Conclusions
This study presents a novel experimental design for assessment of stress combined with an innovative approach for systems-level analyses of molecular phenotypes (DIA-LCMS2). Specific organismal phenotypes (blood osmolality, body condition, feeding rate) were found to indicate extreme salinity stress in O. mossambicus . The rate-independent salinity maximum and critical threshold salinity were determined using a comprehensive set of acclimations and a generalized protocol that is applicable to other species. The DIA-LCMS2 approach was shown to reveal comprehensive molecular phenotypes in ecologically relevant samples. The data generated in this study were used to produce protein networks which elucidate major mechanisms of ionic and mitochondrial protein regulation in response to hypersalinity in general and with respect to the critical threshold. These networks help explain how fish modulate energy metabolism, cell adhesion, and cell structure when experiencing salinity stress below and above the critical limit.