Gill Ionocytes Research Articles

Functional classification of gill ionocytes and spatiotemporal changes in their distribution after transfer from seawater to freshwater in Japanese seabass.

Spatiotemporal changes in branchial ionocyte distribution were investigated following transfer from seawater (SW) to freshwater (FW) in Japanese seabass. The mRNA expression levels of cystic fibrosis transmembrane conductance regulator (CFTR) and Na+/K+/2Cl- cotransporter 1a (NKCC1a) in the gills rapidly decreased after transfer to FW, whereas Na+/H+ exchanger 3 (NHE3) and Na+/Cl- cotransporter 2 (NCC2) expression were upregulated following the transfer. Using quadruple-color whole-mount immunofluorescence staining with anti-Na+/K+-ATPase, anti-NHE3, anti-CFTR and T4 (anti-NKCC1a/NCC2) antibodies, we classified ionocytes into one SW type and two FW types: NHE3 cell and NCC2 cell. Time course observation after transfer revealed an intermediate type between SW-type and FW-type NHE3 ionocytes, suggesting functional plasticity of ionocytes. Finally, on the basis of the ionocyte classification of Japanese seabass, we observed the location of ionocyte subtypes on frozen sections of the gill filaments stained by triple-color immunofluorescence staining. Our observation indicated that SW-type ionocytes transformed into FW-type NHE3 ionocytes and at the same time shifted their distribution from filaments to lamellae. However, FW-specific NCC2 ionocytes appeared mainly in the filaments. Taken together, these findings indicate that ionocytes originated from undifferentiated cells in the filaments and expanded their distribution to the lamellae during FW acclimation.

Comparative analysis of branchial ionocytes in Caspian Sea zander, Sander lucioperca (Linnaeus, 1758) using Na+, K+-ATPase immunolocalisation at different salinities

Gill morphology and branchial ionocyte variability were examined in zander Sander lucioperca (Linnaeus, 1758)fingerlings, having two different body weights (1 and 2 g) for a period of ten days following their transfer from freshwater to two different salinities (7 and 12‰ Caspian Sea water) using Na+, K+-ATPase (NKA) immunolocalisation. Survival rates and plasma osmolarity levels at the end of each salinity challenge were measured. By the end of the experiment, bothweight groups were able to survive and successfully acclimate to salinity change. The number and size of branchial ionocytes in the gills dramatically decreased in both size groups; while, the filament ionocytes in both weight groups reformed at the end of the acclimation period. The results indicated both groups to be in the same osmoregulatory developmental stage, despite difference in their body weight. Also, their blood osmotic pressure and gill ionocyte changes at different salinity were similar when transferred to iso-osmotic condition. Further studies are needed for complete understanding of zander osmoregulatory systems exposed to different salinities.

Effects of fasting and re-alimentation on gill and intestinal morphology and indicators of osmoregulatory capacity in genetically selected sea bass (Dicentrarchus labrax) populations with contrasting tolerance to fasting

Fasting and refeeding occur naturally in predators but this is largely ignored when dealing with farmed fish. Therefore, the effects of 3-week fasting and re-alimentation (2.5% of the individual body mass) were investigated using two genetically selected populations (F2 generation) of 250g juvenile sea bass (Dicentrarchus labrax L.). Blood osmolarity, gill and intestinal morphology and expression of the sodium pump (Na+, K+-ATPase, NKA) were studied on two phenotypes showing different degrees of body mass loss during food deprivation: one group losing body mass rapidly during fasting (F+) and the other one limiting body mass loss during the same period (F−).Blood osmotic pressure significantly decreases due to re-alimentation in both groups, but this is compensated in the F+ group. In this group, gill ionocytes are smaller and less numerous, but a significantly higher NKA gene expression is noted in the gills in comparison to the F− individuals 48 and 72h after re-alimentation, and also in the posterior intestine 72h after re-alimentation. This most probably occurs to compensate for a higher salt intake during nutrient absorption in comparison to the F− group. Furthermore, refed F− fish absorb more lipids along the proximal anterior intestine, and take longer to digest than the F+ group, and show enterocyte vacuolization in the posterior intestine.Therefore, the two selected populations have different postprandial digestive strategies: the F− fish optimize feed efficiency first at the cost of optimal hydromineral adjustment, while the F+ group invests in osmoregulatory performance at the expense of digestive physiology. Statement of relevanceOur paper is highly relevant to the general field of commercial aquaculture. There is an increasing number of research articles dealing with fasting and refeeding in commercial fish and how to improve fish nutrition based on these physiological data and genetic selection.

Osmotic versus adrenergic control of ion transport by ionocytes of Fundulus heteroclitus in the cold

In eurythermic vertebrates, acclimation to the cold may produce changes in physiological control systems. We hypothesize that relatively direct osmosensitive control will operate better than adrenergic receptor mediated control of ion transport in cold vs. warm conditions. Fish were acclimated to full strength seawater (SW) at 21°C and 5°C for four weeks, gill samples and blood were taken and opercular epithelia mounted in Ussing style chambers. Short-circuit current (Isc) at 21°C and 5°C (measured at acclimation temperature), was significantly inhibited by the α2-adrenergic agonist clonidine but the ED50 dose was significantly higher in cold conditions (93.8±16.4nM) than in warm epithelia (47.8±8.1nM) and the maximum inhibition was significantly lower in cold (−66.1±2.2%) vs. warm conditions (−85.6±1.3%), indicating lower sensitivity in the cold. β-Adrenergic responses were unchanged. Hypotonic inhibition of Isc, was higher in warm acclimated (−95%), compared to cold acclimated fish (−75%), while hypertonic stimulations were the same, indicating equal responsiveness to hyperosmotic stimuli. Plasma osmolality was significantly elevated in cold acclimated fish and, by TEM, gill ionocytes from cold acclimated fish had significantly shorter mitochondria. These data are consistent with a shift in these eurythermic animals from complex adrenergic control to relatively simple biomechanical osmotic control of ion secretion in the cold.

The expression of VILL protein is hypoosmotic-dependent in the lamellar gill ionocytes of Otocephala teleost fish, Chanos chanos

Milkfish, a species within the primitive teleost lineage Otocephala, can survive in water conditions ranging from hypo- to hyper-saline. This study explored the effects of environmental salinity on apical morphologies of ionocytes and the expression of villin homologs in the gills of milkfish acclimated to either seawater (SW) or fresh water (FW). Scanning electron microscopy revealed that the ionocytes in the gill filaments of SW and FW milkfish, respectively, cellular apical morphologies were hole-type and squint-type. The flat-type ionocytes were observed in the gill lamellae of FW milkfish. Furthermore, apical surfaces of some lamellar ionocytes exhibited microvilli. Villin 1 is a microvilli marker expressed in the epithelial cells of various vertebrates. In the phylogenetic tree of villin 1 homologs, primitive teleosts exhibit villin 1-like (VILL) and villin 1 proteins. Two mRNA sequences, villin 1 and VILL, were identified from the milkfish transcriptome by next generation sequencing. Low but constant expression of villin 1 (gene and protein) was observed in the gills for both SW and FW fish. VILL gene and protein expression levels in the gills were higher in FW fish, compared to SW fish. Double immunofluorescence staining demonstrated that VILL protein was present in some lamellar ionocytes of FW milkfish, but not in the filament ionocytes of either FW or SW milkfish. Taken together, these findings indicated that the VILL expression of ionocytes is hypoosmotic-dependent. The VILL might be involved in the formation of microvilli in the lamellar ionocytes for hyperosmoregulation of the milkfish.

Prolactin and cortisol mediate the maintenance of hyperosmoregulatory ionocytes in gills of Mozambique tilapia: Exploring with an improved gill incubation system

Endocrine control of osmoregulation is essential for teleosts to adapt to various aquatic environments. Prolactin (PRL) is known as a fundamental endocrine factor for hyperosmoregulation in teleost fishes, acting on ionocytes in the gills to maintain ion concentrations of body fluid within narrow physiological ranges in freshwater conditions. Cortisol is also known as an osmoregulation-related steroid in teleosts; however, its precise function is still controversial. Here, we investigated more detailed effects of PRL and roles of cortisol on ionocytes of Mozambique tilapia (Oreochromis mossambicus) in freshwater, using an improved gill filament incubation system. This incubation system resulted in enhanced cell viability, as evaluated using the dead cell marker propidium iodide. PRL was shown to maintain the density of freshwater-type ionocytes in isolated gill filaments; this effect of PRL is not achieved by the activation of cell proliferation, but by the maintenance of existing ionocytes. Cortisol alone did not show any distinct effect on ionocyte density in isolated gill filaments. We also assessed effects of PRL and cortisol on relative mRNA levels of NCC2, NHE3, NKAa1a, and NKAa1b. PRL maintained relative NCC2 and NKAa1a mRNA abundance, and cortisol showed a stimulatory effect on relative NCC2 and NKAa1a mRNA levels in combination with PRL, though cortisol alone exerted no effect on these genes. An increase in NKAa1b mRNA abundance was detected in cortisol-treated groups. PRL treatment also maintained normal NCC2 localization at the apical membrane of the ionocytes. These results indicate that PRL maintains freshwater-type ionocytes, and that cortisol stimulates the function of ionocytes maintained by PRL.

Distribution and dynamics of branchial ionocytes in houndshark reared in full-strength and diluted seawater environments

In teleost fishes, it is well-established that the gill serves as an important ionoregulatory organ in addition to its primary function of respiratory gas exchange. In elasmobranchs, however, the ionoregulatory function of the gills is still incompletely understood. Although two types of ionocytes, Na+/K+-ATPase (NKA)-rich (type-A) cell and vacuolar-type H+-ATPase (V-ATPase)-rich (type-B) cell, have been found in elasmobranch fishes, these cells were considered to function primarily in acid–base regulation. In the present study, we examined ion-transporting proteins expressed in ionocytes of Japanese-banded houndshark, Triakis scyllium, reared in full-strength seawater (SW) and transferred to diluted (30%) SW. In addition to the upregulation of NKA and Na+/H+ exchanger type 3 (NHE3) mRNAs in the type-A ionocytes, we found that Na+, Cl− cotransporter (NCC, Slc12a3) is expressed in a subpopulation of the type-B ionocytes, and that the expression level of NCC mRNA was enhanced in houndsharks transferred to a low-salinity environment. These results suggest that elasmobranch gill ionocytes contribute to NaCl uptake in addition to the already described function of acid–base regulation, and that NCC is most probably one of the key molecules for hyper-osmoregulatory function of elasmobranch gills. The existence of two types of ionocytes (NHE3- and NCC-expressing cells) that are responsible for NaCl absorption seems to be a common feature in both teleosts and elasmobranchs for adaptation to a low salinity environment. A possible driving mechanism for NCC in type-B ionocytes is discussed.

Acclimation responses of gill ionocytes of red tilapia (Oreochromis mossambicus × O. niloticus) to water salinity and alkalinity

To understand the acclimation strategies of red tilapia to different environments, this study aimed to evaluate different responses of red tilapia (O. mossambicus × O. niloticus) to salinity (10-30‰), alkalinity (1-3 gL -1 NaHCO3) and salinity and alkalinity (10/1-30/3 ‰/gL -1 NaHCO3) environments. Localization, type, size, and numeration of gill ionocytes were investigated on the same specimens by scanning electron microscopy (SEM) and immunohistochemistry (IHC) with antibodies of Na + /K + -ATPase (NKA), Na + /K + /2Cl - contransporter (NKCC), cystic fibrosis transmembrane conductance regulator (CFTR) and carbonic anhydrase (CA). Ionocytes were only located on filaments conducted by SEM. Four types of ionocytes namely pit, convex, concave and transitory types were determined morphologically by their apical openings of which concave and transitory type were not present in freshwater (FW) and saltwater (SW) fish (10). Both ionocytes size and number increased with elevated stress levels. In comparison to FW, density of ionotypes increased to about 4.75, 3.00 and 3.44 fold in SW (30), AW (3) and S&AW (30/3) respectively. Immunoreactive cells on gill filaments confirmed branchial distribution of ionocytes. Immunoreaction of NKA, NKCC and CA appeared in FW except for CFTR while they all appeared in SW (30), AW (3) and S&AW (30/3).

Transcriptomic Analysis of Trout Gill Ionocytes in Fresh Water and Sea Water Using Laser Capture Microdissection Combined with Microarray Analysis.

Fish gills represent a complex organ composed of several cell types that perform multiple physiological functions. Among these cells, ionocytes are implicated in the maintenance of ion homeostasis. However, because the ionocyte represents only a small percent of whole gill tissue, its specific transcriptome can be overlooked among the numerous cell types included in the gill. The objective of this study is to better understand ionocyte functions by comparing the RNA expression of this cell type in freshwater and seawater acclimated rainbow trout. To realize this objective, ionocytes were captured from gill cryosections using laser capture microdissection after immunohistochemistry. Then, transcriptome analyses were performed on an Agilent trout oligonucleotide microarray. Gene expression analysis identified 108 unique annotated genes differentially expressed between freshwater and seawater ionocytes, with a fold change higher than 3. Most of these genes were up-regulated in freshwater cells. Interestingly, several genes implicated in ion transport, extracellular matrix and structural cellular proteins appeared up-regulated in freshwater ionocytes. Among them, several ion transporters, such as CIC2, SLC26A6, and NBC, were validated by qPCR and/or in situ hybridization. The latter technique allowed us to localize the transcripts of these ion transporters in only ionocytes and more particularly in the freshwater cells. Genes involved in metabolism and also several genes implicated in transcriptional regulation, cell signaling and the cell cycle were also enhanced in freshwater ionocytes. In conclusion, laser capture microdissection combined with microarray analysis allowed for the determination of the transcriptional signature of scarce cells in fish gills, such as ionocytes, and aided characterization of the transcriptome of these cells in freshwater and seawater acclimated trout.

In vivo and in vitro effects of high-K+ stress on branchial expression of ROMKa in seawater-acclimated Mozambique tilapia

Recently, a teleost ortholog of renal outer medullary K+ channel (ROMK) expressed in gill ionocytes (ROMKa) has emerged as a primary K+-excreting pathway in fish. However, the mechanisms by which ROMKa expression is regulated in response to perturbations of plasma K+ levels are unknown. In this study, we aimed to identify potential links between the endocrine system and K+ regulation in a euryhaline fish. We assessed time-course changes in multiple endocrine parameters, including plasma cortisol and gene expression of branchial glucocorticoid and mineralocorticoid receptors (GR1, GR2, and MR) and pituitary hormones, in seawater (SW)-acclimated Mozambique tilapia (Oreochromis mossambicus) exposed to high-K+ (H-K) SW. Exposure to H-K SW elicited little effects on plasma cortisol or mRNA levels of GRs and pituitary hormones. Since plasma K+ and branchial ROMKa expression was increased within 6h after H-K treatment in vivo, the effect of high K+ was subsequently tested in a gill filament incubation experiment using media with differing K+ concentrations. ROMKa mRNA levels were induced following incubation of filaments in H-K medium for 6h. The present study is the first to demonstrate that the expression of ROMKa in teleost ionocytes can respond to high K+ conditions independent from systemic signaling.

Histopathological effects of atrazine on gills of Caspian kutum Rutilus frisii kutum fingerlings

The use of chemical pesticides has increased environmental pollution and affects fishes as non-target organisms. To investigate the toxic effects of the widely used herbicide atrazine on Caspian kutum Rutilus frisii kutum fingerlings, fish were exposed to a sublethal concentration of half LC50 for 96 h. The main alterations visible in the gill tissue were detachment of the epithelium of the lamellae, necrosis, lamellar fusion, hyperplasia, club shaped lamellae, collapse of the lamellae, shrinkage and curling of the lamellae, and ultrastructural alterations such as necrosis of the apical microridges of the pavement cells. Results also showed that the gill ionocytes were fewer in number and larger in size in the atrazine-exposed fish. Atrazine appears to be highly toxic to Caspian kutum fingerlings even at a sublethal concentration (12.47 mg l(-1)) and acute exposure. This toxicity could affect gill respiration and ion regulation function of fingerlings by damaging tissue, pavement cells, and ionocytes.

K + secretion by ionocytes in the skin of medaka larve

Epithelial ionocytes in fish gills and skin play a critical role in ionic and acid-base regulation. Molecular mechanisms of sodium, chloride and acid-base regulation in fish ionocytes have been wel...

Occurrence of larval and adult types of ion-secreting ionocytes in Japanese eel Anguilla japonica

The anguillid eels are catadromous fishes that migrate between marine and freshwater habitats. The long migration of eel larvae, called leptocephali, as long as thousands of kilometers in the ocean is important to determine their recruitment successes. The leptocephali in the ocean have a pelagic lifestyle totally different from the benthic one of glass eels and yellow eels in rivers. It is known that eel leptocephali have ionocytes on the body surface that may maintain ionic and osmotic status in the internal environment; however, detailed morphology and function of ionocytes in leptocephali are still unknown. In the present study, we aimed 1) to clarify the morphological features of the epidermis in Japanese eel Anguilla japonica leptocephali cultured in hyper-osmotic condition, and 2) to examine the ion-transporting functions of ionocytes of both leptocephali and yellow eels. Na+/K+-ATPase-immunoreactive ionocytes were distributed all over the body surface of leptocephali. Ionocytes were in contact with external environments through their apical membrane, which was located at the boundary of pavement cells. Na+/K+-ATPase-immunopositive cells were not observed in the skin of seawater-acclimated yellow eels. In ionocytes of the larval skin, the apical membrane appeared as a slightly projecting disk with a microvilli-like structure. Meanwhile, the apical membrane of gill ionocytes of yellow eels formed a concave surface. In ionocytes of leptocephali, mitochondria were enlarged and the tubular system was well developed, as compared with those of the gill of yellow eels. Ionocytes of leptocephali showed CFTR immunoreaction in their apical region and NKCC1 immunoreaction in their basolateral region, suggesting that the skin ionocytes are involved in salt secretion. These results support the notion that Japanese eel maintain their ion balance through skin ionocytes during early life stages, and that the skin ionocytes of leptocephali disappear in yellow eel stages after the formation of functional gills and gill ionocytes.

Diverse mechanisms for body fluid regulation in teleost fishes.

Teleost fishes are the major group of ray-finned fishes and represent more than one-half of the total number of vertebrate species. They have experienced in their evolution an additional third-round whole genome duplication just after the divergence of their lineage, which endowed them with an extra adaptability to invade various aquatic habitats. Thus their physiology is also extremely diverse compared with other vertebrate groups as exemplified by the many patterns of body fluid regulation or osmoregulation. The key osmoregulatory organ for teleosts, whose body fluid composition is similar to mammals, is the gill, where ions are absorbed from or excreted into surrounding waters of various salinities against concentration gradients. It has been shown that the underlying molecular physiology of gill ionocytes responsible for ion regulation is highly variable among species. This variability is also seen in the endocrine control of osmoregulation where some hormones have distinct effects on body fluid regulation in different teleost species. A typical example is atrial natriuretic peptide (ANP); ANP is secreted in response to increased blood volume and acts on various osmoregulatory organs to restore volume in rainbow trout as it does in mammals, but it is secreted in response to increased plasma osmolality, and specifically decreases NaCl, and not water, in the body of eels. The distinct actions of other osmoregulatory hormones such as growth hormone, prolactin, angiotensin II, and vasotocin among teleost species are also evident. We hypothesized that such diversity of ionocytes and hormone actions among species stems from their intrinsic differences in body fluid regulation that originated from their native habitats, either fresh water or seawater. In this review, we summarized remarkable differences in body fluid regulation and its endocrine control among teleost species, although the number of species is still limited to substantiate the hypothesis.

Medaka villin 1-like protein (VILL) is associated with the formation of microvilli induced by decreasing salinities in the absorptive ionocytes

IntroductionVillin 1 is an actin-regulatory protein involved in the formation of microvilli of mammalian enterocytes. The microvilli, finger-like protrusions, are more abundant on the apical surfaces of gill ionocytes in various freshwater (FW) teleosts than in seawater (SW) fishes. However, the plasticity in the mechanisms of microvillus formation in the gill ionocytes are poorly understood, and the actin-regulatory proteins involved in the formation of microvilli have not been identified in fishes. The present study used the euryhaline medaka (Oryzias dancena) as a model to explore the role of a homolog of villin 1 in the actin-organization of cellular morphologies induced by decreasing salinities.ResultsBy ultrastructural observation, there are numerous actin filaments organized on the apical cortex of ion-absorptive ionocytes in the FW-acclimated medaka. From gills of the euryhaline medaka, we have identified the VILL sequence. The phylogenetic tree and functional domains suggest that VILL is the homolog of villin 1 in fishes. Immunofluorescence using a specific antibody revealed that VILL was specifically localized to the apical region of gill ionocytes along with microvilli in the FW medaka, but not in SW fish. The expression levels of Odvill mRNA and VILL protein were higher in the gills of the FW individuals than in the SW group and were induced when fish were transferred from SW to FW. A morpholino oligonucleotide for VILL knockdown eliminated the apical protrusions of ionocytes and pavement cells in the trunk epithelia of embryos.ConclusionsFrom a novel aspect of cytoskeletal functions, our findings highlighted the important role of VILL protein in the ionoregulation of aquatic vertebrates in response to different osmotic challenges. This study is the first to show that the expression of VILL is associated with the formation of microvilli in the absorptive ionocytes of a euryhaline fish. Loss-of-function experiments showed that the distribution of VILL may represent the molecular link between the cytoskeletal organization and cellular morphology of the absorptive ionocytes during hypoosmotic adaptation in aquatic vertebrates.

Claudin-6, -10d, and -10e contribute to seawater acclimation in the euryhaline puffer fishTetraodon nigroviridis

Expression profiles of claudin-6, -10d and -10e in the euryhaline teleost fish Tetraodon nigroviridis revealed claudin-6 in brain, eye, gill and skin tissue, while claudin-10d and -10e were found in brain, gill and skin only. In fishes, the gill and skin are important tissue barriers that interface directly with surrounding water, but these organs generally function differently in osmoregulation. Therefore, roles for gill and skin claudin-6, -10d and -10e in the osmoregulatory strategies of T. nigroviridis were investigated. In the gill epithelium, claudin-6, -10d and -10e co-localized with Na(+)-K(+)-ATPase immunoreactive (NKA-ir) ionocytes, and differences in sub-cellular localization could be observed in hypoosmotic (freshwater, FW) versus hyperosmotic (seawater, SW) environments. Claudin-10d and -10e abundance increased in the gills of fish acclimated to SW versus FW, while claudin-6 abundance decreased in the gills of fish acclimated to SW. Taken together with our knowledge of claudin-6 and -10 function in other vertebrates, data support the idea that in SW-acclimated T. nigroviridis, these claudins are abundant in gill ionocytes, where they contribute to the formation of a Na(+) shunt and 'leaky' epithelium, both of which are characteristic of salt-secreting SW fish gills. Skin claudin-10d and -10e abundance also increased in fish acclimated to SW versus those in FW, but so did claudin-6. In skin, claudin-6 was found to co-localize with NKA-ir cells, but claudin-10d and -10e did not. This study provides direct evidence that the gill epithelium contains salinity-responsive tight junction proteins that are abundant primarily in ionocytes. These same proteins also appear to play a role in the osmoregulatory physiology of the epidermis.

Characterization of Na(+) uptake in the endangered desert pupfish, Cyprinodon macularius (Baird and Girard).

This study provided an initial characterization of Na(+) uptake in saline freshwater by the endangered pupfish, Cyprinodon macularius. This species occurs only in several saline water systems in the southwestern USA and northern Mexico, where salinity is largely controlled by water-management practices. Consequently, understanding the osmoregulatory capacity of this species is important for their conservation. The lower acclimation limit of C. macularius in freshwater was found to be 2 mM Na(+). Fish acclimated to 2 or 7 mM Na(+) displayed similar Na(+) uptake kinetics, with K m values of 4321 and 3672 μM and V max values of 4771 and 3602 nmol g(-1) h(-1), respectively. A series of experiments using pharmacological inhibitors indicated that Na(+) uptake in C. macularius was not sensitive to bumetanide, metolazone, or phenamil. These results indicate the Na(+)-K(+)-2Cl(-) cotransporter, Na(+)-Cl(-) cotransporter, and the Na(+) channel-H(+)-ATPase system are likely not to be involved in Na(+) uptake at the apical membrane of fish gill ionocytes in fish acclimated to 2 or 7 mM Na(+). However, Na(+) uptake was sensitive to 1 × 10(-3) M amiloride (not 1 × 10(-4) or 1 × 10(-5) M), 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), and ethoxzolamide. These data suggest that C. macularius relies on a low-affinity Na(+)-H(+) exchanger for apical Na(+) uptake and that H(+) ions generated via carbonic anhydrase-mediated CO2 hydration are important for the function of this protein.

Differential regulation of sodium–potassium pump isoforms during smolt development and seawater exposure of Atlantic salmon

Freshwater and seawater isoforms of the alpha subunit of Na(+)/K(+)-ATPase (NKA) have previously been identified in gill ionocytes of Atlantic salmon (Salmo salar). In the present study we examine the abundance and cellular localization of these isoforms during the parr-smolt transformation, a developmental process that is preparatory for seawater entry. The abundance of NKAα1a was lower in smolts than in parr, remained relatively constant during spring and decreased in summer. NKAα1b increased tenfold in smolts during spring, peaking in late April, coincident with downstream migration and increased salinity tolerance. NKAα1b increased a further twofold after seawater exposure of smolts, whereas NKAα1a decreased by 98%. The abundance of NKAα1b-positive, and NKAα1b and NKAα1a co-labeled ionocytes increased during smolt development, whereas the number of NKAα1a cells decreased. After seawater exposure of smolts, NKAα1b-positive ionocytes increased, NKAα1a-positive cells decreased, and co-labeled cells disappeared. Plasma growth hormone and cortisol increased during spring in smolts, but not in parr, peaking just prior to the highest levels of NKAα1b. The results indicate that the increase in the abundance of NKAα1b during smolt development is directly linked to the increase in salinity tolerance that occurs at this stage, but that significant changes also occur after seawater exposure. Spring increases in circulating levels of growth hormone and cortisol indicate that these hormones may be instrumental in upregulating NKAα1b during smolt development.

A Fish Out of Water: Gill and Skin Remodeling Promotes Osmo- and Ionoregulation in the Mangrove KillifishKryptolebias marmoratus

The euryhaline, amphibious mangrove killifish Kryptolebias marmoratus is known to survive weeks out of water in moist environments. We tested the hypothesis that the skin is a site of osmo- and ionoregulation in K. marmoratus. We predicted that under terrestrial conditions, gill and skin remodeling would result in an enhanced role for skin and a diminished role for the gills in osmo- and ionoregulation. Fish were exposed to water-either freshwater (FW, 1‰) or hypersaline water (saltwater [SW], 45‰)-or air over a moist surface of FW or SW for 9 d and then recovered in water. When fish were emersed for 9 d, (22)Na and (3)H-H(2)O were exchanged across the cutaneous surface. Homeostasis of whole-body Cl(-) and water levels but not of Na(+) levels was maintained over 9 d in air. In air-exposed fish, there was a significant increase in the size of skin ionocytes (in SW), a decrease in the number of skin mucous cells (in SW), and an increase in the gill interlamellar cell mass relative to those of fish in water. Gill ionocytes were mostly embedded away from the external surface in air-exposed fish, but the number and size of ionocytes increased (in FW). Interestingly, skin ionocytes formed distinct clusters of 20-30 cells. The estimated number of ionocytes over the whole skin surface was comparable to that in the gills. Overall, the findings support the hypothesis that the skin is a site of osmo- and ionoregulation in K. marmoratus in aquatic and terrestrial environments. Reversible cellular and morphological changes to the skin and gills during air exposure probably enhanced the cutaneous contribution to ion and water balance.

Hyperosmotic shock adaptation by cortisol involves upregulation of branchial osmotic stress transcription factor 1 gene expression in Mozambique Tilapia

The Mozambique tilapia ( Oreochromis mossambicus) is a euryhaline species that does not survive direct seawater exposure. Cortisol is involved in re-establishing electrolyte homeostasis in seawater and is thought to play a role in allowing tilapia to cope with abrupt seawater exposure, but the mechanism(s) are far from clear. Recently, osmotic stress transcription factor1 (OSTF1) was identified as a key signaling molecule involved in hyperosmotic stress adaptation in tilapia. Consequently, we tested the hypothesis that upregulation of OSTF1 expression by cortisol is a key response for hyperosmotic stress adaptation in tilapia. Fish were exposed to different salinities over a 24 h period, while a major electrolyte disturbance and mortality was observed only with full-strength seawater exposure. Therefore, we administered cocoa butter implants of cortisol (50 mg/kg) intraperitoneally to tilapia maintained in fresh water and after three days exposed these fish to full-strength seawater. There was 50% mortality in the control fish upon seawater exposure, but this was abolished by cortisol treatment. Abrupt seawater exposure did not affect plasma cortisol levels, while, as expected, exogenous administration of this steroid elevated plasma cortisol levels both in fresh water and seawater. Cortisol treatment significantly induced OSTF1 gene expression in fresh water tilapia, and also enhanced further the seawater-induced OSTF1 mRNA abundance. Plasma osmolality decreased, while gill Na +/K +-ATPase activity was suppressed in the cortisol group in seawater compared to the sham group. This corresponded with a significant reduction in gill ionocyte size and Na +/K +-ATPase activity and protein expression after seawater exposure. Cortisol did not modify liver metabolism, but significantly suppressed gill metabolic capacity in seawater. Overall, cortisol adapts tilapia to a hyperosmotic shock associated with abrupt seawater exposure. This involves upregulation of OSTF1 gene expression and a concomitant suppression of branchial metabolism in tilapia.