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Entomopathogenic nematodes, includingHeterorhabditisandSteinernema, are parasitic to insects and contain mutualistically symbiotic bacteria in their intestines (PhotorhabdusandXenorhabdus,respectively) and therefore offer opportunities to study both mutualistic and parasitic symbiosis. The establishment of genetic tools in entomopathogenic nematodes has been impeded by limited genetic tractability, inconsistent growthin vitro, variable cryopreservation, and low mating efficiency. We obtained the recently describedSteinernema hermaphroditumstrain CS34 and optimized itsin vitrogrowth, with a rapid generation time on a lawn of its native symbiotic bacteriaXenorhabdus griffiniae. We developed a simple and efficient cryopreservation method. Previously,S. hermaphroditumisolated from insect hosts was described as first-generation hermaphroditic and second-generation gonochoristic. We discovered that CS34, when grownin vitro,produced consecutive generations of autonomously reproducing hermaphrodites accompanied by rare males. We performed mutagenesis screens inS. hermaphroditumthat produced mutant lines with visible and heritable phenotypes. Genetic analysis of the mutants demonstrated that this species reproduces by self-fertilization rather than parthenogenesis and that its sex is determined chromosomally. Genetic mapping has thus far identified markers on the X chromosome and three of four autosomes. We report thatS. hermaphroditumCS34 is the first consistently hermaphroditic entomopathogenic nematode and is suitable for genetic model development to study naturally occurring mutualistic symbiosis and insect parasitism.

Microbial symbiotic interactions, mediated by small molecule signaling, drive physiological processes of higher order systems. Metabolic analytic technologies advancements provide new avenues to examine how chemical ecology, or conversion of existing biomass to new forms, changes over a symbiotic lifecycle. We examine such processes using the tripartite relationship between nematode host Steinernema carpocapsae, its obligate mutualist bacterium, Xenorhabdus nematophila, and the insects they infect together. We integrate trophic, metabolomics, and gene regulation analyses to understand insect biomass conversion to nematode or bacterium biomass. Trophic analysis established bacteria as the primary insect consumers, with nematodes at trophic position 4.37, indicating consumption of bacteria and likely other nematodes. Significant, discrete metabolic phases were distinguishable from each other, indicating the insect chemical environment changes reproducibly during bioconversion. Tricarboxylic acid cycle components and amino acids were significantly affected throughout infection. These findings contribute to an ongoing understanding of how symbiont associations shape chemical environments.TeaserEntomopathogenic nematodes act as an apex predator in some ecosystems through altering chemical environments of their prey.