Project description:Entomopathogenic nematodes (EPNs) are unique parasitic nematodes due to their symbiosis with entomopathogenic bacteria and their ability to kill insect hosts quickly after infection. Although it has been widely believed that EPNs rely on their bacterial partners for killing insect hosts, compelling evidence from previous studies challenges this model. We developed an improved method of activating millions of Steinernema carpocapsae infective juveniles (IJs) in vitro to harvest excreted/secreted (ES) proteins for bioactivity tests and proteomics analysis. We found that a low dose of the ES proteins from early activated nematodes is lethal to Drosophila melanogaster adults within 2-6 hours. We analyzed the protein composition of this venom using mass spectrometry and identified 472 proteins. Many of these venom proteins share high homology with those of vertebrate-parasitic nematodes. Among many different families of proteins found in the venom, proteases and protease inhibitors are especially abundant. Some toxin-related proteins such as Shk domain-containing proteins were also detected. We further analyzed the transcriptomes of individual non-activated IJs and nematodes that were activated in vitro and in vivo, which revealed a dramatic shift in gene expression during IJ activation. By comparing the whole transcriptomes and the genes encoding venom proteins between the in vitro and in vivo activated nematodes, we confirmed that the in vitro activation is a good approximation of the in vivo process. 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 ES products.
Project description:Entomopathogenic nematodes from the genus Steinernema are lethal insect parasites that quickly kill their insect hosts with the help of their symbiotic bacteria. Steinernema carpocapsae is one of the most studied entomopathogens due to its broad lethality to diverse insect species and its effective commercial use as a biological control agent for insect pests, as well as a genetic model for studying parasitism, pathogenesis, and symbiosis. In this study, we used long-reads from the Pacific Biosciences platform and BioNano Genomics Irys system to assemble the best genome of S. carpocapsae ALL strain to date, comprising 84.5 Mb in 16 scaffolds, with an N50 of 7.36Mb. The largest scaffold, with 20.9Mb, was identified as chromosome X based on sex-specific genome sequencing. The high level of contiguity allowed us to characterize gene density, repeat content, and GC content. RNA-seq data from 17 developmental stages, spanning from embryo to adult, were used to predict 30,957 gene models. Using this new genome, we performed a macrosyntenic analysis to Caenorhabditis elegans and Pristionchus pacificus and found S. carpocapsae’s chromosome X to be primarily orthologous to C. elegans’ and P. pacificus’ chromosome II and IV. We also investigated the expansion of protein families and gene expression differences between male and female stage nematodes. This new genome and more accurate set of annotations provide a foundation for new comparative genomic and gene expression studies within the Steinernema clade and across the Nematode phylum.
Project description:Entomopathogenic nematode infective juveniles activate when they infect a host and begin releasing excreted-secreted products. These are the excreted-secreted proteins detected by mass spec from non-activated/naive Steinernema feltiae infective juveniles(infective juveniles not exposed to host or host tissue).
Project description:Entomopathogenic nematode infective juveniles activate when they infect a host and begin releasing excreted-secreted products. These are the excreted-secreted proteins detected by mass spec from activated Steinernema feltiae infective juveniles. The infective juveniles were activated in vitro by exposure to insect-host tissue for 6 hours.
Project description:We report small RNA sequencing of the entomopathogenic nematode Steinernema carpocapsae. The nematodes were grown in liquid culture in homogenates of pig kidney/fat and infective juveniles were gathered. Then Galleria mellonella insect haemolymph was added to simulate insect infection, control nematodes weren't added haemolymph. Nematodes were collected after two hours after haemolymph addition.
Project description:We report small RNA sequencing of the entomopathogenic nematode Steinernema carpocapsae. The nematodes were grown in liquid culture in homogenates of pig kidney/fat and infective juveniles were gathered. Then Galleria mellonella insect haemolymph was added to simulate insect infection, control nematodes weren't added haemolymph. Nematodes were collected after two hours after haemolymph addition. infective juveniles S. carpocapsae were incubated with and without haemolymph, three replicates
Project description:The infective juveniles (IJs) of entomopathogenic nematodes such as Steinernema carpocapsae are the only stage in their life cycle that is free living before infecting new hosts. When the IJs enter the host, they activate and secrete proteins to modulate the host immune system. The secreted proteins are identified by mass spectrometry in this study.
Project description: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). 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. 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.
Project description: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, S. glaseri) distantly related to Caenorhabditis elegans. We used these genomes to establish phylogenetic relationships, explore gene conservation across species, identify genes uniquely expanded in insect parasites, and to identify conserved non-coding regulatory motifs that influence similar biological processes. Protein domain analysis of these genomes reveals a striking expansion of numerous putative parasitism genes including certain protease and protease inhibitor families as well as fatty acid- and retinol-binding proteins. We identify rapid evolution and expansion of the important developmental Hox gene cluster and identify novel conserved non-coding regulatory motifs associated with orthologous genes in Steinernema and Caenorhabditis. The deep conservation of the network of non-coding DNA motifs between these two genera for a subset of orthologous genes involved in neurogenesis and embryonic development suggests that a kernel of protein-DNA relationships is conserved through nematode evolution. We analyzed the gene expression of a total of 24 RNA-seq samples from 3 nematode species( S. carpocapsae, S. feltiae, and C. elegans) for comparative analysis. We collected the RNA at four developmental time points (mixed embryo, L1, infective juvenile/dauer, young adult) for each species in replicates.