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).