Regulation of protein networks in hypersalinity
The largest and most highly connected cluster in the network of significantly regulated proteins in all treatments included mitochondrial proteins involved in the ETC. Multiple previous proteomic studies with Oreochromis species have shown the widespread upregulation of mitochondrial proteins during acclimation to higher salinity levels, especially as these levels approach the upper range of salinity tolerance [29], [38], [39]. Microscopy has shown that ionocytes, the main site of transepithelial ion transport in fish gills [40], increase in number within 12 hours and remain elevated following transfer of O. mossambicus from FW to SW [41]. Additionally, SW-specific ionocyte subtypes are significantly larger than ionocytes from fish in FW [42]. Ionocytes, once called mitochondrial rich cells (MRCs) [43], are characterized by high concentrations of mitochondria, which is reflected in protein abundance patterns from this and previous studies.
In addition to ETC proteins, the hypersalinity response network combining all treatments has a strong representation of glycolysis and the TCA cycle proteins, emphasizing the importance of increased energy production in response to hypersaline conditions in initial and long-term stages of exposure. These proteomic responses combined with decreasing body condition/growth indicate that one of the dominant adaptive mechanisms of O. mossambicus to hypersaline conditions is to increase energy production and allocation to meet increased osmoregulatory energy requirements. Osmoregulation can account for 20-50% of basal metabolic cost across a range of taxa in fish [44]. Comparing O. mossambicus oxygen consumption rates, fish acclimated to hypersaline water at 1.6X SW salinity had higher oxygen consumption than in FW or SW [6]. Evidence is scant for salinity levels as high as those used in this study, but it is reasonable to suggest that increasing hypersalinity requires greater energy devoted to osmoregulation, especially given that much of the active ion transport is ATP-dependent.
Proteins directly related to ion regulation in the network of significant proteins in all treatments, specifically ion transporters (Na+/K+ ATPase, NH4+ transporter) and compatible osmolyte synthesis enzymes (IMPase, sorbitol dehydrogenase), are present but are peripheral in the network map and do not contain many members. Small numbers of significant proteins combined with a high degree of regulation (many are among the most highly regulated proteins), indicate that ion balance is controlled through highly targeted regulation of specific proteins and subunits. This contrasts with the regulation of energy production, which is comprehensive and involves a large network cluster. Targeted regulation of ion transport includes isoform switching in Na+/K+ATPase subunits, as the α-1 isoform X1 increased by an average of 10-fold greater in all treatments while α-1 isoform X4 decreased by 20-fold on average. Isoform switching in Na+/K+ ATPase subunit α has been documented in O. mossambicus [45] and other fish species [46] during salinity acclimation. In O. mossambicus ,myo -inositol is synthesized to counteract increased intracellular electrolyte concentration through a two-step metabolic path from D-glucose by the enzymes myo -inositol-3-phosphate synthase (MIPS) and IMPase [47], and as stated IMPase 1 isoform X1 was the most highly upregulated protein on average across treatments. MIPS was also significantly upregulated in all treatments except the extended 75g/kg exposure. Myo -inositol concentration is also regulated inO. niloticus kidney during salinity acclimation, although here the mechanism is to reduce degradation by downregulatingmyo -inositol oxidase[39]. Interestingly, nomyo -inositol related proteins were significantly regulated by salinity in gills of O. niloticus, which has an upper salinity tolerance limit near 25 g/kg [29].
The hypersalinity response network also includes a cluster (Figure 4, inset 2) which includes proteins involved in fatty acid β-oxidation and detoxification. Acetyl-CoA acyltransferase is involved in producing acetyl-CoA through β-oxidation to be processed in the TCA cycle. Aldehyde dehydrogenase (ALDH) is involved in fatty acid metabolism but also neutralizes carbonyl compounds resulting from lipid peroxidation [48]. Lipid peroxidation is one result of oxidative stress causing turnover in lipid membranes and the formation of toxic fatty aldehydes, which ALDH plays a large role in converting into fatty acids [49]. ALDH was also highly upregulated in O. niloticus kidney indicating that this response is conserved across species and tissues [39]. Upregulation of acetyl-CoA acyltransferase has also been observed in other organisms exposed to toxic compounds such as in mice exposed to perflourooctane sulfonate [50], diphenylarsinic acid [51], and in bacteria exposed to hydrocarbon spills [52].