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.