Direct sampling of organisms from the field and knowledge of their phenotype: key recommendations for environmental metabolomics.

Critical questions must be addressed to evaluate the potential of metabolomics for studying free-living wildlife. First, can metabolomics identify stress-induced phenotypes in animals experiencing a highly variable environment or must animals be stabilized in a controlled laboratory prior to sampling? Second, is knowledge of species and phenotype (gender and age) required to interpret metabolomics data? To address these questions, we characterized the metabolic variability of the mussel and determined if inherent variability masked the metabolic response to an environmental stressor, hypoxia. Specifically, we compared metabolic fingerprints of adductor muscle and mantle from four groups of Mytilus galloprovincialis: animals sampled directly from the field with and without hypoxia and those stabilized in a laboratory for 60 h, also with and without hypoxia. Contrary to expectation, laboratory stabilization increased metabolic variability in adductor muscle, thereby completely masking the response to hypoxia. The principal source of metabolic variability in mantle was shown to be gender-based, highlighting the importance of phenotypic anchoring of samples to known life history traits. We conclude that direct field sampling is recommended for environmental metabolomics since it minimizes metabolic variability and enables stress-induced phenotypic changes to be observed. Furthermore, we recommend that species and phenotype of the study organism must be known for meaningful interpretation of metabolomics data.