Untargeted mass spectrometry to investigate ocean acidification in Cancer borealis using optimized metabolite extraction methods

Ocean acidification (OA) refers to the ongoing decline in ocean pH caused by the cascading effects of increased atmospheric CO2, which has significant negative impacts on various marine organisms, particularly crustaceans with calcified shells. However, research on the metabolic responses of crustaceans remains limited. In this study, we performed untargeted metabolomics on hemolymph samples from Cancer borealis (Jonah crab), a crustacean species well known for its tolerance to temperature and pH changes, to investigate its metabolic responses to OA. Two extraction methods-isopropanol (IPA) and acidified methanol (AcMeOH)-were employed to capture a broad range of metabolites and small peptides. Both methods enabled comprehensive detection; however, IPA extraction yielded more consistent and extensive metabolite coverage, identifying 43 lipids compared to only 15 with AcMeOH. We identified 15 metabolites that responded significantly to OA. Several metabolites, including the potential neuropeptide cycloprolylglycine and the exogenous compound curcumin, exhibited concentration changes under OA exposure, suggesting their potential relevance in stress response pathways triggered by environmental stress. Overall, we highlight IPA as a more effective extraction method for untargeted metabolomics of crustacean hemolymph. Our study elucidates metabolic dynamics that enhance our understanding of the physiological adaptability of marine crustaceans under environmental stress and provides a comprehensive dataset for future OA research.