Discovery of a new highly pathogenic toxin involved in insect sepsis.

Insect sepsis is a severe consequence that arises from the invasion of the hemocoel by symbionts of entomopathogenic nematodes and bacteria. In the present study, we unveiled the heightened virulence of the entomopathogenic nematode and the entomopathogenic bacteria , which operate symbiotically, against the wax moth . Maximum mortality was observed at 25°C while the optimal infestation efficiency was 20 nematodes per host. After infestation, displayed rapid darkening and softening, accompanied by an escalated esterase activity at 9 h. The , released by , underwent substantial proliferation and discharged toxins that attacked hemocytes, thus triggering extensive hemolysis and sepsis. The host was usually killed within 24 h due to disseminated septicemia. Additionally, infestation led to the upregulation of metabolites like 3-hydroxyanthranilic acid. Strikingly, we identified the perilous actinomycin D, generated through kynurenine metabolites, representing a novel biomarker of insect sepsis. Furthermore, a comprehensive transcriptomic analysis unveiled a noteworthy upregulation of gene expression associated with actinomycin D. Overall, induced apoptosis and sepsis through actinomycin D production, indicating its pivotal role in infestation activity. These findings open up new avenues for studying the mechanism of sepsis and developing innovative biotic pesticides. IMPORTANCE As a current biocontrol resource, entomopathogenic nematodes and their symbiotic bacterium can produce many toxin factors to trigger insect sepsis, having the potential to promote sustainable pest management. In this study, we found and were highly virulent against the insects. After infective juvenile injection, quickly turned black and softened with increasing esterase activity. Simultaneously, attacked hemocytes and released toxic components, resulting in extensive hemolysis and sepsis. Then, we applied high-resolution mass spectrometry-based metabolomics and found multiple substances were upregulated in the host hemolymph. We found extremely hazardous actinomycin D produced via 3-hydroxyanthranilic acid metabolites. Moreover, a combined transcriptomic analysis revealed that gene expression of proteins associated with actinomycin D was upregulated. Our research revealed actinomycin D might be responsible for the infestation activity of , indicating a new direction for exploring the sepsis mechanism and developing novel biotic pesticides.