Characterization of Heterorhabditis bacteriophora response to insect-derived and non-biological stimuli: Insights into nematode recovery and released proteins

Entomopathogenic nematodes (EPNs) are biological control agents that naturally kill insect pests, providing an eco-friendly alternative to chemical pesticides. Despite extensive research, the mechanisms behind the recovery process, where infective juveniles (IJs) transition to a parasitic state upon contact with the host, remain unclear. This study investigates the stimulatory effect of insect-derived materials on the recovery of Heterorhabditis bacteriophora IJs. Three materials from Galleria mellonella larvae-bioactive homogenates from live and frozen larvae, and heat-inactivated homogenate-were tested, along with non-host stimuli including filtered water and phosphate-buffered saline (PBS). While none of the materials induced complete recovery of IJs, all triggered the release of excreted/secreted products (ESPs), with consistent protein concentrations across treatments. However, mass spectrometry revealed significant differences in ESP protein composition. IJs exposed to PBS released the highest number of proteins, while bioactive homogenates induced the fewest. Proteins linked to host-parasite interactions, such as alpha-2-macroglobulins and trypsin inhibitor-like proteins, were more abundant in ESPs following exposure to insect-derived materials and PBS. Interestingly, nematodes exposed to water released a substantial number of proteins, comparable to stimulation by heat-inactivated homogenates, though their protein profiles were distinct, reflecting stress responses in the former and host-parasite interaction-related proteins in the latter. Our findings demonstrate that both host-derived and non-biological stimuli can trigger IJs recovery and ESPs release, underscoring the complexity of host-nematode interactions. These results provide novel insights into molecular mechanisms underlying H. bacteriophora parasitism and may contribute to optimizing biocontrol strategies through a better understanding of nematode activation and released ESPs.