Acclimations of Oreochromis mossambicus to elevated salinity were conducted with multiple rates of salinity increase and durations of exposure to determine the rate-independent maximum salinity limit and the incipient lethal salinity. Quantitative proteomics of over 3000 gill proteins simultaneously was performed to analyze molecular phenotypes associated with treatments representative of key zones in the salinity-level x duration matrix. For this purpose, a species- and tissue-specific data-independent acquisition (DIA) assay library of MSMS spectra was created. From these DIA data, protein networks representing complex molecular phenotypes associated with salinity acclimation were generated. Organismal performance indicators of salinity tolerance were then correlated with salinity-regulated protein networks. O. mossambicus was determined to have a wide “zone of resistance” from approximately 75g/kg salinity to 120g/kg, which fish survive for a limited period with eventual loss of function. Crossing the critical threshold salinity into the zone of resistance corresponds with blood osmolality increasing beyond 400 mOsm, significantly reduced body condition factor, and cessation of feeding. Gill protein networks impacted at extreme salinity levels both above and below the critical salinity threshold include increased energy metabolism, especially upregulation of electron transport chain proteins, and regulation of specific osmoregulatory proteins. Cytoskeletal, cell adhesion, and extracellular matrix proteins are enriched in regulation network patterns that are sensitive to the critical salinity threshold. Network analysis of these patterns provides deep insight into specific mechanisms of energy homeostasis during salinity stress.

Interactions of organisms with their environment are complex and environmental regulation at different levels of biological organization is often non-linear. Therefore, the genotype to phenotype continuum requires study at multiple levels of organization. While studies of transcriptome regulation are now common for many species, quantitative studies of environmental effects on proteomes are needed. Here we report the generation of a data-independent acquisition (DIA) assay library that enables simultaneous targeted proteomics of thousands of Oreochromis niloticus kidney proteins using a label- and gel-free workflow that is well suited for ecologically relevant field samples. We demonstrate the usefulness of this DIA assay library by discerning environmental effects on the kidney proteome of O. niloticus. Moreover, we demonstrate that the DIA assay library approach generates data that are complimentary rather than redundant to transcriptomics data. Transcript and protein abundance differences in kidneys of tilapia acclimated to freshwater and brackish water (25 g/kg) were correlated for 2114 unique genes. A high degree of non-linearity in salinity-dependent regulation of transcriptomes and proteomes was revealed suggesting that the regulation of O. niloticus renal function by environmental salinity relies heavily on post-transcriptional mechanisms. The application of functional enrichment analyses using STRING and KEGG to DIA assay datasets is demonstrated by identifying myo-inositol metabolism, antioxidant and xenobiotic functions, and signaling mechanisms as key elements controlled by salinity in tilapia kidneys. The DIA assay library resource presented here can be adopted for other tissues and other organisms to study proteome dynamics during changing ecological contexts.