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Parasitic helminths release molecular effectors into their hosts and these effectors can directly damage host tissue and modulate host immunity. Excreted/secreted proteins (ESPs) are one category of parasite molecular effectors that are critical to their success within the host. However, most studies of nematode ESPs rely on  in vitro  stimulation or culture conditions to collect the ESPs, operating under the assumption that  in vitro  conditions mimic actual  in vivo  infection. This assumption is rarely if ever validated. Entomopathogenic nematodes (EPNs) are lethal parasites of insects that produce and release toxins into their insect hosts and are a powerful model parasite system. We compared transcriptional profiles of individual  Steinernema feltiae  nematodes at different time points of activation under  in vitro  and  in vivo  conditions and found that some but not all time points during  in vitro  parasite activation have similar transcriptional profiles with nematodes from  in vivo  infections. These findings highlight the importance of experimental validation of ESP collection conditions. Additionally, we found that a suite of genes in the neuropeptide pathway were downregulated as nematodes activated and infection progressed  in vivo , suggesting that these genes are involved in host-seeking behavior and are less important during active infection. We then characterized the ESPs of activated  S .  feltiae  infective juveniles (IJs) using mass spectrometry and identified 266 proteins that are released by these nematodes. In comparing these ESPs with those previously identified in activated  S .  carpocapsae  IJs, we identified a core set of 52 proteins that are conserved and present in the ESPs of activated IJs of both species. These core venom proteins include both tissue-damaging and immune-modulating proteins, suggesting that the ESPs of these parasites include both a core set of effectors as well as a specialized set, more adapted to the particular hosts they infect.

A core set of venom proteins is released by entomopathogenic nematodes in the genus Steinernema