ABSTRACT
Microbial symbiotic interactions, mediated in part by small molecule signaling, drive physiological processes of higher order systems, including the acquisition and consumption of nutrients that support symbiotic partner reproduction. Advances in metabolic analytic technologies provide new avenues to examine how chemical ecology, or the conversion of existing biomass to new forms, changes over a symbiotic lifecycle. Here we examine such processes using the tripartite relationship involving the nematode host Steinernema carpocapsae, its obligate mutualist bacterium, Xenorhabdus nematophila, and the insects they infect together. The nematode infective juveniles infect insects into which they release bacteria that help suppress insect immunity and kill the insect. The nematode-bacterium pair consume the insect cadaver and reproduce until nutrients are depleted, causing a new generation of infective juvenile nematodes, colonized by the bacterial symbiont, to leave the cadaver in search of insect prey. To begin to understand the processes by which insect biomass is converted over time to either nematode or bacterium biomass, we took a three-pronged approach integrating information from trophic, metabolomics, and gene regulation analyses. Trophic analysis established bacteria as the primary insect consumers, with nematodes at a trophic position of 4.37, indicating consumption of bacteria and likely also other nematodes. Metabolic changes associated with bioconversion of Galleria mellonella insects were assessed using multivariate statistical analyses of metabolomics datasets derived from sampling over an infection time course. Statistically significant, discrete phases were distinguishable from each other, indicating the insect chemical environment changes reproducibly during bioconversion. Tricarboxylic acid (TCA) cycle components and amino acids such as proline and leucine were significantly affected throughout the infection. Hierarchical clustering revealed a similar molecular abundance fluctuation pattern for nucleic acid, amino acid, and lipid biosynthesis metabolites. Together, these findings contribute to an ongoing understanding of how symbiont associations shape chemical environments.
IMPORTANCE The biological world depends on microbial processes, including symbiotic exchanges among mutualists and antagonists. Technical advances have enabled investigation of microbial trophic positions within communities and the identification of metabolites exchanged between microbes and their hosts. These tools were applied to the tripartite Xenorhabdus bacteria-Steinernema nematode-Galleria insect symbiosis. Trophic analyses demonstrate the primary consumer of the insect are the bacteria, and in turn the nematode consumes the bacteria. This suggests the Steinernema-Xenorhabdus mutualism is a form of agriculture in which the nematode cultivates their bacterial food source by inoculating them into insect hosts. Metabolomics analysis revealed a shift in biological material throughout progression of the lifecycle: active infection, insect death, and conversion of cadaver tissues into bacterial biomass and nematode tissue. We show that each phase of the lifecycle is metabolically distinct, with significant differences in tricarboxylic acid cycle and amino acid metabolism. Our findings demonstrate that symbiotic lifecycles can be defined by reproducible stage-specific chemical signatures, enhancing our broad understanding of metabolic processes that underpin a three-way symbiosis.
Competing Interest Statement
The authors have declared no competing interest.
