physiology, and the resistance and/or tolerance responses employed by the host to allow persistence of the microbiota while maintaining vigilance to infection are not well understood; on the other hand, the bacterial characteristics that permit persistence within the gut and homeostasis of the gut ecosystem in different nutritional states remain to be addressed. This chapter aims to provide insights into some of these important questions by examining the mosquito gut and its microbiota as a highly integrated system. The first part of this chapter reviews our understanding of the mosquito immune system, and the second part consolidates our knowledge of the mosquito microbiota. The third and last parts examine what is known about the interactions between the immune system and the microbiota.

Peptidoglycan recognition proteins (PGRPs) form a family of immune regulators that is conserved from insects to mammals. In the malaria vector mosquito Anophelescoluzzii, the peptidoglycan receptor PGRPLC activates the immune-deficiency (Imd) pathway limiting both the microbiota load and Plasmodium infection. Here, we carried out an RNA interference screen to examine the role of all 7 Anopheles PGRPs in infections with Plasmodium berghei and P. falciparum. We show that, in addition to PGRPLC, PGRPLA and PGRPS2/PGRPS3 also participate in antiparasitic defenses, and that PGRPLB promotes mosquito permissiveness to P. falciparum. We also demonstrate that following a mosquito blood feeding, which promotes growth of the gut microbiota, PGRPLA and PGRPLB positively and negatively regulate the activation of the Imd pathway, respectively. Our data demonstrate that PGRPs are important regulators of the mosquito epithelial immunity and vector competence.

Malaria reduction is most efficiently achieved by vector control whereby human populations at high risk of contracting and transmitting the disease are protected from mosquito bites. Here, we identify the presence of antibiotics in the blood of malaria-infected people as a new risk of increasing disease transmission. We show that antibiotics in ingested blood enhance the susceptibility of Anopheles gambiae mosquitoes to malaria infection by disturbing their gut microbiota. This effect is confirmed in a semi-natural setting by feeding mosquitoes with blood of children naturally infected with Plasmodium falciparum. Antibiotic exposure additionally increases mosquito survival and fecundity, which are known to augment vectorial capacity. These findings suggest that malaria transmission may be exacerbated in areas of high antibiotic usage, and that regions targeted by mass drug administration programs against communicable diseases may necessitate increased vector control., The gut microbiota of malaria-transmitting mosquitoes contributes to the insects’ resistance to the parasite. Here, Gendrin et al. show that antibiotics in ingested human blood alter the mosquito gut microbiota and increase the insect’s survival, fecundity and susceptibility to the parasites.

Manipulation of the mosquito gut microbiota can lay the foundations for novel methods for disease transmission control. Mosquito blood feeding triggers a significant, transient increase of the gut microbiota, but little is known about the mechanisms by which the mosquito controls this bacterial growth whilst limiting inflammation of the gut epithelium. Here, we investigate the gut epithelial response to the changing microbiota load upon blood feeding in the malaria vector Anopheles coluzzii. We show that the synthesis and integrity of the peritrophic matrix, which physically separates the gut epithelium from its luminal contents, is microbiota dependent. We reveal that the peritrophic matrix limits the growth and persistence of Enterobacteriaceae within the gut, whilst preventing seeding of a systemic infection. Our results demonstrate that the peritrophic matrix is a key regulator of mosquito gut homeostasis and establish functional analogies between this and the mucus layers of the mammalian gastrointestinal tract., Author summary When a female mosquito takes a blood meal from a human, the bacteria residing within its gut grow significantly. Following a blood meal, female mosquitoes produce a barrier within their gut, known as the peritrophic matrix, which physically separates the blood meal from the cells of the epithelium. Here, we show that the presence of bacteria in the gut is required for the synthesis of the peritrophic matrix. By experimentally disrupting this barrier, we find that this structure plays a role in limiting the extent to which bacteria of one particular family are able to grow and persist in the mosquito gut. We also find that the peritrophic matrix ensures that bacteria remain within the gut, preventing them from invading the mosquito body cavity. These results will be useful in designing disease control strategies that depend on the ability of bacteria to colonize and persist in relevant tissues in the mosquito host.

Ensembl Genomes (https://www.ensemblgenomes.org) provides access to non-vertebrate genomes and analysis complementing vertebrate resources developed by the Ensembl project (https://www.ensembl.org). The two resources collectively present genome annotation through a consistent set of interfaces spanning the tree of life presenting genome sequence, annotation, variation, transcriptomic data and comparative analysis. Here, we present our largest increase in plant, metazoan and fungal genomes since the project's inception creating one of the world's most comprehensive genomic resources and describe our efforts to reduce genome redundancy in our Bacteria portal. We detail our new efforts in gene annotation, our emerging support for pangenome analysis, our efforts to accelerate data dissemination through the Ensembl Rapid Release resource and our new AlphaFold visualization. Finally, we present details of our future plans including updates on our integration with Ensembl, and how we plan to improve our support for the microbial research community. Software and data are made available without restriction via our website, online tools platform and programmatic interfaces (available under an Apache 2.0 license). Data updates are synchronised with Ensembl's release cycle.