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].