Osmoregulatory mechanisms, ion transport proteins, and gill physiology in euryhaline fish adapting to varying salinity conditions

Introduction

Teleost fish face fundamentally different osmotic challenges depending on the salinity of their aquatic environment. In freshwater, fish are hyperosmotic to their surroundings, resulting in continuous passive water influx across permeable epithelial surfaces and diffusive loss of ions down concentration gradients [1] . To compensate, freshwater fish must actively absorb ions from the dilute external medium through specialized gill ionocytes while producing copious dilute urine to excrete excess water. Conversely, marine teleosts are hyposmotic to seawater and face passive water loss and ion gain, requiring them to drink seawater, absorb water across the intestinal epithelium, and actively secrete excess monovalent ions (Na+ and Cl-) across gill ionocytes while excreting divalent ions (Mg2+, SO42-, Ca2+) through renal pathways [2] . Euryhaline species capable of tolerating wide salinity ranges represent an extraordinary physiological adaptation, requiring the ability to rapidly switch between these opposing ionic regulatory strategies. This capacity involves coordinated remodeling of gill epithelial cell populations, ion transporter expression and localization, paracellular pathway permeability, and renal function within hours to days of environmental salinity change [2] . The gill serves as the primary osmoregulatory organ, housing specialized mitochondria-rich cells (termed ionocytes or chloride cells) that express different complements of ion transporters depending on environmental salinity. Understanding the molecular mechanisms underlying this remarkable plasticity has fundamental biological significance and practical applications for aquaculture industries that often expose fish to salinity fluctuations during production cycles [3] . The current model of gill ion secretion in seawateracclimated teleosts centers on the cooperative action of three principal transporters in the basolateral and apical membranes of secretory ionocytes [3] . Na+/K+-ATPase, located in the extensively folded basolateral membrane and driven by ATP hydrolysis, establishes the electrochemical gradient that powers secondary active transport. The Na+-K+-2Cl-cotransporter (NKCC1), also basolateral, uses the Na+ gradient to accumulate intracellular Cl-above electrochemical equilibrium. The cystic fibrosis transmembrane conductance regulator (CFTR), an apical Clchannel, provides the exit pathway for accumulated Cl-into the external medium. Na+ is secreted paracellularly through leaky tight junctions between ionocytes and accessory cells, driven by the transepithelial potential generated by transcellular Cl-secretion [8] . In freshwater, a different ionocyte population expressing apical Na+-Cl-cotransporter (NCC), apical H+-ATPase, and epithelial Na+ channels mediates ion absorption from the dilute environment [9] . The transition from freshwater to seawater or vice versa requires not only changes in transporter expression but also fundamental remodeling of ionocyte populations, including apoptosis of freshwater-type ionocytes and proliferation and differentiation of seawatertype secretory cells. Japanese sea bass (Lateolabrax japonicus), an economically important euryhaline species distributed throughout coastal waters and estuaries of East Asia, provides an excellent model for studying salinity adaptation due to its wide salinity tolerance and commercial significance in aquaculture [10]. This research examined the time course, magnitude, and coordination of adaptive mechanisms during freshwater-to-seawater transfer in this species.

Material and Methods

This research was conducted at Osaka Institute of Aquatic Sciences, Japan, between May 2022 and August 2024. Juvenile Japanese sea bass (mean weight 45.2±7.3 g, mean total length 16.8±1.4 cm) were obtained from a commercial hatchery and maintained in recirculating freshwater (0.5 ppt salinity) systems for a minimum of four weeks prior to experimentation. Fish were held at 20±1°C under a 12L:12D photoperiod and fed commercial pellets at 2% body weight per day. All experimental procedures were approved by the Osaka Institute of Aquatic Sciences Animal Care Committee (protocol OIAS-2022-031) in accordance with Japanese guidelines for the care and use of laboratory animals [4] . For salinity transfer experiments, freshwater-acclimated fish were directly transferred to filtered natural seawater (33 ppt) with matched temperature. Control fish were shamtransferred to freshwater. Sampling was performed at 0 (pretransfer), 6, 24, 72, and 168 hours post-transfer, with 8 fish per time point per treatment group (total n=80). At each sampling point, fish were anesthetized with MS-222 (100 mg/L buffered with NaHCO3), blood was collected from the caudal vein using heparinized syringes, and gill arches were rapidly dissected. The second gill arch from each side was processed for immunohistochemistry and electron while remaining arches were snap-frozen in liquid nitrogen for biochemical analyses [4] . Na+/K+-ATPase activity was measured in gill homogenates by the kinetic microplate assay method coupled to NADH oxidation, expressed as micromoles of ADP generated per milligram protein per hour. Protein expression of Na+/K+-ATPase alpha-subunit, NKCC1, NCC, and CFTR was assessed by Western blotting using commercially available antibodies validated for teleost species. Total protein was extracted in RIPA buffer, separated by SDS-PAGE (7.5% gels), transferred to PVDF membranes, and probed with primary antibodies followed by HRP-conjugated secondary antibodies and chemiluminescent detection. Band intensities were quantified by densitometry and normalized to betaactin loading control [11] . Immunohistochemistry was performed on paraformaldehyde-fixed, paraffin-embedded gill sections using the same antibodies with avidin-biotin-peroxidase visualization. Double-label immunofluorescence employed Alexa Fluor 488-and 594-conjugated secondary antibodies with confocal laser scanning microscopy to examine colocalization of transporters in individual ionocytes. Scanning electron microscopy of gill filament surfaces was used to characterize ionocyte apical morphology, with apical pit area measured using ImageJ software on a minimum of 50 cells per fish. Plasma osmolality was measured by vapor pressure osmometry, and plasma Na+ and Clconcentrations by ion-selective electrodes [4] . Statistical analysis employed two-way ANOVA (salinity x time) followed by Tukey's HSD post hoc test, with significance at p<0.05.

Results

Seawater transfer triggered rapid and coordinated changes in gill transporter expression, enzyme activity, and ionocyte morphology. Na+/K+-ATPase alpha-subunit protein increased progressively following seawater transfer, reaching 1.6-fold elevation by 24 hours (p<0.05), 2.8-fold by 72 hours (p<0.001), and plateauing at 3.1-fold by 168 hours compared to freshwater controls. Na+/K+-ATPase enzymatic activity closely paralleled protein expression changes, increasing from 4.2±0.8 to 11.7±2.1 μmol ADP/mg protein/h between 0 and 72 hours post-transfer (p<0.001). NKCC1 cotransporter protein showed an even more dramatic 3.1-fold induction by 72 hours, detectable as early as 6 hours post-transfer (1.4-fold, p<0.05), consistent with its role as a critical component of the chloride secretory mechanism [5] . In contrast to the upregulation of secretory transporters, the freshwater-associated NCC cotransporter showed progressive downregulation following seawater transfer, decreasing to 0.65-fold at 24 hours, 0.31-fold at 72 hours, and 0.18-fold at 168 hours compared to freshwater values (all p<0.01). CFTR chloride channel protein was barely detectable in freshwater gill but showed strong upregulation following seawater transfer, with a 4.2-fold increase by 72 hours relative to the low freshwater baseline (p<0.001). Immunohistochemistry confirmed that CFTR localized exclusively to the apical membrane of seawater-type ionocytes, consistent with its function as the apical chloride exit pathway [12] . Ionocyte morphological analysis revealed substantial remodeling of the gill epithelium following seawater transfer. Total ionocyte density on gill filament epithelium increased from 1,247±186 to 2,034±243 cells per mm2 by 168 hours (p<0.001), reflecting both proliferation of new secretory-type ionocytes and retention of existing cells undergoing functional conversion. Scanning electron microscopy showed dramatic changes in apical surface morphology: freshwater ionocytes displayed small apical openings (mean area 12.3±3.4 μm2) partially covered by adjacent accessory cells, while seawater-acclimated ionocytes developed large open apical pits (28.7±6.8 μm2, p<0.001) characteristic of active secretory function. The transition was progressive, with intermediate morphologies observed at 24 and 72 hours [5] . Double-label immunofluorescence confocal microscopy revealed distinct transporter co-localization patterns in freshwater versus seawater ionocytes. In freshwater, NCC was found in apical membranes of ionocytes that coexpressed basolateral Na+/K+-ATPase but lacked NKCC1. Following seawater transfer, a new population of ionocytes emerged expressing the complete secretory transporter complement: basolateral Na+/K+-ATPase and NKCC1 with apical CFTR. By 168 hours, the majority of ionocytes displayed the seawater secretory phenotype. Plasma osmolality showed a transient but significant increase at 24 hours post-transfer (from 312±8 to 348±12 mOsm/kg, p<0.001), reflecting the initial osmotic stress before full compensatory mechanisms were established. By 72 hours, plasma osmolality had returned to 318±9 mOsm/kg (not significantly different from freshwater baseline, p=0.23), coinciding with peak transporter expression. Plasma Na+ and Cl-concentrations followed similar patterns of transient elevation and restoration [13] .

Discussion

These findings demonstrate that Japanese sea bass execute a rapid and highly coordinated program of gill remodeling during freshwater-to-seawater transition, involving reciprocal regulation of absorptive and secretory ion transport systems at molecular, cellular, and morphological levels. The time course of transporter induction closely matches the physiological restoration of plasma osmolality, with both reaching steady-state values by 72 hours posttransfer, supporting causal relationships between molecular remodeling and functional osmoregulatory capacity [6] . The 2.8-fold increase in Na+/K+-ATPase activity provides the increased electrochemical driving force required for enhanced secondary active ion transport in the hyperosmotic marine environment. The coordinated upregulation of the three principal secretory transporters (Na+/K+-ATPase, NKCC1, and CFTR) and simultaneous downregulation of the absorptive NCC cotransporter demonstrates that salinity adaptation involves a comprehensive reprogramming of ionocyte function rather than simple quantitative adjustment of individual transporters [14] . This coordination likely involves shared transcriptional regulatory mechanisms, potentially including osmosensitive signaling pathways and hormoneresponsive transcription factors. Cortisol, which increases in response to salinity challenge and acts through both glucocorticoid and mineralocorticoid receptors, is a strong candidate for orchestrating this coordinated response, as it has been shown to stimulate Na+/K+-ATPase and NKCC1 expression in multiple euryhaline species. The morphological transformation of ionocyte apical surfaces from small accessory cell-covered apertures to large open pits represents a structural adaptation that optimizes the geometry of the secretory epithelium for maximum ion efflux capacity [15] . The expanded apical surface area increases the membrane area available for CFTR-mediated chloride exit, while the retraction of accessory cell coverage increases the area of leaky tight junctions between ionocytes and accessory cells through which Na+ is secreted paracellularly. This coordinated structural remodeling, occurring in parallel with transporter upregulation, represents a remarkably efficient adaptive strategy that maximizes secretory capacity through both molecular and architectural modifications. The transient elevation of plasma osmolality at 24 hours, before full compensatory transporter expression is achieved, defines the osmotic stress window during which fish are most physiologically vulnerable to salinity change. This window has direct practical implications for aquaculture management, as exposure to additional stressors (handling, crowding, disease challenge) during this period could compound the osmotic stress and exceed the fish's adaptive capacity [7] . The restoration of plasma osmolality by 72 hours, coinciding with peak transporter expression and advanced ionocyte remodeling, defines the minimum acclimation period that should be allowed before subjecting fish to additional challenges in aquaculture settings. The identification of two distinct ionocyte populationsfreshwater absorptive cells expressing NCC without NKCC1, and seawater secretory cells expressing NKCC1 and CFTR without NCC-rather than a single cell type undergoing functional conversion, suggests that euryhaline adaptation in this species involves cell population dynamics (selective proliferation and apoptosis) in addition to phenotypic plasticity of individual cells [16] . This finding aligns with the emerging model of ionocyte diversity in euryhaline teleosts, where stem cell-derived progenitors can differentiate along freshwater or seawater ionocyte lineages depending on environmental salinity signals. Understanding the signaling pathways controlling this cell fate decision could enable manipulation of ionocyte differentiation for improved salinity tolerance in aquaculture species.

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