Apex Predator Nematodes and Meso-Predator Bacteria Consume Their Basal Insect Prey through Discrete Stages of Chemical Transformations

The processes by which organic life is consumed and reborn in a complex ecosystem were investigated through a multiomics approach applied to the tripartite Xenorhabdus bacterium-Steinernema nematode-Galleria insect symbiosis. Trophic analyses demonstrate the primary consumers of the insect are the bacteria, and the nematode in turn consumes the bacteria. ABSTRACT Microbial symbiosis drives physiological processes of higher-order systems, including the acquisition and consumption of nutrients that support symbiotic partner reproduction. Metabolic analytics provide new avenues to examine how chemical ecology, or the conversion of existing biomass to new forms, changes over a symbiotic life cycle. We applied these approaches to the nematode Steinernema carpocapsae, its mutualist bacterium, Xenorhabdus nematophila, and the insects they infect. The nematode-bacterium pair infects, kills, and reproduces in an insect until nutrients are depleted. To understand the conversion of insect biomass over time into either nematode or bacterium biomass, we integrated information from trophic, metabolomic, and gene regulation analyses. Trophic analysis established bacteria as meso-predators and primary insect consumers. Nematodes hold a trophic position of 4.6, indicative of an apex predator, consuming bacteria and likely other nematodes. Metabolic changes associated with Galleria mellonella insect bioconversion were assessed using multivariate statistical analyses of metabolomics data sets derived from sampling over an infection time course. Statistically significant, discrete phases were detected, indicating the insect chemical environment changes reproducibly during bioconversion. A novel hierarchical clustering method was designed to probe molecular abundance fluctuation patterns over time, revealing distinct metabolite clusters that exhibit similar abundance shifts across the time course. Composite data suggest bacterial tryptophan and nematode kynurenine pathways are coordinated for reciprocal exchange of tryptophan and NAD+ and for synthesis of intermediates that can have complex effects on bacterial phenotypes and nematode behaviors. Our analysis of pathways and metabolites reveals the chemistry underlying the recycling of organic material during carnivory. IMPORTANCE The processes by which organic life is consumed and reborn in a complex ecosystem were investigated through a multiomics approach applied to the tripartite Xenorhabdus bacterium-Steinernema nematode-Galleria insect symbiosis. Trophic analyses demonstrate the primary consumers of the insect are the bacteria, and the nematode in turn consumes the bacteria. This suggests the Steinernema-Xenorhabdus mutualism is a form of agriculture in which the nematode cultivates the bacterial food sources by inoculating them into insect hosts. Metabolomics analysis revealed a shift in biological material throughout progression of the life cycle: active infection, insect death, and conversion of cadaver tissues into bacterial biomass and nematode tissue. We show that each phase of the life cycle is metabolically distinct, with significant differences including those in the tricarboxylic acid cycle and amino acid pathways. Our findings demonstrate that symbiotic life cycles can be defined by reproducible stage-specific chemical signatures, enhancing our broad understanding of metabolic processes that underpin a three-way symbiosis.