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

Activated entomopathogenic nematode infective juveniles release lethal venom proteins

<div><p>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 <i>in vitro</i> activated infective juveniles (IJs) of <i>Steinernema carpocapsae</i> (a well-studied EPN species) release venom proteins that are lethal to several insects including <i>Drosophila melanogaster</i>. We confirmed that the <i>in vitro</i> activation is a good approximation of the <i>in vivo</i> process by comparing the transcriptomes of individual <i>in vitro</i> and <i>in vivo</i> 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 <i>S</i>. <i>carpocapsae</i> and likely other <i>Steinernema</i> 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.</p></div

Activated <i>S</i>. <i>carpocapsae</i> ESPs contain lethal proteins.

<p><b>(A)</b><i>In vitro</i> activation rates of IJs over a time course. Error bars represent standard errors. <b>(B)</b> A silver-stained protein gel of ESPs from nematodes that have been activated for different lengths of time, and ESPs from axenic nematodes. The left-hand side of the gel shows the secreted proteins from symbiont-associated IJs activated for different amounts of time. The right-hand side of the gel shows the proteins secreted from symbiont-associated (S) and axenic (A) IJs that were exposed to waxworm homogenate for 12 h. Each lane contains 1% of the total ESP volume. The 0h sample was collected from non-activated IJs. L, protein ladder; S, symbiotic IJs; A, axenic IJs. <b>(C)</b> Survival curves within 72 hrs of <i>Drosophila</i> injected with 20 ng of ESPs from nematodes having been activated for different lengths of time. The 30 hr curve (blue) mostly overlaps with the 6 hr curve (red). <b>(D)</b> Survival curves over 40 days of <i>Drosophila</i> injected with 20 ng of ESPs from nematodes having been activated for different lengths of time. <b>(E)</b> The average amount of ESPs secreted by individual IJs in 3 hours. <b>(F)</b> Survival curves of 2^nd instar <i>Bombyx mori</i> larvae injected with 650 ng of ESPs from axenic nematodes that were activated for 12 h. <b>(G)</b> Phenotype curves of last instar <i>Galleria mellonella</i> larvae injected with 4 μg of ESPs from axenic nematodes that were activated for 12 h. The dotted line shows the number of larvae that were either killed or paralyzed after the injection. Paralyzed waxworms recovered over time.</p

The gene expression profile of the 12 hour <i>in vitro</i> activated nematodes is most similar to that of 15 hour <i>in vivo</i> activated nematodes.

<p><b>(A)</b> Scatterplot comparing the average gene expression counts of non-activated IJs and 12 hr <i>in vitro</i> activated IJs. Genes colored in orange and blue are differentially expressed (FDR < 0.05 and fold change > 2) and have higher expression in non-activated IJs and 12 hr <i>in vitro</i> activated IJs respectively. <b>(B)</b> Scatterplot comparing the average gene expression counts between 12 hr <i>in vitro</i> and 15 hr <i>in vivo</i> activated IJs. Genes colored in orange and blue are differentially expressed (FDR < 0.05 and fold change > 2) and have higher expression in 12 hr <i>in vitro</i> and 15 hr <i>in vivo</i> activated IJs respectively. <b>(C)</b> Venn diagrams showing genes that are upregulated and downregulated in the 12 hr <i>in vitro</i> and 15 hr <i>in vivo</i> activated IJs relative to non-activated IJs. The total number of upregulated and downregulated genes and their genome percentages are shown in the upward and downward block arrows, respectively. Representative GO terms and p-values for each category are listed on the right. Categories that do not have GO terms had no significant enrichment results.</p

A large proportion of some protein families are secreted venom proteins.

<p>A large proportion of some protein families are secreted venom proteins.</p

Gene expression dynamics during <i>in vivo</i> IJ activation.

<p><b>(A)</b> Gene expression correlation matrix comparing the Spearman’s rank correlation coefficients between activated and non-activated IJs. Yellow colored cells indicate high correlations while blue cells indicate low correlations. Gene expression was transformed (log₂(counts+1)) prior to calculating the correlation coefficients. Samples were K-means clustered by their Spearman’s rank correlation coefficients. <b>(B)</b> Activated and non-activated IJ transcriptomes plotted in the space of the first two principal components (which comprises 46% of the variation). A short description of what each principal component (PC) explains is included next to the PCs. The green line show the trajectory of IJ activation. <b>(C)</b> Scatterplots comparing the expression of all genes between non-activated IJs and 9 hr, 12 hr, 15 hr <i>in vivo</i> activated IJs. Genes labeled in orange and blue are differentially expressed (FDR < 0.05 and fold change > 2) between the stages compared, while grey genes are not. Expression for each gene was averaged across the biological replicates. <b>(D)</b> maSigPro plots showing the average expression of clusters of genes that are differentially expressed over the <i>in vivo</i> activation time course. The GO terms are representative of these expression clusters.</p

Expression of venom protein genes during IJ activation.

<p><b>(A)</b> A heatmap showing the mean-centered gene expression counts of 472 venom protein genes across activated and non-activated IJs. Protein products of these genes were detected with mass spectrometry in the venom from 12 hr <i>in vitro</i> activated IJs. DE, differentially expressed. The green and red colored rows in the DE column represent the upregulated and downregulated genes in activated IJs, which are used in (B). <b>(B)</b> Venn diagrams showing the venom protein genes that are transcriptionally upregulated or downregulated in activated IJs relative to non-activated IJs, and their breakdown by activation method (12 hr <i>in vitro</i> and 15 hr <i>in vivo</i>). <b>(C)</b> maSigPro plots showing venom protein genes that are differentially expressed over the 15 hr <i>in vivo</i> activation time course.</p