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).