Fluctuating salinity is symptomatic of climate change challenging aquatic species. The melting of polar ice, rising sea levels, coastal surface and groundwater salinization, and increased evaporation in arid habitats alter salinity world-wide. Moreover, the frequency and intensity of extreme weather events such as rainstorms and floods increase, causing rapid shifts in brackish and coastal habitat salinity. Such salinity alterations disrupt homeostasis, and ultimately diminish fitness, of aquatic organisms by interfering with metabolism, reproduction, immunity, and other critical aspects of physiology. Proteins are central for these physiological mechanisms. They represent the molecular building blocks of phenotypes that govern organismal responses to environmental challenges. Environmental cues regulate proteins in concerted fashion, necessitating holistic analyses of proteomes for comprehending salinity stress responses. Proteomics approaches reveal molecular causes of population declines and enable holistic bioindication geared towards timely interventions to prevent local extinctions. Proteomics analyses of salinity effects on aquatic organisms have been performed since the mid-1990s, propelled by the invention of two-dimensional protein gels, soft ionization techniques for mass spectrometry, and nano-liquid chromatography in the 1970s and 1980s. This review summarizes the current knowledge on salinity regulation of proteomes from aquatic organisms, including key methodological advances over the past decades.

Botryllus schlosseri, is a model marine invertebrate for studying immunity, regeneration, and stress-induced evolution. Conditions for validating its predicted proteome were optimized using nanoElute® 2 deep-coverage LCMS, revealing up to 4,930 protein groups and 20,984 unique peptides per sample. Spectral libraries were generated and filtered to remove interferences, low-quality transitions, and only retain proteins with >3 unique peptides. The resulting DIA assay library enabled label-free quantitation of 3,426 protein groups represented by 22,593 unique peptides. Quantitative comparisons of a laboratory-raised with two field-collected populations revealed (1) a more unique proteome in the laboratory-raised population, and (2) proteins with high/low individual variabilities in each population. DNA repair/replication, ion transport, and intracellular signaling processes were unique in laboratory-cultured colonies. Spliceosome and Wnt signaling proteins were the least variable (highly functionally constrained) in all populations. In conclusion, we present the first colonial tunicate’s deep quantitative proteome analysis, identifying functional protein clusters associated with laboratory conditions, different habitats, and strong versus relaxed abundance constraints. These results empower research on B. schlosseri with proteomics resources and enable quantitative molecular phenotyping of changes associated with transfer from in situ to ex situ and from in vivo to in vitro culture conditions.

Acclimations of Oreochromis mossambicus to hypersalinity 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 hypersalinity. 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. O. mossambicus was determined to have a wide “zone of resistance” from approximately 75g/kg salinity to 120g/kg, which is tolerated for a limited period with eventual loss of organismal function. Crossing the critical threshold salinity into the zone of resistance corresponds with blood osmolality increasing beyond 400 mOsm/kg, significantly reduced body condition factor, and cessation of feeding. Gill protein networks impacted by hypersalinity 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 networks 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.

All organisms encounter environmental changes that lead to physiological adjustments and drive evolutionary adaptations. These, in turn, induce behavioral, physiological and molecular changes that affect each other. Deciphering the role of molecular adjustments in physiological changes will help to understand how multiple levels of biological organization are synchronized during adaptations. Transmembrane transporters are prime targets for molecular studies of environmental effects, as they facilitate the ability of cells to interact with the external surrounding. Fish are subjected to fluctuations of environmental factors of their aquatic surrounding and exhibit different coping mechanisms. To study the molecular adjustments of fish proteins to their unique external surrounding, suitable experimental systems must be established. Mozambique tilapia (Oreochromis mossambicus) is an excellent model for environmental stress studies due to its extreme osmotolerance. We established a homologues cellular-based expression system, and an uptake assay, that allowed us to study effects of environmental conditions on transmembrane transport. We applied it to study the effects of environmental conditions on the activity of PepT2, a widely studied transporter due to its importance in absorption of dietary peptides and drugs. We created a stable, modified fish cell-line, exogenously expressing the tilapia PepT2 and tested the effects of temperature and water salinity on the uptake of fluorescent di-peptide, β-Ala-Lys-AMCA. While temperature affected the Vmax of the transport, salinity affected both the Vmax and the Km. These assays demonstrate the importance of suitable experimental systems for fish ecophysiology studies. The presented tools and methods can be adapted to study other transporters in-vitro.

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.