Introduction:
Abiotic factors are key in determining the extent of suitable habitat
for teleost fish (Somero, G., Lockwood, B., Tomanek, L., 2016). Fish
living in freshwater and brackish systems often have limited migration
options due to geographic constraints. Habitat shifts include
exploration of areas that vary in salinity, e.g., brackish coastal areas
and salt lakes. Euryhaline tilapia species regularly occupy freshwater
environments but these fish also venture into extremely hypersaline
habitats which can exceed 100g/kg in salinity (Panfili et al., 2004;
Whitfield et al., 2006). Adaptations that enable euryhaline fish to
explore such habitats also improve tolerance to other stressors (Elliott
& Quintino, 2007). Holistic systems level approaches can be utilized to
identify molecular and organismal phenotypes to habitat tolerance
adaptations.
Salinity tolerance under relevant ecological conditions is dependent on
the level of salinity of exposure, the rate of salinity increase, the
ionic composition of saline water, and time spent at high salinity.
Acute salinity challenges involving direct transfer are common in
environmental physiology research (Amoudi et al., 1996; Fuadi et al.,
2021; Iwama et al., 1997; Kammerer et al., 2010), but acute salinity
tolerance is often less than the salinity to which a fish can acclimate
over time and may not represent a common salinity challenge fish have
evolved to survive. For example, O. mossambicus cannot survive
direct transfer to seawater (SW), which has a standard salinity of 35
g/kg (Lewis & Perkin, 1978), but can be experimentally acclimated to
several times SW salinity, while O. niloticus has similar
capacity for direct transfer salinity tolerance as O. mossambicusbut cannot acclimate to salinity levels greater than SW (Avella et al.,
1993; Basiao et al., 2005; Ronkin et al., 2015). Researchers have used
survival at different lengths of time from days to weeks as indicatory
of long term survival (Blackburn, 1987; Christensen et al., 2019;
Langston et al., 2010; Schultz & McCormick, 2012; Watanabe et al.,
1985), despite evidence that degeneration of biological function and
mortality can occur in Oreochromis species following extended
periods in hypersaline conditions (Sardella, 2004).
Energy homeostasis theory provides a framework for understanding the
relationship between the intensity of a stress and the duration of
exposure using a three-tiered system of biological function. Within the
“optimum” range of an environmental parameter a basal amount of energy
is required to maintain internal homeostasis (Sokolova et al., 2012). In
the “pejus” range, energy demand increases linearly with the stress to
maintain homeostasis and manage the impacts of stress-related
macromolecular damage, while the “pessimum” range is reached when
energy expenditure and macromolecular damage increase in a non-linear
relationship with the stress until loss of biological function (death).
The boundary between pejus (zone of tolerance) and pessimum (zone of
resistance) ranges is called the “critical threshold” or the incipient
lethal level (Brett, 1956). Salinity levels in the pejus range are
tolerable in the long term but result in reduced reproduction and/or
growth due to reduced free energy, whereas exposure to pessimum range
salinity is temporarily survivable but fish cannot increase energy
expenditure sufficiently to maintain homeostasis, eventually resulting
in death if conditions do not improve (Sokolova et al., 2012). Reaching
the endpoint of biological function, called the Critical Salinity
Maximum (CSMAX), will occur some period of time after
crossing the critical salinity threshold. Energy homeostasis theory was
developed predominantly using thermal stress response (Pörtner, 2010),
in which case the critical threshold is defined physiologically by a
transition to partial anaerobic metabolism. However, it is not clear if
this indicator is applicable to salinity stress because the relationship
between salinity and dissolved oxygen concentration is less direct than
for temperature. Physiological indicators of fish transitioning from
pejus to pessimum salinity ranges are undefined but crucial to assess
the threat of salinity change on natural populations.
Whole organism measurements can describe the physiological state of a
fish, but understanding the mechanisms of acclimation requires
tissue-specific analysis of the interactions of molecular components.
Proteomic analysis is a particularly promising approach for this purpose
because proteins are linked directly to specific genomic loci via their
accession numbers (Keerthikumar & Mathivanan, 2017). Proteins define
the structures and enact the majority of biochemical processes of each
level of biological function (Ebhardt et al., 2015), and are thus the
primary source of phenotypic variability which enables natural selection
(Clarke, 1971; Mularoni et al., 2010). Careful choice of environmental
challenges and time points allow the capture of relevant snapshots of
protein signatures, which provide systems-level insight of organismal
adaptation to maintain biological function (Kültz et al., 2007, 2016).
In the current study, Data-Independent Acquisition Liquid Chromatography
Mass Spectrometry (DIA-LCMS2) was used to capture gill protein
signatures at key points in the salinity-level x duration matrix and
identify biochemical networks that are indicative of adaptation in the
pejus and pessimum ranges. The gill epithelium is particularly important
for supporting organismal salinity tolerance of fish in addition to its
critical role for respiration, acid-base regulation, and nitrogenous
waste excretion (Sardella & Brauner, 2007).