Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology
Metabolic costs associated with seawater acclimation in a euryhaline teleost, the fourspine stickleback (Apeltes quadracus)
Graphical abstract
Introduction
Environmental salinity impacts physiological processes in organisms, and strongly influences species and life-stage distribution of fishes (Nelson, 1968; Baker, 1971; Audet et al., 1985a; Glover et al., 2012). Highly soluble ions, such as Na+, K+, Cl‐, and Ca2+, affect hydration, blood pH, and function of all cellular processes (Laurent and Perry, 1991; Glitsch, 2001). Teleost fishes have evolved an osmoregulatory strategy in which blood osmotic pressure is maintained at approximately one-third the concentration of seawater. Most teleosts are stenohaline and therefore physiologically restricted to a narrow range of salinity values, either freshwater (FW) or seawater (SW). Euryhaline fishes, accounting for approximately 5 % of all teleosts, tolerate a large range of salinity, often ranging from FW to full-strength SW (Schultz and McCormick, 2013).
Regulating internal ion content requires mechanisms to counteract passive fluxes and maintain water and ion homeostasis within the body. In fishes, the gills, kidneys, and intestines are the three major osmoregulatory tissues, which have different function in FW and SW (McCormick et al., 1989b). Freshwater fishes live in hyposmotic conditions, maintaining blood osmolality above the levels found in the environment (~300 mOsmol kg‐1 versus 0 mOsmol kg‐1, Edwards and Marshall, 2013). To maintain these concentrations, multiple osmoregulatory strategies in FW teleosts are utilized, including increased ion uptake through the gills (Foskett et al., 1983; McCormick, 2001), reabsorption of filtered ions in the kidney, and reduced drinking (Foskett et al., 1983; Wood and Marshall, 1994). In contrast, marine teleosts are in a hyperosmotic environment (~320 mOsmol kg‐1 versus 1100 mOsmol kg‐1, Edwards and Marshall, 2013). Osmoregulation in SW occurs via an increased drinking rate (Foskett et al., 1983; Wood and Marshall, 1994; McCormick, 2001), uptake of ions and water in the intestine, reduced glomerular filtration in the kidneys (Takvam et al., 2021), and an active secretion of ions by the gills (Foskett et al., 1983).
Ionocytes in the gill, also known as “mitochondrion-rich” or “chloride” cells, use a number of transporter and channel proteins, including Na+/K+-ATPase (NKA), to create concentration and electrical gradients that facilitate ion movements in both FW and SW (Wood and Marshall, 1994; Evans et al., 2005). The energy needed to produce the conformational changes in NKA that result in translocation of Na+ and K+ across the cell membrane is made primarily through aerobic metabolism. For most (but not all) species of teleosts examined, the levels of gill NKA and mitochondrial abundance are greater after acclimation in SW compared to FW (Edwards and Marshall, 2013), suggesting that the metabolic costs of the gill may also be greater in SW. Citrate synthase (CS) is the rate-limiting enzyme of the citric acid cycle and has been widely used as an index of respiratory capacity following exposure to SW, with some species showing little to no change (i.e. Atlantic salmon, Salmo salar, McCormick et al., 1989b; mummichog, Fundulus heteroclitus, Marshall et al., 1999), while others have seen significant increases (Mozambique tilapia, Oreochromis mossambicus, Tseng et al., 2008).
It has long been argued that osmoregulation must be metabolically demanding since it requires cellular energy reserves for synthesizing and activating enzymes and transport proteins (Whittam, 1962; McCairns and Bernatchez, 2010, Tseng and Hwang, 2008; Ern et al., 2014). Energy spent on osmoregulation is unavailable for alternative uses, and some investigators have argued that this cost can affect the growth and fitness of teleost fishes (e.g. Wood and Marshall, 1994; Boeuf and Payan, 2001; Sampaio and Bianchini, 2002; Altinok and Grizzle, 2003). Theoretical calculations have estimated the cost of ion transport to be quite low, only 0.5–1.6% of routine metabolic rate (Kirschner, 1993; Ern et al., 2014). In contrast, direct measures of the effect of salinity on metabolic rate are often much higher, ranging from undetectable up to 30% of routine metabolic rate (Boeuf and Payan, 2001; Ern et al., 2014). Ern et al. (2014) have suggested that metabolic costs of osmoregulation are highly species dependent; for most species, metabolic rate is lowest in the habitat in which they normally reside or at isosmotic salinity conditions. For instance, Mozambique tilapia captured in brackish water and acclimated to FW had metabolic rates approximately 2.3-fold greater than fish acclimated to SW (Iwama et al., 1997). Alternatively, measures of metabolic costs of osmoregulation in FW rainbow trout (Oncorhynchus mykiss) account for approximately 20% of the energy budget whereas in SW they are approximately 27%, perhaps reflecting that FW is the predominant habitat for this species (Rao, 1971). Plaut (1999) found that the freshwater blenny (Salaria fluviatilis), a euryhaline species which inhabits FW, had significantly higher metabolic rates in FW compared to SW. Examination of more euryhaline species will help establish whether there are patterns in the relative metabolic rates in FW and SW that can be related to their normal or preferred halohabitats.
Most studies like those cited above examine the costs of osmoregulation in fish that have been fully acclimated to any given salinity. There is reason to suspect, however, that there are added costs associated with the process of acclimation. Branchial ionocyte remodeling occurs after exposure to SW as FW ion uptake mechanisms are lost and SW ion secretory mechanisms are gained (Inokuchi and Kaneko, 2012). It has been estimated that the theoretical energetic cost of new protein synthesis in fish ranges from 50 to 100 mmol ATP · g protein synthesized (Jobling, 1985). When expanded theoretically to oxygen consumption of a whole animal, protein synthesis accounts for approximately 20–40% (Smith and Houlihan, 1995). To date, only a few studies have examined changes in metabolic rate during the SW acclimation process, which includes an adaptive phase that occurs in the first 3–5 days and a chronic regulatory phase that follows where osmoregulatory traits reach homeostasis after 2 weeks (Soengas et al., 2007; McCormick et al., 2022). One study showed that FW-acclimated Mozambique tilapia had 20% greater oxygen consumption in SW compared to FW and isosmotic conditions 4 days after salinity changes (Morgan et al., 1997). Kammerer et al. (2010) found similar results that showed oxygen consumption was significantly greater after 24 h in FW-acclimated tilapia exposed to 25 ppt SW but went down 3 and 5 days after exposure. In contrast, Leray et al. (1981) found that FW-acclimated rainbow trout had a significant decrease in oxygen consumption 6 and 10 days after SW exposure. Finally, Kidder et al. (2006) found no effect of salinity on oxygen consumption in mummichog within the first 24 h of exposure to FW, isosmotic, and SW conditions. To our knowledge these are the only studies that have examined costs of osmoregulation during acclimation from FW to SW conditions. Thus, both the energetic costs of steady-state osmoregulation and the costs of acclimation to changes in salinity are still unclear.
The natural habitats of each species of the family Gasterosteidae are well described, with some species generally confined to marine and brackish (sea stickleback, Spinachia spinachia and blackspotted stickleback, Gasterosteus wheatlandi) or FW environments (brook stickleback, Culaea inconstans), while other species are capable of surviving broad salinity ranges from 0 ppt to 60 ppt (threespine stickleback, Gasterosteus aculeatus; ninespine stickleback, Pungitus pungitius; fourspine stickleback, Apeltes quadracus; Nelson, 1968). However, ionoregulation and osmoregulation have not been thoroughly investigated in most stickleback species other than the threespine stickleback (Grøtan et al., 2012; Divino et al., 2016; Li and Kültz, 2020). This lack of information on basic osmoregulatory physiology of stickleback is somewhat surprising given their use as an evolutionary model for FW invasions (Divino et al., 2016). The fourspine stickleback is a euryhaline species with populations generally in brackish water and SW, with a some resident populations occupying in FW, from Newfoundland to Virginia (Blouw and Hagen, 1984; Nelson, 1968). Fourspine sticklebacks were observed to have a near-isosmotic preference (7 ppt) in a horizontal gradient from 0 ppt to 35 ppt (Audet et al., 1985a). Further investigation showed that cortisol injections stimulated high salinity preference of 28 ppt while prolactin injections stimulated low salinity preference of 14 ppt in fourspine stickleback previously acclimated to 20 ppt (Audet et al., 1985b). Feeding in fourspine stickleback occurred at 38 ppt, which was higher than 24.5 and 28 ppt for observed for brook and ninespine stickleback, respectively (Nelson, 1968). These studies indicate the wide range of salinity tolerance and preference of fourspine stickleback.
The euryhaline capabilities of fourspine stickleback and their stability as a laboratory animal made it appealing as a model species to examine the metabolic costs of osmoregulation in teleosts. In the present study we measured routine metabolic rate in fourspine sticklebacks throughout a 2-week period following SW exposure, allowing us to assess both short-term and long-term metabolic rates. We also examined physiological responses, including survival, tissue water content, and gill NKA and CS activity to monitor the acute response (days 1 and 3) and acclimation process (days 7 and 14). A period of 2 weeks is generally recognized as sufficient for complete acclimation to altered salinity (McCormick et al., 2022). We expected that there would be higher metabolic rates during initial exposure to elevated salinity, but due to the high euryhalinity of this species there would be only small differences in animals fully acclimated to SW.
Section snippets
Fish collection and maintenance
Adult fourspine sticklebacks (n = 198) were collected in the upper Quinnipiac River estuary (salinity ≤1 ppt), North Haven, CT, USA between August 12 to September 14, 2014; all fish were transported to laboratory facilities at the University of New Haven, West Haven, CT, USA. Fish were divided into four 150-L recirculating, aerated, filtered tanks. The fish were acclimated to 20 °C water temperature, 0.5 ppt salinity (equivalent to hard FW), and a natural photoperiod during the time of fish
Results
There was significant mortality of 18% in the SW-exposed group, primarily within 3 days after SW exposure (Fig. 1). SW day 1 had significantly higher mortality than SW day 2 (p < 0.01). There was a significant difference in mortality between SW and FW on day 1 (p < 0.01). No mortality was observed in the FW group.
There was a significant time (F = 20.00, p < 0.01), salinity (F = 55.68, p < 0.01), and interaction (F = 15.54, p = 0.01) effect on gill NKA activity. Enzyme activity levels increased
Discussion
To date, only a few studies have examined the metabolic rates of osmoregulation over a time course, and there have been no studies of oxygen consumption and routine metabolic rate in fourspine sticklebacks, a common euryhaline species in the eastern United States. In this study, the exposure of FW-acclimated fourspine stickleback to SW resulted in: 1) mortality in the first 3 days, 2) transient loss of water content, 3) increase in gill NKA activity within 3 days and that remained elevated, 4)
Conclusions
Understanding the energetic costs of SW acclimation furthers our understanding of osmoregulation in fishes in general and provides insight into the evolution of euryhaline fishes (Schultz and McCormick, 2013). With natural selection placing pressures on high-cost activities, it was previously thought that euryhalinity was too expensive to be maintained unless there was a clear benefit to having the trait (Kültz, 2015). Yet, euryhalinity has evolved repeatedly in most major families of teleosts (
Declaration of interests
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Acknowledgments
This work was made possible by the University of New Haven's undergraduate Marine Biology program and Honors Program. We would like to thank R. Flick and S. Bishop for assistance with fish collection and A. Weinstock for assistance with enzyme assays. We thank J. Lonthair for reviewing an early version of the manuscript. Graphical abstract was created with BioRender.com (agreement number MW23YQ4B2P).
Any use of trade, firm, or product names is for descriptive purposes only and does not imply
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Present address: 204C French Hall, University of Massachusetts Amherst, 230 Stockbridge Rd, Amherst, MA 01003, USA.
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Present address: California Department of Fish and Wildlife, Fisheries Branch, 1010 Riverside Parkway, West Sacramento, CA 95605, USA.