Applying Tissue Separation and Untargeted Metabolomics to Understanding Lipid Saturation Kinetics of Host Mitochondria and Symbiotic Algae in Corals Under High Temperature Stress

Untargeted metabolomics is a powerful tool for profiling the biochemical phenotypes of organisms and discovering new metabolites that drive biological function and might be exploited as pharmaceutical leads. Yet, connecting physiological processes to metabolites detected remains a challenge due to the lack of structural and activity annotations and the underlying complexity of mixed samples (e.g., multiple microorganisms, organelles, etc.). To simplify this biological complexity, we separated coral holobionts into host mitochondria and their algal symbionts prior to LC-MS/MS-based untargeted metabolomic analysis followed by molecular networking. We found distinct metabolomic profiles between tissue fractions. Notably, 14% of metabolites detected were only observed in the mitochondria and algal symbionts, not in the holobiont, and thus were masked when the bulk (holobiont) sample was analyzed. The utility of tissue separation for hypothesis testing was assessed using a simple temperature experiment. We tested the hypothesis that membrane lipids of the coral mitochondria and algal symbionts become more saturated at higher temperatures to maintain membrane rigidity. While the holobiont metabolite profiles showed little change in response to elevated temperature, there was a change in lipid saturation of both fractions through time. The fatty acid saturation of both the coral mitochondria and the algal symbionts shifted upon exposure to higher temperatures (1 h) then returned to ambient saturation levels by 4 h, indicating rapid acclimatization to warmer water. Surprisingly, the fractions deviated in opposite directions: during the first hour of the experiment, the mitochondria showed an increase in saturated lipid concentrations, while the algal symbionts showed an increase in unsaturated lipids. Partitioning the holobiont prior to untargeted metabolomic analysis revealed disparate responses to environmental stress that would have gone undetected if only the holobiont/bulk tissue was analyzed. This work illustrates rapid physiological acclimatization to environmental changes in specific host organelles and symbionts, though via different paths.