Activated entomopathogenic nematode infective juveniles release lethal venom proteins

Abstract

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.

Author summary

Steinernema carpocapsae belongs to a special group of insect-parasitic nematodes known as entomopathogenic nematodes (EPNs). These differ from other insect parasites in at least two ways; first they kill their hosts quickly (within 2–3 days), and second they associate with bacteria to facilitate their parasitic lifestyle. The infective stage of these parasites, the infective juvenile (IJ) stage, is the only free-living stage and these IJs are developmentally arrested and only reinitiate development once they are inside a suitable host. Little is known about the early stages of parasitism and how these parasites initiate the parasitic phase of their life cycle and reinitiate development. Here we characterized the changes that occur to the nematodes' physical morphology, gene expression, and the release of protein molecules that accompany the transition from developmentally arrested IJ to active, developing parasite. We showed that contrary to long-held assumptions, the nematodes are not merely transporting pathogenic bacteria but that the nematodes contribute to parasitism by releasing toxic proteins into the host. Many of the S. carpocapsae toxins are also found in species of human-parasitic nematodes, and S. carpocapsae may serve as a valuable model for understanding the specific function of these toxins.