Introduction:
Abiotic factors are key determinants of suitable habitat for teleost
fish [1]. 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 regularly occupy
freshwater environments but these fish also venture into extremely
hypersaline habitats which can exceed 100g/kg in salinity [2],
[3]. Salinity tolerance is dependent not only on the salinity of
exposure, but also the rate of salinity increase, the ionic composition
of saline water, and time and frequency spent at high salinity. Acute
salinity challenges involving direct transfer from freshwater to a
target salinity are common in environmental physiology research
[4]–[7], but acute salinity tolerance is often lower than the
salinity to which a fish can acclimate over time and may not represent
an ecologically relevant salinity challenge. For example, O.
mossambicus cannot survive direct transfer from freshwater to seawater
(SW) [8], but can be experimentally acclimated to several times SW
salinity. Survival for different lengths of time from days to weeks has
been used as indicatory of long term survival [9]–[13], despite
evidence that degeneration of biological function and mortality can
occur in Oreochromis species following longer time periods in
hypersaline conditions [14].
Energy homeostasis theory provides a framework for understanding the
relationship between the intensity of a stress and the duration of
exposure using a tiered system of biological function. Within the
“optimum” range of an environmental parameter, a basal amount of
energy is required to maintain internal homeostasis [15]. In the
“pejus” range, energy demand increases linearly with the intensity of
the stressor to maintain homeostasis and manage the impacts of
stress-related macromolecular damage. The “pessimum” range is reached
when energy expenditure and macromolecular damage increase in a
non-linear relationship with the stress until loss of 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 [16]. Stressor levels in the pejus range are tolerable
for long periods but result in reduced reproduction and/or growth due to
energy reallocation to stress responses. The pessimum range is only
temporarily survivable and will eventually result in death if conditions
do not improve [15]. Biological function will cease after crossing
the critical salinity threshold, i.e. the Critical Salinity Maximum
(CSMAX), if conditions do not improve. Energy
homeostasis theory was developed predominantly in the context of thermal
stress [17], with the critical threshold defined physiologically by
a transition to partial anaerobic metabolism. This indicator may not
apply to salinity stress and other physiological indicators of fish
transitioning from pejus to pessimum salinity ranges are not yet
defined.
Indicators of this transition may be found in whole organism physiology
and/or 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 [18] and they define the structures
and enact the majority of biochemical processes of each level of
biological function [19]. Proteins are thus the primary source of
phenotypic variability enabling natural selection [20], [21].
Careful choice of environmental challenges and time points allow the
capture of protein signatures, which provide systems-level insight of
organismal adaptation to biological function maintenance [22],
[23]. 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. Gills are one of the most important sites of fish
osmoregulation, in addition to their roles forrespiration, acid-base
regulation, and nitrogenous waste excretion [24]. The hypothesis
driving this study is that pejus and pessimum ranges of salinity
tolerance can be identified based on physiological assessments and
corresponding changes in dynamic gill proteome networks.