Regulation 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 species specific salinity tolerance (Kültz et al., 2013; Root et al., 2021a, 2021b). Microscopy has shown that ionocytes, the main site of transepithelial ion transport in fish gills (Evans et al., 2005), increase in number within 12 hours and the total number of ionocytes remains elevated following transfer of O. mossambicus from FW to SW (Hiroi et al., 2005). Additionally, SW-specific ionocyte subtypes are significantly larger than ionocytes from fish in FW (Kültz et al., 1995). Ionocytes, once called mitochondrial rich cells (MRCs) (Inokuchi et al., 2009), 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 generalized hypersalinity response network 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 (Bœuf & Payan, 2001). ComparingO. mossambicus oxygen consumption rates, which is linked to metabolic rate, fish acclimated to SW consumed less oxygen than in FW, but fish acclimated to hypersaline water at 1.6X SW salinity had higher oxygen consumption than in FW or SW (Iwama et al., 1997). 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 production, 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 the 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 (Tipsmark et al., 2011) and other fish species (Richards et al., 2003) during salinity acclimation. IMPase 1 isoform X1 was the most highly upregulated protein on average across treatments, which is consistent with previous proteomic analyses. InO. mossambicus , myo -inositol is synthesized to counteract increased intracellular electrolyte concentration through a two-step metabolic path from D-glucose by the enzymesmyo -inositol-3-phosphate synthase (MIPS) and IMPase (Gardell et al., 2013). MIPS was also significantly upregulated in all treatments except the extended 75g/kg exposure. Myo -inositol concentration is also regulated in O. niloticus kidney during salinity acclimation, although here the mechanism is to reduce degradation by downregulating myo -inositol oxidase(Root et al., 2021b). Interestingly, no myo -inositol related proteins were significantly regulated by salinity in gills of O. niloticus,which has an upper salinity tolerance limit near 25 g/kg (Root et al., 2021a).
A novel cluster is found in the lower portion of the generalized salinity response network connected to the TCA cycle and ETC clusters 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 (Laskar & Younus, 2019). Increased oxidative phosphorylation and other metabolic processes create harmful molecules such as reactive oxygen species (ROS) and carbonyl compounds (Bazil et al., 2016). Lipid peroxidation is one result of oxidative stress causing turnover in lipid membranes and the formation of toxic fatty aldehydes. ALDH plays a large role in converting these fatty aldehydes into fatty acids (Zeng et al., 2021), and was also highly upregulated in O. niloticus kidney indicating that this response is conserved across species and tissues (Root et al., 2021b). Upregulation of acetyl-CoA acyltransferase has also been observed in other organisms exposed to toxic compounds such as in mice exposed to perflourooctane sulfonate (Rosen et al., 2010), diphenylarsinic acid (Yamaguchi et al., 2019), and in bacterial communities exposed to hydrocarbon spills in nature (Edet & Antai, 2018).