Adapting the Smart-seq2 Protocol for Robust Single Worm RNA-seq.

Most nematodes are small worms that lack enough RNA for regular RNA-seq protocols without pooling hundred to thousand of individuals. We have adapted the Smart-seq2 protocol in order to sequence the transcriptome of an individual worm. While developed for individual Steinernema carpocapsae and Caenorhabditis elegans larvae as well as embryos, the protocol should be adaptable for other nematode species and small invertebrates. In addition, we describe how to analyze the RNA-seq results using the Galaxy online environment. We expect that this method will be useful for the studying gene expression variances of individual nematodes in wild type and mutant backgrounds

The Draft Genome and Transcriptome of Panagrellus redivivus Are Shaped by the Harsh Demands of a Free-Living Lifestyle

Nematodes compose an abundant and diverse invertebrate phylum with members inhabiting nearly every ecological niche. Panagrellus redivivus (the “microworm”) is a free-living nematode frequently used to understand the evolution of developmental and behavioral processes given its phylogenetic distance to Caenorhabditis elegans. Here we report the de novo sequencing of the genome, transcriptome, and small RNAs of P. redivivus. Using a combination of automated gene finders and RNA-seq data, we predict 24,249 genes and 32,676 transcripts. Small RNA analysis revealed 248 microRNA (miRNA) hairpins, of which 63 had orthologs in other species. Fourteen miRNA clusters containing 42 miRNA precursors were found. The RNA interference, dauer development, and programmed cell death pathways are largely conserved. Analysis of protein family domain abundance revealed that P. redivivus has experienced a striking expansion of BTB domain-containing proteins and an unprecedented expansion of the cullin scaffold family of proteins involved in multi-subunit ubiquitin ligases, suggesting proteolytic plasticity and/or tighter regulation of protein turnover. The eukaryotic release factor protein family has also been dramatically expanded and suggests an ongoing evolutionary arms race with viruses and transposons. The P. redivivus genome provides a resource to advance our understanding of nematode evolution and biology and to further elucidate the genomic architecture leading to free-living lineages, taking advantage of the many fascinating features of this worm revealed by comparative studies

Comparative genomics of Steinernema reveals deeply conserved gene regulatory networks

Background: Parasitism is a major ecological niche for a variety of nematodes. Multiple nematode lineages have specialized as pathogens, including deadly parasites of insects that are used in biological control. We have sequenced and analyzed the draft genomes and transcriptomes of the entomopathogenic nematode Steinernema carpocapsae and four congeners (S. scapterisci, S. monticolum, S. feltiae, and S. glaseri). Results: We used these genomes to establish phylogenetic relationships, explore gene conservation across species, and identify genes uniquely expanded in insect parasites. Protein domain analysis in Steinernema revealed a striking expansion of numerous putative parasitism genes, including certain protease and protease inhibitor families, as well as fatty acid- and retinol-binding proteins. Stage-specific gene expression of some of these expanded families further supports the notion that they are involved in insect parasitism by Steinernema. We show that sets of novel conserved non-coding regulatory motifs are associated with orthologous genes in Steinernema and Caenorhabditis. Conclusions: We have identified a set of expanded gene families that are likely to be involved in parasitism. We have also identified a set of non-coding motifs associated with groups of orthologous genes in Steinernema and Caenorhabditis involved in neurogenesis and embryonic development that are likely part of conserved protein–DNA relationships shared between these two genera

Host- and Helminth-Derived Endocannabinoids That Have Effects on Host Immunity Are Generated during Infection.

Helminths have coevolved with their hosts, resulting in the development of specialized host immune mechanisms and parasite-specific regulatory products. Identification of new pathways that regulate helminth infection could provide a better understanding of host-helminth interaction and may identify new therapeutic targets for helminth infection. Here we identify the endocannabinoid system as a new mechanism that influences host immunity to helminths. Endocannabinoids are lipid-derived signaling molecules that control important physiologic processes, such as feeding behavior and metabolism. Following murine infection with Nippostrongylus brasiliensis, an intestinal nematode with a life cycle similar to that of hookworms, we observed increased levels of endocannabinoids (2-arachidonoylglycerol [2-AG] or anandamide [AEA]) and the endocannabinoid-like molecule oleoylethanolamine (OEA) in infected lung and intestine. To investigate endocannabinoid function in helminth infection, we employed pharmacological inhibitors of cannabinoid subtype receptors 1 and 2 (CB1R and CB2R). Compared to findings for vehicle-treated mice, inhibition of CB1R but not CB2R resulted in increased N. brasiliensis worm burden and egg output, associated with significantly decreased expression of the T helper type 2 cytokine interleukin 5 (IL-5) in intestinal tissue and splenocyte cultures. Strikingly, bioinformatic analysis of genomic and transcriptome sequencing (RNA-seq) data sets identified putative genes encoding endocannabinoid biosynthetic and degradative enzymes in many parasitic nematodes. To test the novel hypothesis that helminth parasites produce their own endocannabinoids, we measured endocannabinoid levels in N. brasiliensis by mass spectrometry and quantitative PCR and found that N. brasiliensis parasites produced endocannabinoids, especially at the infectious larval stage. To our knowledge, this is the first report of helminth- and host-derived endocannabinoids that promote host immune responses and reduce parasite burden

SQANTI : extensive characterization of long-read transcript sequences for quality control in full-length transcriptome identification and quantification

High-throughput sequencing of full-length transcripts using long reads has paved the way for the discovery of thousands of novel transcripts, even in well-annotated mammalian species. The advances in sequencing technology have created a need for studies and tools that can characterize these novel variants. Here, we present SQANTI, an automated pipeline for the classification of long-read transcripts that can assess the quality of data and the preprocessing pipeline using 47 unique descriptors. We apply SQANTI to a neuronal mouse transcriptome using Pacific Biosciences (PacBio) long reads and illustrate how the tool is effective in characterizing and describing the composition of the full-length transcriptome. We perform extensive evaluation of ToFU PacBio transcripts by PCR to reveal that an important number of the novel transcripts are technical artifacts of the sequencing approach and that SQANTI quality descriptors can be used to engineer a filtering strategy to remove them. Most novel transcripts in this curated transcriptome are novel combinations of existing splice sites, resulting more frequently in novel ORFs than novel UTRs, and are enriched in both general metabolic and neural-specific functions. We show that these new transcripts have a major impact in the correct quantification of transcript levels by state-of-the-art short-read-based quantification algorithms. By comparing our iso-transcriptome with public proteomics databases, we find that alternative isoforms are elusive to proteogenomics detection. SQANTI allows the user to maximize the analytical outcome of long-read technologies by providing the tools to deliver quality-evaluated and curated full-length transcriptomes

Comparative genomics of Steinernema

Nematodes comprise one of the most diverse bilaterian phyla, having colonized nearly every imaginable ecological niche on earth. They are major parasites of plants, animals, and humans, despite sharing a relatively conserved body plan. The Steinernema genus comprises over 70 characterized species that are lethal parasites of insects, which have different foraging strategies and host ranges, and are distantly related to the model organism C. elegans. To better understand the evolution of parasitism and development in nematodes, we sequenced and analyzed the genomes as well as transcriptomes of five key members of the Steinernema genus (S. carpocapsae, S. scapterisci, S. monticolum, S. glaseri, and S. feltiae). In chapter 2, using available ecological and molecular data, we explore genomic differences likely to be involved in insect parasitism, particularly in host-range and specificity of these five species. We find surprising gene family evolution of proteases, protease inhibitors, proteolytic cascade proteins, GPCRs, transposon and retroviral content, and even protein-protein interaction domains, many of which correlate excitingly with known differences in host range and specificity among these parasites. The combination of multiple closely related genomes in a non-Caenorhabidtis clade and accompanying deeply sequenced transcriptomes allows for powerful comparisons to other genera such as Caenorhabditis. In particular, comparisons in gene expression at defined stages show surprising plasticity of timing across one-to-one orthologous genes in the five genomes when compared to C. elegans. Our conservation analysis shows that approximately 20 Mb are conserved across the Steinernema species, with 5.1 Mb of this comprising non-coding regions. Our analysis of the conserved non-coding regions combined with stage-specific gene expression data reveals that a limited number of regulatory motifs are associated with conservation of stage-specific ortholog expression in Steinernema and Caenorhabditis, which suggests that several underlying gene regulatory relationships controlling development are conserved in the two genera. In Chapter 3, we investigate embryonic development in Steinernema by comparing the expression of orthologous genes at eleven different embryonic stages of two Steinernema species with two Caenorhabditis species. We found that zygotic transcription initiates at different developmental stages in each species, with the Steinernema species initiating transcription at earlier developmental stages than Caenorhabditis. Surprisingly, we also found that gene expression conservation during development is highest at the later embryonic stages than at the earlier ones, indicating that ortholog expression divergence across distantly related species follows a funnel-shaped model in contrast to the hourglass model of nematode development that has been previously proposed. Thus, this work provides novel insight into embryonic development across distantly related nematode species and demonstrates that the mechanisms controlling early development are more diverse than previously thought

Comparative genomics of Steinernema

Nematodes comprise one of the most diverse bilaterian phyla, having colonized nearly every imaginable ecological niche on earth. They are major parasites of plants, animals, and humans, despite sharing a relatively conserved body plan. The Steinernema genus comprises over 70 characterized species that are lethal parasites of insects, which have different foraging strategies and host ranges, and are distantly related to the model organism C. elegans. To better understand the evolution of parasitism and development in nematodes, we sequenced and analyzed the genomes as well as transcriptomes of five key members of the Steinernema genus (S. carpocapsae, S. scapterisci, S. monticolum, S. glaseri, and S. feltiae). In chapter 2, using available ecological and molecular data, we explore genomic differences likely to be involved in insect parasitism, particularly in host-range and specificity of these five species. We find surprising gene family evolution of proteases, protease inhibitors, proteolytic cascade proteins, GPCRs, transposon and retroviral content, and even protein-protein interaction domains, many of which correlate excitingly with known differences in host range and specificity among these parasites. The combination of multiple closely related genomes in a non-Caenorhabidtis clade and accompanying deeply sequenced transcriptomes allows for powerful comparisons to other genera such as Caenorhabditis. In particular, comparisons in gene expression at defined stages show surprising plasticity of timing across one-to-one orthologous genes in the five genomes when compared to C. elegans. Our conservation analysis shows that approximately 20 Mb are conserved across the Steinernema species, with 5.1 Mb of this comprising non-coding regions. Our analysis of the conserved non-coding regions combined with stage-specific gene expression data reveals that a limited number of regulatory motifs are associated with conservation of stage-specific ortholog expression in Steinernema and Caenorhabditis, which suggests that several underlying gene regulatory relationships controlling development are conserved in the two genera. In Chapter 3, we investigate embryonic development in Steinernema by comparing the expression of orthologous genes at eleven different embryonic stages of two Steinernema species with two Caenorhabditis species. We found that zygotic transcription initiates at different developmental stages in each species, with the Steinernema species initiating transcription at earlier developmental stages than Caenorhabditis. Surprisingly, we also found that gene expression conservation during development is highest at the later embryonic stages than at the earlier ones, indicating that ortholog expression divergence across distantly related species follows a funnel-shaped model in contrast to the hourglass model of nematode development that has been previously proposed. Thus, this work provides novel insight into embryonic development across distantly related nematode species and demonstrates that the mechanisms controlling early development are more diverse than previously thought

Activated entomopathogenic nematode infective juveniles release lethal venom proteins.

Entomopathogenic nematodes (EPNs) are unique parasites due to their symbiosis with entomopathogenic bacteria and their ability to kill insect hosts quickly after infection. It is widely believed that EPNs rely on their bacterial partners for killing hosts. Here we disproved this theory by demonstrating that the in vitro activated infective juveniles (IJs) of Steinernema carpocapsae (a well-studied EPN species) release venom proteins that are lethal to several insects including Drosophila melanogaster. We confirmed that the in vitro activation is a good approximation of the in vivo process by comparing the transcriptomes of individual in vitro and in vivo activated IJs. We further analyzed the transcriptomes of non-activated and activated IJs and revealed a dramatic shift in gene expression during IJ activation. We also analyzed the venom proteome using mass spectrometry. Among the 472 venom proteins, proteases and protease inhibitors are especially abundant, and toxin-related proteins such as Shk domain-containing proteins and fatty acid- and retinol-binding proteins are also detected, which are potential candidates for suppressing the host immune system. Many of the venom proteins have conserved orthologs in vertebrate-parasitic nematodes and are differentially expressed during IJ activation, suggesting conserved functions in nematode parasitism. In summary, our findings strongly support a new model that S. carpocapsae and likely other Steinernema EPNs have a more active role in contributing to the pathogenicity of the nematode-bacterium complex than simply relying on their symbiotic bacteria. Furthermore, we propose that EPNs are a good model system for investigating vertebrate- and human-parasitic nematodes, especially regarding the function of excretory/secretory products

Adapting the Smart-seq2 Protocol for Robust Single Worm RNA-seq.

Most nematodes are small worms that lack enough RNA for regular RNA-seq protocols without pooling hundred to thousand of individuals. We have adapted the Smart-seq2 protocol in order to sequence the transcriptome of an individual worm. While developed for individual Steinernema carpocapsae and Caenorhabditis elegans larvae as well as embryos, the protocol should be adaptable for other nematode species and small invertebrates. In addition, we describe how to analyze the RNA-seq results using the Galaxy online environment. We expect that this method will be useful for the studying gene expression variances of individual nematodes in wild type and mutant backgrounds