Expression and distribution of Na, K-ATPase in gill and kidney of the spotted green pufferfish, Tetraodon nigroviridis, in response to salinity challenge

Freshwater (FW) spotted green pufferfish (Tetraodon nigroviridis) were transferred directly from a local aquarium to fresh water (FW; 0 per thousand ), brackish water (BW; 15 per thousand ), and seawater (SW; 35 per thousand ) conditions in the laboratory and reared for at least two weeks. No mortality was found. To investigate the efficient mechanisms of osmoregulation in the euryhaline teleost, distribution and expression of Na,K-ATPase (NKA) in gill and kidney of the pufferfish were examined and the osmolality, [Na+] and [Cl-] of the blood were assayed. The lowest levels of both relative protein abundance and activity were found to be exhibited in the BW group, and higher levels in the SW group than FW group. In all salinities, branchial NKA immunoreactivity was found in epithelial cells of the interlamellar region of the filament and not on the lamellae. Relative abundance of kidney NKA alpha-subunit, as well as the NKA activity, was found to be higher in the FW pufferfish than fish in BW or SW. Renal NKA appeared in the epithelial cells of distal tubules, proximal tubules, and collecting tubules, but not in glomeruli, in fish groups of various salinities. Plasma osmolality and chloride levels were significantly lower in FW pufferfish than those in BW and SW, whereas plasma sodium did not differ among the groups. Although identical distributions of NKA were found in either gill or kidney of FW-, BW- or SW-acclimated spotted green pufferfish, differential NKA expression in fish of various salinity groups was associated with physiological homeostasis (stable blood osmolality), and illustrated the impressive osmoregulatory ability of this freshwater and estuarine species in response to salinity challenge.     

Elevated Na+/K+-ATPase responses and its potential role in triggering ion reabsorption in kidneys for homeostasis of marine euryhaline milkfish (Chanos chanos) when acclimated to hypotonic fresh water

The milkfish (Chanos chanos) is an economic species in Southeast Asia. In Taiwan, the milkfish are commercially cultured in environments of various salinities. Na⁺/K⁺-ATPase (NKA) is a key enzyme for fish iono- and osmoregulation. When compared with gills, NKA and its potential role were less examined by different approaches in the other osmoregulatory organs (e.g., kidney) of euryhaline teleosts. The objective of this study was to investigate the correlation between osmoregulatory plasticity and renal NKA in this euryhaline species. Muscle water contents (MWC), plasma, and urine osmolality, kidney histology, as well as distribution, expression (mRNA and protein), and specific activity of renal NKA were examined in juvenile milkfish acclimated to fresh water (FW), seawater (SW 35‰), and hypersaline water (HSW 60‰) for at least two weeks before experiments. MWC showed no significant difference among all groups. Plasma osmolality was maintained within the range of physiological homeostasis in milkfish acclimated to different salinities, while, urine osmolality of FW-acclimated fish was evidently lower than SW- and HSW-acclimated individuals. The renal tubules were identified by staining with periodic acid Schiff’s reagent and hematoxylin. Moreover, immunohistochemical staining showed that NKA was distributed in the epithelial cells of proximal tubules, distal tubules, and collecting tubules, but not in glomeruli, of milkfish exposed to different ambient salinities. The highest abundance of relative NKA α subunit mRNA was found in FW-acclimated milkfish rather than SW- and HSW-acclimated individuals. Furthermore, relative protein amounts of renal NKA α and β subunits as well as NKA-specific activity were also found to be higher in the FW group than SW and the HSW groups. This study integrated diverse levels (i.e., histological distribution, gene, protein, and specific activity) of renal NKA expression and illustrated the potential role of NKA in triggering ion reabsorption in kidneys of the marine euryhaline milkfish when acclimated to a hypotonic FW environment.     

Branchial osmoregulatory response to salinity in the gilthead sea bream, Sparus auratus

The branchial osmoregulatory response of gilthead sea bream (Sparus auratus L.) to short-term (2-192 hr) and long-term (2 weeks) exposure to different environmental salinities (5 per thousand, 15 per thousand, 25 per thousand, 38 per thousand and 60 per thousand) was investigated. A "U-shaped" relationship was observed between environmental salinity and gill Na+,K+ -ATPase activity in both long- and short-term exposure to altered salinity, with the increase in activity occurring between 24 and 96 hr after the onset of exposure. Plasma osmolality and plasma ions (sodium, chloride, calcium and potassium) showed a tendency to increase in parallel with salinity. These variables only differed significantly (P<0.05) in fish adapted to 60 per thousand salinity with respect to fish adapted to full-strength sea-water (SW). Plasma glucose remained unchanged whereas plasma lactate was elevated at 5 per thousand and 60 per thousand. Muscle water content (MWC) was significantly lower in fish adapted to 60 per thousand. Chloride cells (CC) were only present on the surface of the gill filaments and absent from the secondary lamellae. CC distribution was not altered by external salinity. However, the number and size of CC were significantly increased at salinity extremes (5 per thousand and 60 per thousand), whereas fish exposed to intermediate salinities (15 per thousand and 25 per thousand) had fewer and smaller cells. Furthermore, the CC of fish exposed to diluted SW became rounder whereas they were more elongated in fish in full-strength and hypersaline SW. This is consistent with previous reports indicating the existence of two CC types in euryhaline fish. At likely environmental salinities, gilthead sea bream show minor changes in plasma variables and the effective regulation of gill Na+,K+ -ATPase. However, at very low salinities both haemodilution and up-regulation of gill Na+,K+ -ATPase predict a poor adaptation most likely related to deficiency or absence of specific components of the CC important for ion xuptake.     

Differential expression of branchial Na+/K(+)-ATPase of two medaka species, Oryzias latipes and Oryzias dancena, with different salinity tolerances acclimated to fresh water, brackish water and seawater

Previous studies on non-diadromous euryhaline teleosts introduced a hypothesis that the lowest level of gill Na⁺/K⁺-ATPase (NKA) activity occurs in the environments with salinity close to the primary natural habitats of the studied species. To provide more evidence of the hypothesis, two medaka species, Oryzias latipes and O. dancena, whose primary natural habitats are fresh water (FW) and brackish water (BW) environments, respectively, were compared from levels of mRNA to cells in this study. The plasma osmolalities of O. latipes and O. dancena were lowest in the FW individuals. The muscle water contents of O. latipes decreased with elevated external salinities, but were constant among FW-, BW-, and seawater (SW)-acclimated O. dancena. Expression of NKA, the primary driving force of ion transporters in gill ionocytes, revealed different patterns in the two Oryzias species. The highest NKA α-subunit mRNA abundances were found in the gills of the SW O. latipes and the FW O. dancena, respectively. The pattern of NKA activity and α-subunit protein abundance in the gills of O. latipes revealed that the FW group was the lowest, while the pattern in O. dancena revealed that the BW group was the lowest. Immunohistochemical staining showed similar profiles of NKA immunoreactive (NKIR) cell activities (NKIR cell number × cell size) in the gills of these two species among FW, BW, and SW groups. Taken together, O. latipes exhibited better hyposmoregulatory ability, while O. dancena exhibited better hyperosmoregulatory ability. Our results corresponding to the hypothesis indicated that the lowest branchial NKA activities of these two medaka species were found in the environments with salinities similar to their natural habitats.     

Branchial chloride cells in sea bass (Dicentrarchus labrax) adapted to fresh water,seawater, and doubly concentrated seawater

Branchial chloride cells (CC) were studied in sea bass (Dicentrarchus labrax) maintained in seawater (SW: 35 per thousand) or gradually adapted to and subsequently maintained in fresh water (0.2 per thousand) or doubly concentrated seawater (DSW: 70 per thousand). Changes were observed in the location, number, and structure of CCs, that were discriminated by light, scanning, and transmission electron microscopy, as well as by immunofluorescence on the basis of their high Na(+)/K(+)-ATPase antigen content. The number of CCs increased in both fresh water and doubly concentrated seawater compared to control fish maintained in SW. In both experimental conditions, these cells were found on the gill filament (as in control fish) and even on the lamellae, especially in hypersaline conditions. Structural changes concerned the shapes and sizes of CCs and their apical outcrops and particularly the structures of their functional complexes (mitochondria, tubular system, and endoplasmic reticulum), which developed significantly in DSW adapted fish. The changes in the expression of the Na(+)/K(+)-ATPase were evaluated by assessing the enzyme's density at the ultrastructural level following immunogold labeling. This parameter was significantly higher in doubly concentrated seawater. The adaptative significance of the quantitative and morphofunctional changes in branchial chloride cells is discussed in relation to the original osmoregulatory strategy of this marine euryhaline teleost.     

Short-term effects of hyposmotic shock on Na+/K+ -ATPase expression in gills of the euryhaline milkfish, Chanos chanos

Changes in expression of gill Na+/K+ -ATPase (NKA) on a short-term (96 h) time-course following hyposmotic shock (direct transfer to fresh water) of the euryhaline, marine milkfish were studied on gene, protein, and cell levels in this paper. Plasma osmolality and [Na+] responded with rapid declines in 3 h post-transfer yet, thereafter, remained constant. Plasma [Cl-] gradually fell to a significantly lower level at 6 h post-transfer. Gills responded to hyposmotic shock by a dual phase enhancement of NKA activity and protein abundance; (a) Before 24 h: NKA activity increased as early as 3 h and reached a maximum level from 6 to 12 h post-transfer coincided with the sustained lower levels of plasma osmolality, [Na+], and [Cl-] since 3 h post-transfer. This was followed by a gradual rise in alpha-subunit protein levels that peaked at 12 h post-transfer. Meanwhile, alpha-mRNA of NKA did no show significant change. (b) After 24 h: NKA activity as well as the amounts of alpha-subunit mRNA and protein increased significantly. Direct freshwater transfer induced a prompt and significant decrease of NKA immunoreactive (NKIR) cell abundance in filaments before 24 h, followed by a significant increase after 24 h due to their development in filaments and lamellae. Increased number of NKIR cells after 24 h of hyposmotic shock may occur in conjunction with rise of NKA activity as well as alpha-subunit mRNA and protein abundance. In conclusion, milkfish is able to avoid an excessive drop in plasma ions immediately upon hyposmotic shock and maintain plasma ions on a marginal lower level in fresh water. Notably, the initial increase in NKA activity (adjustive phase; 3-12 h) and delayed increase in NKA mRNA and protein abundance (regulatory phase; 48-96 h) indicate the importance of a higher level of the gill enzyme in milkfish upon hyposmotic shock.     

Age/size effects on juvenile green sturgeon, Acipenser medirostris, oxygen consumption, growth, and osmoregulation in saline environments

The green sturgeon, Acipenser medirostris, is an anadromous species that migrates from freshwater (FW) to seawater (SW) relatively early in its life history, although the ages and sizes of juveniles at SW entry are not known. Developmental constraints of osmoregulatory organs may either prohibit (i.e., due to salinity tolerance limits) or minimize (i.e., due to substantial osmoregulatory or ionoregulatory energetic costs) SW entry in small fish. Interestingly, larger green sturgeon are often encountered in brackish water (BW) estuaries, perhaps due to an energetic advantage in occupying these near-isosmotic environments. To test hypotheses concerning fish-size effects on the energetic costs of occupying habitats of different salinities, we measured oxygen consumption rates in green sturgeon representing three age groups (100, 170, and 533 days post hatch; dph), which were acclimated for 5 weeks to one of three salinities (FW, <3‰; BW, 10‰; or SW, 33‰). Also, after 7 weeks, final wet masses were compared and blood and muscle tissue samples were taken to assess osmoregulatory abilities. There were no differences in body-mass-adjusted oxygen consumption rates between any salinities or ages, indicating that the energetic costs were not prohibitively high to occupy any of these salinities. The only mortalities occurred in the 100 dph SW group, where 23% of the fish died, from apparent starvation. Final wet masses were comparable between FW and BW for each age group and with the 533 dph SW group, but were lower in SW groups at 100 and 170 dph. Similarly, osmoregulatory abilities, in terms of plasma osmolality, Na⁺, K⁺, lactate, and protein concentrations, and muscle water content, were comparable in FW and BW groups at all ages, and with the SW group at 533 dph. These results indicated an age/body size effect in hyperosmotic adaptability, and that juvenile green sturgeon may be found in FW or BW at any age, but only have the ability to enter SW by 1.5 years (75 cm, 1.5 kg) of age.     

Effect of salinity on expression of branchial ion transporters in striped bass (Morone saxatilis)

The time course of osmoregulatory adjustments and expressional changes of three key ion transporters in the gill were investigated in the striped bass during salinity acclimations. In three experiments, fish were transferred from fresh water (FW) to seawater (SW), from SW to FW, and from 15-ppt brackish water (BW) to either FW or SW, respectively. Each transfer induced minor deflections in serum [Na+] and muscle water content, both being corrected rapidly (24 hr). Transfer from FW to SW increased gill Na+,K+-ATPase activity and Na+,K+,2Cl- co-transporter expression after 3 days. Abundance of Na+,K+-ATPase alpha-subunit mRNA and protein was unchanged. Changes in Na+,K+,2Cl- co-transporter protein were preceded by increased mRNA expression after 24 hr. Expression of V-type H+-ATPase mRNA decreased after 3 days. Transfer from SW to FW induced no change in expression of gill Na+,K+-ATPase. However, Na+,K+,2Cl- co-transporter mRNA and protein levels decreased after 24 hr and 7 days, respectively. Expression of H+-ATPase mRNA increased in response to FW after 7 days. In BW fish transferred to FW and SW, gill Na+,K+-ATPase activity was stimulated by both challenges, suggesting both a hyper- and a hypo-osmoregulatory response of the enzyme. Acclimation of striped bass to SW occurs on a rapid time scale. This seems partly to rely on the relative high abundance of gill Na+,K+-ATPase and Na+,K+,2Cl- co-transporter in FW fish. In a separate study, we found a smaller response to SW in expression of these ion transport proteins in striped bass when compared with the less euryhaline brown trout. In both FW and SW, NEM-sensitive gill H+-ATPase activity was negligible in striped bass and approximately 10-fold higher in brown trout. This suggests that in striped bass Na+-uptake in FW may rely more on a relatively high abundance/activity of Na+,K+-ATPase compared to trout, where H+-ATPase is critical for establishing a thermodynamically favorable gradient for Na+-uptake.     

Arginine vasotocin, isotocin and melatonin responses following acclimation of gilthead sea bream (Sparus aurata) to different environmental salinities

Gilthead sea bream (Sparus aurata) is a euryhaline species with a capacity to cope with demands in a wide range of salinities and thus is a perfect model-fish to study osmoregulatory responses to salinity-adaptive processes and their hormonal control. Immature sea bream acclimated to different salinities, i.e. SW (38 per thousand), LSW (5 per thousand) and HSW (55 per thousand), were kept at 18 degrees C under natural photoperiod. Arginine vasotocin (AVT) and isotocin (IT) in plasma and pituitary were determined by HPLC. Plasma melatonin (Mel) was assayed by RIA. Plasma osmolality, ion concentrations (Na(+), K(+), Ca(2+), Cl(-)) and Na(+),K(+)-ATPase activity in gill were measured. A steady increase in plasma AVT, along with increasing water salinity was observed. Pituitary IT concentration in HSW-acclimated fish was significantly higher than that in LSW group. AVT/IT secretory system of sea bream does appear to be involved in the mechanism of long-term acclimation to different salinities. The distinct roles and control mechanisms of both nonapeptides are suggested. Plasma Mel was significantly higher in LSW compared with both HSW and SW groups. Data indicate that the changes in Mel level are linked to osmoregulation. Further studies are required to elucidate a complex role of AVT, IT and Mel in sea bream osmoregulation.     

Phenotypic changes in mitochondrion-rich cells and responses of Na+/K+-ATPase in gills of tilapia exposed to deionized water

The aim of this study was to illustrate the phenotypic modification of mitochondrion-rich (MR) cells and Na(+)/K(+)-ATPase (NKA) responses, including relative protein abundance, specific activity, and immunolocalization in gills of euryhaline tilapia exposed to deionized water (DW) for one week. The plasma osmolality was not significantly different between tilapia of the local fresh water (LFW) group and DW group. Remodeling of MR cells occurred in DW-exposed fish. After transfer to DW for one week, the relative percentage of subtype-I (wavy-convex) MR cells with apical size ranging from 3 to 9 microm increased and eventually became the dominant MR cell subtype. In DW tilapia gills, relative percentages of lamellar NKA immunoreactive (NKIR) cells among total NKIR cells increased to 29% and led to significant increases in the number of NKIR cells. In addition, the relative protein abundance and specific activity of NKA were significantly higher in gills of the DW-exposed fish. Our study concluded that tilapia require the development of subtype-I MR cells, the presence of lamellar NKIR cells, and enhancement of NKA protein abundance and activity in gills to deal with the challenge of an ion-deficient environment.     

Time-course changes in the expression of Na, K-ATPase and the morphometry of mitochondrion-rich cells in gills of euryhaline tilapia (Oreochromis mossambicus) during freshwater acclimation

Changes in expression of Na, K-ATPase (NKA) and morphometry of mitochondrion-rich (MR) cells in gills of tilapia were investigated on a 96-hr time course following transfer from seawater (SW) to fresh water (FW). A transient decline in plasma osmolality and Na+, Cl- concentrations occurred from 3 hrs onward. Gills responded to FW transfer by decreasing NKA activity as early as 3 hrs from transfer. This response was followed by a significant decrease in the NKA isoform alpha1-mRNA abundance, which was detected by real-time PCR at 6 hrs post transfer. Next, a decrease of alpha1-protein amounts were observed from 6 hrs until 24 hrs post transfer. Additionally, during the time course of FW transfer, modifications in number and size of subtypes of gill MR cells were observed although no significant difference was found in densities of all subtypes of MR cells. These modifications were found as early as 3 hrs, evident at 6 hrs (exhibition of 3 subtypes of MR cells), and mostly completed by 24 hrs post transfer. Such rapid responses (in 3 hrs) as concurrent changes in branchial NKA expression and modifications of MR cell subtypes are thought to improve the osmoregulatory capacity of tilapia in acclimation from hypertonic SW to hypotonic FW.     

The effect of environmental salinity on the protein expression of Na+/K+-ATPase, Na+/K+/2Cl- cotransporter, cystic fibrosis transmembrane conductance regulator, anion exchanger 1, and chloride channel 3 in gills of a euryhaline teleost, Tetraodon nigroviridis

Chloride transport mechanisms in the gills of the estuarine spotted green pufferfish (Tetraodon nigroviridis) were investigated. Protein abundance of Na(+)/K(+)-ATPase (NKA) and the other four chloride transporters, i.e., Na(+)/K(+)/2Cl(-) cotransporter (NKCC), cystic fibrosis transmembrane conductance regulator (CFTR), Cl(-)/HCO(3)(-) anion exchanger 1 (AE1), and chloride channel 3 (CLC-3) in gills of the seawater- (SW; 35 per thousand) or freshwater (FW)-acclimatized fish were examined by immunoblot analysis. Appropriate negative controls were used to confirm the specificity of the antibodies to the target proteins. The relative protein abundance of NKA was higher (i.e., 2-fold) in gills of the SW group compared to the FW group. NKCC and CFTR were expressed in gills of the SW group but not in the FW group. In contrast, the levels of relative protein abundance of branchial AE1 and CLC-3 in the FW group were 23-fold and 2.7-fold higher, respectively, compared to those of the SW group. This study is first of its kind to provide direct in vivo evidence of the protein expression of CLC-3 in teleostean gills, as well as to examine the simultaneous protein expression of the Cl(-) transporters, especially AE1 and CLC-3 of FW- and SW-acclimatized teleosts. The differential protein expression of NKA, chloride transporters in gills of the FW- and SW-acclimatized T. nigroviridis observed in the present study shows their close relationship to the physiological homeostasis (stable blood osmolality), as well as explains the impressive ionoregulatory ability of this euryhaline species in response to salinity challenges.     

Sodium or potassium ions activate different kinetics of gill Na, K-ATPase in three seawater- and freshwater-acclimated euryhaline teleosts

The effects of [Na(+)] or [K(+)] on Na, K-ATPase activity of FW-acclimated and SW-acclimated tilapia, puffer and milkfish were examined in gill homogenates. [Na(+)] or [K(+)] stimulated Na, K-ATPase hydrolyzing ATP in all experimental groups. ATP hydrolysis stimulated by [Na(+)] or [K(+)] followed Michaelian-Menten kinetics. Km values for [K(+)] (i.e., Km(K)), were lower in SW- than FW-acclimated tilapia and puffer fishes (tilapia: 8.69+/-0.22 vs. 11.93+/-1.17 mM; puffer: 13.51+/-1.39 vs. 30.52+/-2.66 mM). Km values for [Na(+)] (i.e., Km(Na)), were lower in FW- than SW-acclimated milkfish (3.76+/-0.54 vs. 7.55+/-1.08 mM). These data suggest that [K(+)] stimulates ATP hydrolysis to rates higher in SW- than FW-acclimated tilapia and puffer fishes, while [Na(+)] stimulated ATP hydrolysis at rates higher in FW- than SW-acclimated milkfish. This is the first demonstration that Na, K-ATPase activity of euryhaline tilapia, puffer, and milkfish modulated by [Na(+)] or [K(+)] have different effects between FW- and SW-acclimated groups. Such responses as changes in properties of branchial Na, K-ATPase may contribute to improve the osmoregulatory capacity of tilapia, puffer and milkfish to acclimate in seawater and fresh water.     

The ClC-3 chloride channel and osmoregulation in the European Sea Bass, Dicentrarchus labrax

Dicentrarchus labrax migrates between sea (SW), brackish and fresh water (FW) where chloride concentrations and requirements for chloride handling change: in FW, fish absorb chloride and restrict renal losses; in SW, they excrete chloride. In this study, the expression and localization of ClC-3 and Na⁺/K⁺-ATPase (NKA) were studied in fish adapted to SW, or exposed to FW from 10 min to 30 days. In gills, NKA-α1 subunit expression transiently increased from 10 min and reached a stabilized intermediate expression level after 24 h in FW. ClC-3 co-localized with NKA in the basolateral membrane of mitochondria-rich cells (MRCs) at all conditions. The intensity of MRC ClC-3 immunostaining was significantly higher (by 50 %) 1 h after the transfer to FW, whereas the branchial ClC-3 protein expression was 30 % higher 7 days after the transfer as compared to SW. This is consistent with the increased number of immunopositive MRCs (immunostained for NKA and ClC-3). However, the ClC-3 mRNA expression was significantly lower in FW gills. In the kidney, after FW transfer, a transient decrease in NKA-α1 subunit expression was followed by significantly higher stable levels from 24 h. The low ClC-3 protein expression detected at both salinities was not observed by immunocytochemistry in the SW kidney; ClC-3 was localized in the basal membrane of the collecting ducts and tubules 7 and 30 days after transfer to FW. Renal ClC-3 mRNA expression, however, seemed higher in SW than in FW. The potential role of this chloride channel ClC-3 in osmoregulatory and osmosensing mechanisms is discussed.     

Morphological and physiological traits of Mediterranean sticklebacks living in the Camargue wetland (Rhone river delta)

Three-spined sticklebacks (Gasterosteus aculeatus L.) living at the southern limit of the species distribution range could possess specific morphological and physiological traits that enable these fish to live at the threshold of their physiological capacities. Morphological analysis was carried out on samples of sticklebacks living in different saline habitats of the Camargue area (Rhone delta, northern Mediterranean coast) obtained from 1993 to 2017. Salinity acclimation capacities were also investigated using individuals from freshwater-low salinity drainage canals and from mesohaline–euryhaline lagoons. Fish were maintained in laboratory conditions at salinity values close to those of their respective habitats: low salinity (LS, 5‰) or seawater (SW, 30‰). Fish obtained from a mesohaline brackish water lagoon (BW, 15‰) were acclimated to SW or LS. Oxygen consumption rates and branchial Na⁺/K⁺-ATPase (NKA) activity (indicator of fish osmoregulatory capacity) were measured in these LS or SW control fish and in individuals subjected to abrupt SW or LS transfers. At all the studied locations, only the low-plated “leiurus” morphotype showed no spatial or temporal variations in their body morphology. Gill rakers were only longer and denser in fish sampled from the LS–freshwater (FW) drainage canals. All fish presented similar physiological capacities. Oxygen consumption rates were not influenced by salinity challenge except in SW fish transferred to LS immediately and 1 h after transfer. However, and as expected, gill NKA activity was salinity dependent. Sticklebacks of the Camargue area sampled from habitats with contrasted saline conditions are homogenously euryhaline, have low oxygen consumption rates and do not appear to experience significantly greater metabolic costs when challenged with salinity. However, an observed difference in gill raker length and density is most probably related to the nutritional condition of their habitat, indicating that individuals can rapidly acclimatize to different diets.     

The effect of environmental hypercapnia and salinity on the expression of NHE-like isoforms in the gills of a euryhaline fish (Fundulus heteroclitus)

The current models for branchial acid excretion in fishes include Na(+)/H(+) exchange and the electrogenic excretion of H+ via H+-ATPase. The predominant route of acid excretion in some freshwater fishes is thought to be via the H+-ATPase/Na+ channel system. The euryhaline Fundulus heteroclitus may not fit this profile even when adapted to freshwater (FW). We hypothesize that the Na+/H+ exchanger (NHE) in this species may play a predominant role in acid-base regulation for both marine and FW adapted animals. Acidosis induced by ambient hypercapnia (1% CO2 in air), resulted in an increase in net H+ excretion to the water in F. heteroclitus pre-adapted to FW, brackish (isoosmotic; BW) and seawater (SW). Both FW and SW adapted mummichogs were tested for NHE protein expression using mammalian NHE antibodies, and we identified NHE-like immunoreactive proteins in gill membrane preparations from both groups. Hypercapnia induced a approximately three-fold elevation in gill NHE2-like protein in FW animals but SW adapted fish showed inconsistent NHE3-like protein expression. There was no change in NHE-1 levels in FW fish. In contrast, SW animals demonstrated a significant increase in both NHE1 and NHE3-like proteins following hypercapnia but limited expression of the NHE2 protein. We hypothesize that different isoforms of NHE may be preferentially expressed depending on the salinity to which the animals are adapted. Net H+ transfers during acidosis may be driven, at least in part by the action of these transporters.     

The ultrastructural characterization of mitochondria‐rich cells as a response to variations in salinity in two types of teleostean pseudobranch: milkfish (Chanos chanos) and Mozambique tilapia (Oreochromis mossambicus)

The pseudobranchs of two euryhaline teleost species, the milkfish (Chanos chanos ) and the Mozambique tilapia (Oreochromis mossambicus ), were studied after acclimization to different salinities using optical and electron microscopy. The milkfish pseudobranch was the lamellae‐free type, with separate lamellae along the filaments containing two groups of mitochondria (Mt)‐rich cells: chloride cells (CCs) and pseudobranch type cells (PSCs). Conversely, the tilapia pseudobranch was the embedded type, covered with connective tissues and with only one group of Mt‐rich PSCs. Chloride cells were identified according to the apical openings and branched tubular networks around randomly distributed and diversely shaped Mt. Pseudobranchs type cells, however, were characterized according to the orderly arrangement of parallel tubules around closely packed Mt; both the tubules and the Mt were distributed in the vascular side of the cell, but were absent from the apical region. Compared with those of seawater (SW)‐acclimated milkfish, the pseudobranchial lamellae of freshwater (FW) specimens were longer on average, and the Mt of the CCs had fewer cristae, were less electron‐dense, and were often vacuolated. The Mt in the PSCs of FW‐acclimated milkfish and tilapia were larger and more electron‐dense than those of their SW‐acclimated counterparts; in addition, more tubules were found to aggregately surround the Mt and basolateral membranes in the PSCs of fish from the hypo‐osmotic environment. Conversely, the PSCs of tilapia were periodic acid‐Schiff (PAS)‐positive, and Mt in PSCs were concentrated with more parallel arrays of the tubule system than those of milkfish. Therefore, salinity‐dependent changes in the ultrastructures of PSCs suggest their potential role in energy metabolism of both lamellae‐free and embedded pseudobranchs, whereas the PAS‐positive staining characteristics suggest a role in releasing or storaging polysaccharides in the embedded pseudobranch. J. Morphol. 278:390–402, 2017. © 2017 Wiley Periodicals, Inc.