Expression of aquaporin 3 in gills of the Atlantic killifish (Fundulus heteroclitus): Effects of seawater acclimation

Estuarine fish, such as the Atlantic killifish (Fundulus heteroclitus), are constantly and rapidly exposed to changes in salinity. Although ion transport in killifish gills during acclimation to increased salinity has been studied extensively, no studies have examined the role of aquaglyceroporin 3 (AQP3), a water, glycerol, urea, and ammonia transporter, during acclimation to increased salinity in this sentinel environmental model organism. The goal of this study was to test the hypothesis that transfer from freshwater to seawater decreases AQP3 gene and protein expression in the gill of killifish. Transfer from freshwater to seawater decreased AQP3 mRNA in the gill after 1 day, but had no effect on total gill AQP3 protein abundance as determined by western blot. Quantitative confocal immunocytochemistry confirmed western blot studies that transfer from freshwater to seawater did not change total AQP3 abundance in the gill; however, immunocytochemistry revealed that the amount of AQP3 in pillar cells of secondary lamellae decreased in seawater fish, whereas the amount of AQP3 in mitochondrion rich cells (MRC) in primary filaments of the gill increased in seawater fish. This response of AQP3 expression is unique to killifish compared to other teleosts. Although the role of AQP3 in the gill of killifish has not been completely elucidated, these results suggest that AQP3 may play an important role in the ability of killifish to acclimate to increased salinity.     

Effects of spironolactone and RU486 on gene expression and cell proliferation after freshwater transfer in the euryhaline killifish

We have explored the possible mechanisms by which mineralocorticoid (MR) and glucocorticoid (GR) receptors regulate the response to freshwater transfer in the gills of the euryhaline killifish Fundulus heteroclitus. Killifish were implanted with RU486 (GR antagonist) or spironolactone (MR antagonist) at doses of 0.1–1.0 mg g⁻¹, and subsequently transferred from 10‰ brackish water to freshwater. Compared to brackish water sham fish, mRNA expression of CFTR and NKCC1 decreased in the gills of sham fish transferred to freshwater, whereas Na⁺,K⁺–ATPase α_1a mRNA expression and α protein abundance, as well as cell proliferation (detected using BrdU) increased. Spironolactone inhibited the normal increase in cell proliferation and Na⁺,K⁺-ATPase expression after freshwater transfer. RU486 increased plasma cortisol levels and may have slightly inhibited Na⁺,K⁺–ATPase activity, but did not change α _1a expression. RU486 had no effect on cell proliferation in the non-lamellar region of the gills, but increased proliferation in the lamellar region. Neither antagonist inhibited the suppression of CFTR or NKCC1 expression after freshwater transfer. Glucocorticoid receptor expression was reduced in all sham and antagonist treatments compared to untreated controls, but no other consistent differences were observed. The effects of spironolactone suggest that MR is important for regulating ion transport in killifish gills after freshwater transfer.     

Recruitment and degeneration of mitochondrion-rich cells in the gills of Mozambique tilapia Oreochromis mossambicus during adaptation to a hyperosmotic environment

Cellular recruitment and degeneration of branchial mitochondrion-rich (MR) cells were examined in Mozambique tilapia transferred from hypoosmotic to hyperosmotic water. To examine apoptosis in the gills associated with salinity change, tilapia were directly transferred from freshwater to 70% seawater. The TUNEL assay showed that apoptotic cells in the gills were significantly increased at 1 day after transfer, which was supported by an electron-microscopic observation that gill MR cells underwent morphological changes characteristic of apoptosis such as an irregularly shaped electron-dense nucleus and distension of the tubular system. To further examine MR-cell recruitment, freshwater-acclimated tilapia were transferred either to freshwater or to 70% seawater after BrdU injection. Immunohistochemical detection of BrdU-labeled nuclei and Na(+)/K(+)-ATPase-rich MR cells allowed us to classify BrdU-labeled MR cells into two subtypes: a single MR cell and an MR-cell complex. Although newly generated single MR cells were observed similarly in both freshwater and 70% seawater-transferred fish, the density of MR-cell complexes was much higher in 70% seawater than in freshwater. Our findings indicated that transfer from hypoosmotic to hyperosmotic water enhanced apoptosis of freshwater-type MR cells, resulting in reduction in hyperosmoregulatory ability for freshwater adaptation, and stimulated the recruitment of MR-cell complexes to develop hypoosmoregulatory ability for seawater adaptation.     

Changes in the levels of chloride cells and (Na+ + K+)-dependent ATPase in the gills of yellow and silver eels adapting to seawater.

Changes were measured in the numbers of chloride cells and the levels of (Na+ + K+)-DEPENDENT ATPase in the gills of immature, yellow eels and mature, silver eels during adaptation from freshwater to seawater. The percentage of chloride cells in yellow eels more than doubled after six days in seawater; at this time the specific activity and concentration of (Na+ + K+)-dependent ATPase in gills start to increase in parallel to reach maxima after two weeks that are 2.5 times the starting values. It is concluded that adaptation of yellow eels to seawater involves an increase in the numbers of chloride cells in gills as well as an increased amount of (Na+ + K+)-dependent ATPase per chloride cell. Mature silver eels in freshwater had essentially the same numbers of chloride cells and the same specific activity of the enzyme in the gills as yellow eels fully adapted to seawater. Transferring silver eels to seawater did not alter the percentage of chloride cells in gills although the level of (Na+ + K+)-dependent ATPase and its specific activity increased slightly. Thus, although the silver eel is better prepared for life in seawater than the yellow eel, it still has to attain an increased level of (Na+ + K+)-dependent ATPase in its chloride cells to be fully adapted to seawater.     

Swimming performance and energetics as a function of temperature in killifish Fundulus heteroclitus

Populations of the common killifish Fundulus heteroclitus are found along a latitudinal temperature gradient in habitats with high thermal variability. The objectives of this study were to assess the effects of temperature and population of origin on killifish swimming performance (assessed as critical swimming speed, U(crit)). Acclimated fish from northern and southern killifish populations demonstrated a wide zone (from 7 degrees to 33 degrees C) over which U(crit) showed little change with temperature, with performance declining significantly only at lower temperatures. Although we observed significant differences in swimming performance between a northern and a southern population of killifish in one experiment, with northern fish having an approximately 1.5-fold-greater U(crit) than southern fish across all acclimation temperatures, we were unable to replicate this finding in other populations or collection years, and performance was consistently high across all populations and at both low (7 degrees C) and high (23 degrees C) acclimation temperatures. The poor swimming performance of southern killifish from a single collection year was correlated with low muscle [glycogen] rather than with other indicators of fuel stores or body condition. Killifish acclimated to 18 degrees C and acutely challenged at temperatures of 5 degrees , 18 degrees , 25 degrees , or 34 degrees C showed modest thermal sensitivity of U(crit) between 18 degrees and 34 degrees C, with performance declining substantially at 5 degrees C. Thus, much of the zone of relative thermal insensitivity of swimming performance is intrinsic in this species rather than acquired as a result of acclimation. These data suggest that killifish are broadly tolerant of changing temperatures, whether acute or chronic, and demonstrate little evidence of local adaptation in endurance swimming performance in populations from different thermal habitats.     

Characterization of Na/K-ATPase in Macrobrachium rosenbergii and the effects of changing salinity on enzymatic activity

A ouabain-sensitive Na/K-ATPase kinetic assay system based on the hydrolysis of ATP and the oxidation of NADH was adapted in order to characterize enzymatic activity in gills and examine the effects of changing salinity in Macrobrachium rosenbergii. Maximum inhibition by ouabain occurred at a concentration of 1.4 mM, and the K(m) of the reaction was 0.2 mM. In a first experiment, animals were acclimated to freshwater, 1/3 seawater, 2/3 seawater and full seawater for up to 1 week. Na/K-ATPase activity in front gills was 1. 62+/-0.19 micromol ADP/mg protein per h in freshwater, and was seen to increase slightly in 1/3 seawater (1.88+/-0.19 micromol ADP/mg protein per h) and 2/3 seawater (2.09+/-0.24 micromol ADP/mg protein per h), decreasing slightly in full seawater (1.92+/-0.43 micromol ADP/mg protein per h); however, differences were not significant. Back gills showed slightly higher levels, and a similar pattern of Na/K-ATPase activity. In a second experiment, animals were acclimated to 1/3 seawater and 2/3 seawater, and then transferred to freshwater. However, no changes in activity were seen, indicating that exposure to dilute media did not effect enzymatic activity. Whereas Na/K-ATPase is important in osmoregulatory function in marine euryhaline crustaceans, it may not play a significant role in adaptation in freshwater crustaceans that inhabit a more narrow range of salinities.     

Comparative ionic flux and gill mucous cell histochemistry: effects of salinity and disease status in Atlantic salmon (Salmo salar L.)

Two experiments were conducted to assess the physiological effects of freshwater exposure and amoebic gill disease (AGD) in marine Atlantic salmon (Salmo salar L.). The first experiment monitored marine salmon during a 3 h freshwater exposure, the standard treatment for AGD in Tasmania. The second experiment described the gill mucous cell histochemistry for freshwater adapted and seawater acclimated fish (AGD affected and unaffected) for possible correlations to ionoregulation. When exposed to freshwater, marine Atlantic salmon experienced a minor ionoregulatory dysfunction represented by a net efflux of Cl(-) ions at 3 h. AGD affected fish experienced the net efflux of Cl(-) ions 1 h sooner, and had a significantly greater net efflux of total ammonia. Changes to gill mucous cell populations corresponded to differing salinity and the presence of AGD. In AGD affected fish, these populations significantly differed between lesion and non-lesion associated areas of the gill filament. Our results have shown changes in the ionoregulatory capacity of Atlantic salmon due to freshwater exposure and AGD. Gill mucous cell histochemistry indicates the potential importance of the mucous layer in ionoregulation and disease. In comparison to previous studies on rainbow trout, these results suggest that Atlantic salmon have a greater short-term ionoregulatory capacity.     

Intestinal transport following transfer to increased salinity in an anadromous fish (Oncorhynchus mykiss)

The ability to transition from freshwater to seawater environments is an intrinsic requirement of the life history of some fish species, including the anadromous rainbow trout (Oncorhynchus mykiss). The differences between hyper- and hypoosmoregulation are developed quickly (in hours to days), and at all scales, from gene expression to organ function. In this study, intestinal ion and water transport was examined in O. mykiss following acute transfer from freshwater (FW) to 70% seawater (SW). Plasma [Mg2+] increased at 24h post-transfer but recovered by 72 h. In the intestinal fluids, total CO? was found to increase with SW exposure/acclimation, while [Na+] decreased after 24h of SW exposure. Overall, in vitro experiments demonstrated the importance of base secretion to epithelial water uptake, and suggested that the primary physiological adjustments occurred 24-72 h after acute SW transfer. The mRNA expression of ion transporters important for intestinal osmoregulation and maintenance of acid-base balance was also investigated. A Na+/H+ exchanger (NHE2) and anion exchanger (SLC26a6) were hypothesized to be involved in the transport of acid-base equivalents, Na+, and Cl?, but were not uniformly expressed across tissue samples, and expression, where present, did not change following salinity transfer. NHE1, however, was expressed in all examined tissues (gill, kidney, anterior intestine, and pyloric cecae), but exhibited no changes in expression following acute salinity transfer.     

Differential design for hopping in two species of wallabies

We explored molecular and morphological alteration in gill mitochondria-rich (MR) cells of Mozambique tilapia, Oreochromis mossambicus, acclimated to deionized freshwater (DFW), freshwater (FW), 1/3-diluted seawater (1/3 SW) and seawater (SW). Scanning electron microscopic observations revealed that the apical membrane of MR cells appeared as a flat or slightly projecting disk in DFW and FW, being larger in DFW than in FW. In contrast, the apical membrane typically formed a pit structure in 1/3 SW and SW. The mRNA expression levels of Na(+)/H(+) exchanger-3 (NHE3) and Na(+)/Cl(-) cotransporter (NCC) in the gills were increased with decreasing environmental salinity, whereas Na(+)/K(+)/2Cl(-) cotransporter-1a (NKCC1a) expression was upregulated by increasing salinity. Immunofluorescence staining showed that the MR cell population of DFW- and FW-acclimated tilapia consisted mostly of MR cells with apical NHE3 and those with apical-NCC; MR cells with basolateral NKCC1a dominated in SW-acclimated tilapia. These results indicated that apical-NHE3 and apical-NCC MR cells were ion-absorbing cells, and that basolateral-NKCC1a MR cells were ion-secreting cells. In fish acclimated to 1/3 SW, both ion-absorbing and secreting cells existed in the gills, suggesting that fish in near-isotonic water were equipped with mechanisms of both hyper- and hypoosmoregulation to prepare for environmental salinity changes.     

Wild Arctic char (Salvelinus alpinus) upregulate gill Na+,K+-ATPase during freshwater migration

The successful acclimation of eurhyhaline fishes from seawater to freshwater requires the gills to stop actively secreting ions and start actively absorbing ions. Gill Na(+),K(+)-ATPase is known to be an integral part of the active ion secretion model of marine fishes, but its importance in the active ion uptake model of freshwater fishes is less clear. This study, conducted in the high Arctic, examines gill Na(+),K(+)-ATPase regulation in wild anadromous arctic char returning to freshwater from the ocean. Gill Na(+),K(+)-ATPase activity, protein expression, and mRNA expression of Na(+),K(+)-ATPase isoforms alpha 1a and alpha 1b were monitored in arctic char at three points along their migration route to and from Somerset Island, Nunavut, Canada: out at sea (Whaler's Point), in seawater near the river mouth (Nat's Camp), and after entering the Union River. Arctic char collected from the Union River had more than twofold greater gill Na(+),K(+)-ATPase activity. This was associated with a significant increase (threefold) in Na(+),K(+)-ATPase isoform alpha 1a mRNA expression and a significant increase in plasma sodium and osmolality levels compared with seawater char. Compared with char sampled from Whaler's Point, Na(+),K(+)-ATPase isoform alpha 1b mRNA expression was decreased by approximately 50% in char sampled at Nat's Camp and the Union River. These results suggest that the upregulation of gill Na(+),K(+)-ATPase activity is involved in freshwater acclimation of arctic char and implicate a role for Na(+),K(+)-ATPase isoform alpha 1a in this process. In addition, we discuss evidence that arctic char go through a preparatory phase, or "reverse smoltification," before entering freshwater.     

Ambient salinity modulates the expression of sodium pumps in branchial mitochondria-rich cells of Mozambique tilapia,Oreochromis mossambicus

Na,K-ATPase (sodium pumps) provide the primitive driving force for ion transport in branchial epithelial cells. Immunoblots of epithelial homogenates of both seawater (SW)- and freshwater (FW)-adapted tilapia gills as well as rat brain homogenate, a positive control, revealed one major band with a molecular weight of about 100 kDa. SW-adapted tilapia gills possessed larger (about 2-fold) amounts of sodium pumps compared with FW-adapted tilapia gills. (3)H-ouabain binding representing functional binding sites of Na,K-ATPase was also higher (about 3.5-fold) in gills of SW-adapted tilapia compared to that of FW-adapted fish. Moreover, specific activities of SW fish were higher (about 2-fold) than those of FW fish. Double labeling of Na,K-ATPase and Con-A, a fluorescent marker of MR cells, in tilapia gills followed by analysis with confocal microscopy showed that sodium pumps were localized mainly in MR cells, including the SW type and different FW types. Although more-active expression of Na,K-ATPase was demonstrated in gills of SW-adapted tilapia, no significant differences in densities of apical openings of MR cells were found between SW- and FW-adapted fish. These results indicate that, during salinity challenge, tilapia develop more "functional" Na,K-ATPase in SW-type MR cells to meet physiological demands.     

Transient receptor potential vanilloid 4 in the European sea bass Dicentrarchus labrax: a candidate protein for osmosensing

The Transient Receptor Potential Vanilloid 4 (TRPV4) protein is a member of the TRP ion channels superfamily that has been proposed as a potential fish osmosensor in previous studies. TRPV4 has been widely studied in mammals, particularly for its involvement in sensing the hypotonicity. The European sea bass, Dicentrarchus labrax, is a euryhaline teleost that is exposed to salinity changes due to its migrations between the sea and estuaries/lagoons. TRPV4 expression and localization in D. labrax was studied in seawater (SW)-adapted fish and in fish exposed to freshwater (FW) over different time-courses from 10 min to 30 days. TRPV4 mRNA expression was detected in gills, kidney and brain. In gills, the expression increased significantly in FW from 24 h to 30 d. In contrast, in the kidney, the TRPV4 expression decreased from 10 min to 7d of exposure to FW and then it increased at 30 d. In the brain, its expression was relatively low in SW compared to other organs and a significant decrease occurred in FW. The TRPV4 protein was localized in the basement membranes in branchial lamellae, the cartilage of gills, the posterior pituitary gland and in the collecting ducts. Possible roles of TRPV4 are discussed.     

Morpholino gene knockdown in adult Fundulus heteroclitus: role of SGK1 in seawater acclimation

The Atlantic killifish (Fundulus heteroclitus) is an environmental sentinel organism used extensively for studies on environmental toxicants and salt (NaCl) homeostasis. Previous research in our laboratory has shown that rapid acclimation of killifish to seawater is mediated by trafficking of CFTR chloride channels from intracellular vesicles to the plasma membrane in the opercular membrane within the first hour in seawater, which enhances chloride secretion into seawater, thereby contributing to salt homeostasis. Acute transition to seawater is also marked by an increase in both mRNA and protein levels of serum glucocorticoid kinase 1 (SGK1) within 15 minutes of transfer. Although the rise in SGK1 in gill and its functional analog, the opercular membrane, after seawater transfer precedes the increase in membrane CFTR, a direct role of SGK1 in elevating membrane CFTR has not been established in vivo. To test the hypothesis that SGK1 mediates the increase in plasma membrane CFTR we designed two functionally different vivo-morpholinos to knock down SGK1 in gill, and developed and validated a vivo-morpholino knock down technique for adult killifish. Injection (intraperitoneal, IP) of the splice blocking SGK1 vivo-morpholino reduced SGK1 mRNA in the gill after transition from fresh to seawater by 66%. The IP injection of the translational blocking and splice blocking vivo-morpholinos reduced gill SGK1 protein abundance in fish transferred from fresh to seawater by 64% and 53%, respectively. Moreover, knock down of SGK1 completely eliminated the seawater induced rise in plasma membrane CFTR, demonstrating that the increase in SGK1 protein is required for the trafficking of CFTR from intracellular vesicles in mitochondrion rich cells to the plasma membrane in the gill during acclimation to seawater. This is the first report of the use of vivo-morpholinos in adult killifish and demonstrates that vivo-morpholinos are a valuable genetic tool for this environmentally relevant model organism.     

Time-course changes in the expression of Na+, K+-ATPase in gills and pyloric caeca of brown trout (Salmo trutta) during acclimation to seawater

Changes in protein and mRNA expression of Na(+),K(+)-ATPase in gills and pyloric caeca of brown trout were investigated on a detailed time course after transfer from freshwater to 25 ppt seawater (SW). A transient deflection in plasma osmolality and muscle water content lasting from 4 h until day 3 was followed by restoration of hydromineral balance from day 5 onward. Gills and pyloric caeca responded to SW transfer by increasing Na(+),K(+)-ATPase activity from days 5 and 3, respectively, onward. In both tissues, this response was preceded by an increase in alpha-subunit Na(+), K(+)-ATPase mRNA as early as 12 h posttransfer. The similarity of the response in these two organs suggests that they both play significant physiological roles in restoring hydromineral balance after abrupt increase in salinity. Further, SW transfer induced a slight, though significant, increase in primary gill filament Na(+), K(+)-ATPase immunoreactive (NKIR) cell abundance. This was paralleled by a marked (50%) decrease in secondary lamellar NKIR cell abundance after less than 1 d in SW. Thus, SW acclimation in brown trout is characterised by a lasting decrease in overall NKIR cell abundance in the gill. We propose that SW transfer stimulates Na(+),K(+)-ATPase enzymatic activity within individual chloride cells long before (<1 d) it becomes apparent in measurements of whole-gill homogenate enzymatic activity. This is supported by the early stabilisation (12 h) of hydromineral balance.     

Impact of environmental DDT concentrations on gill adaptation to increased salinity in the tilapia Sarotherodon melanotheron

Estuaries of tropical developing countries suffering from severe droughts induced by climate change are habitats to fish, which face drastic salinity variations and the contact with pollutants. The Western Africa tilapia Sarotherodon melanotheron is highly resistant to hypersalinity, but the effect of human-released xenobiotics on its adaptation is barely known. Controlled experiments were conducted to observe S. melanotheron gill adaptation to abrupt salinity variations in the presence of waterborne DDT, at concentrations detected in their natural habitat. The gills appeared as an important site of DDT conversion to DDD and/or depuration. A 12-days DDT exposure resulted in decreased gill epithelium thickness at all salinities (from fresh- to hypersaline-water), and the structure of gills from freshwater fish was particularly altered, relative to controls. No unbalance in tilapia blood osmolality was observed following DDT exposure, which however caused a decrease in branchial Na(+)-K(+)-ATPase (NKA) activity. Gill cellular NKA expression was reduced in salt-water, together with the expression of the CFTR chloride channel in hypersaline water. Although S. melanotheron seems very resistant (especially in seawater) to short-term waterborne DDT contamination, the resulting alterations of the gill tissue, cells and enzymes might affect longer term respiration, toxicant depuration and/or osmoregulation in highly fluctuating salinities.     

Fundulus heteroclitus acutely transferred from seawater to high salinity require few adjustments to intestinal transport associated with osmoregulation

The common killifish, Fundulus heteroclitus, has historically been a favorite organism for the study of euryhalinity in teleost fish. Despite the species' large range of salinity tolerance, studies of osmoregulation in high salinity are rare, with most previous studies focused on fish transferred between freshwater and seawater. Similarly, while branchial transport properties have been studied extensively, there are relatively few studies investigating the role of the intestine in osmoregulation in killifish. This study sought to characterize the fluid and ion transport occurring in the intestinal tract of killifish adapted to seawater, and furthermore to investigate the adjustments that occur to these mechanisms following acute transfer to high salinity (70ppt). In vivo samples of blood plasma and intestinal fluids of seawater-acclimated killifish indicated absorption of Na(+), Cl(-), and water, the relative impermeability of the intestine to Mg(2+) and SO(4)(2-), and active secretion of HCO(3)(-) into the intestinal lumen. The details of these processes were investigated further using in vitro techniques of isolated intestinal sac preparations and an Ussing chamber pH-stat titration system. However, these methods were discovered to be of limited utility under physiologically relevant conditions due to tissue deterioration. Results that could be validly interpreted suggested that there are few changes to intestinal transport following transfer to high salinity, and that adjustments to epithelial permeability occur in the first 24h post-transfer.     

Heat shock protein (Hsp70) induced by a mild heat shock slightly moderates plasma osmolarity increases upon salinity transfer in rainbow trout (Oncorhynchus mykiss)

We have investigated whether mild heat shock, and resulting Hsp70 expression, can confer cross-protection against the stress associated with transfer from freshwater (FW) to seawater (SW) in juvenile rainbow trout (Oncorhynchus mykiss). In experimental Series I, juvenile trout reared in FW were transferred from 13.5 degrees C to 25.5 degrees C in FW, held for 2 h, returned to 13.5 degrees C for 12 h, and then transferred to 32 ppt SW at 13.5 degrees C. Branchial Hsp70 increased approximately 10-fold in the heat-shocked fish relative to the control by the end of recovery and remained high 2, 8, and 24 h post-salinity transfer. However, no clear differences could be detected in blood parameters (blood hemoglobin, hematocrit, MCHC, plasma Na(+) and plasma osmolarity) or muscle water content between heat-shocked and sham-shocked fish in SW at any sampling interval (0, 2, 8, 24, 48, 120, 240 and 360 h post-SW transfer). In experimental Series II, trout acclimated to 8 degrees C were heat-shocked at 22 degrees C for 2 h, allowed to recover 18 h, and exposed to a more severe salinity transfer (either 36 or 45 ppt) than in Series I. Branchial Hsp70 levels increased approximately 6-fold in heat-shocked fish, but had declined to baseline after 120 h in SW. Plasma osmolarity and chloride increased in both groups upon transfer to 36 ppt; however, the increase was significantly less in heat-shocked fish when compared to the increase observed in sham-shocked fish at 24 h. No significant differences could be detected in branchial Na(+)/K(+)-ATPase activity or Na(+)/K(+)-ATPase alpha1a and alpha1b mRNA expression between the two groups. Our data indicate that a mild temperature shock has only modest effects on the ability of rainbow trout to resist osmotic stress during FW to SW transfer.