Combined metabolomics and histological analysis of the tissues from cuttlefish Sepia pharaonis exposed to inking stress

Inking is part of a defensive stress response in cephalopods (cuttlefish, squid, and octopus). Some individual cuttlefish (Sepia pharaonis) die after continued stress and inking; however, the physiological effects of cephalopods in response to stress and inking remain unknown. The present study investigated the metabolic profile and discussed the physiological roles of S. pharaonis tissues in response to continuous inking using the 1H NMR spectroscopy coupled with multivariate data analysis. A total of 50 metabolites, including amino acids, organic osmolytes, nucleotides, energy storage compounds, and obvious tissue-specific metabolites induced by inking stress, were identified in S. pharaonis tissues. Exposure to inking stress had different effects on the levels of the studied metabolites, for example, the levels of isoleucine, trimethylamine-N-oxide, and betaine increased, but those of arginine and ATP decreased in the liver; inosine and lactate were accumulated whereas glutamate and choline were depleted in the gill; the levels of lactate and isoleucine were elevated but those of arginine and glycogen were depleted in the muscle tissue. Furthermore, the corresponding metabolic pathways of the characteristic metabolites indicated major changes in the functions of these metabolites. Histological changes in the studied tissues revealed liver lobule damage immediately after inking, with the presence of disordered epithelial cells and partial cell necrosis in the gill. Our results demonstrated that a combination of metabolomics and histological analyses could provide molecular-level insights for elucidating the defense response of cuttlefish against predators.