Freshwater-adapted Fish Research Articles

Salinity-responsive histone PTMs identified in the gills and gonads of Mozambique tilapia (Oreochromis mossambicus)

BackgroundHistone post-translational modifications (PTMs) are epigenetic marks that can be induced by environmental stress and elicit heritable patterns of gene expression. To investigate this process in an ecological context, we characterized the influence of salinity stress on histone PTMs within the gills, kidney, and testes of Mozambique tilapia (Oreochromis mossambicus). A total of 221 histone PTMs were quantified in each tissue sample and compared between freshwater-adapted fish exposed to salinity treatments that varied in intensity and duration.ResultsFour salinity-responsive histone PTMs were identified in this study. When freshwater-adapted fish were exposed to seawater for two hours, the relative abundance of H1K16ub significantly increased in the gills. Long-term salinity stress elicited changes in both the gills and testes. When freshwater-adapted fish were exposed to a pulse of severe salinity stress, where salinity gradually increased from freshwater to a maximum of 82.5 g/kg, the relative abundance of H1S1ac significantly decreased in the gills. Under the same conditions, the relative abundance of both H3K14ac and H3K18ub decreased significantly in the testes of Mozambique tilapia.ConclusionsThis study demonstrates that salinity stress can alter histone PTMs in the gills and gonads of Mozambique tilapia, which, respectively, signify a potential for histone PTMs to be involved in salinity acclimation and adaptation in euryhaline fishes. These results thereby add to a growing body of evidence that epigenetic mechanisms may be involved in such processes.

Evidence for nitric oxide role in the caudal neurosecretory system of the European flounder, Platichthys flesus

A neuromodulatory role for nitric oxide has been reported for magnocellular neuroendocrine cells in mammalian hypothalamus. We examined its potential as a local intercellular messenger in the neuroendocrine Dahlgren cell population of the caudal neurosecretory system (CNSS) of the euryhaline flounder. Immunocytochemistry using an antibody raised against human neuronal nitric oxide synthase (NOS) indicated the presence of NOS in the Dahlgren cells. Quantitative RT-PCR, using a flounder-specific probe, revealed NOS mRNA expression in the CNSS. In July, though not in September, NOS mRNA expression was significantly higher in fish fully adapted to seawater, compared to freshwater-adapted fish. Following acute transfer of fish from freshwater to seawater, NOS mRNA expression was elevated at 8 h and then recovered by 24 h. In pharmacological experiments in vitro, application of NO donors (SNAP, SNP) caused an increase in electrical activity (firing frequency) of Dahlgren cells, recruitment of previously silent cells, together with a greater proportion of cells showing phasic (irregular) activity. The NOS substrate, l-arginine, led to increased firing frequency, cell recruitment and enhanced bursting activity. However, this effect was not blocked by the NOS inhibitor L-NAME. These findings suggest that NO acts as a modulator within the CNSS, potentially enhancing electrical activity and hence secretory output. A role in supporting adaptation to hyperosmotic conditions is also indicated.

Distribution of peptidase activity in teleost and rat tissues

Peptides play important roles in cell regulation and signaling in many tissues. The actions of peptides are regulated by peptidases. Although the activity of these enzymes has been thoroughly characterized in mammals, little is known about their presence or function in fish. In the present study, we compared the activity of several peptidases in selected tissues (pituitary gland, different brain areas, kidney and gills) of the gilthead sea bream and rainbow trout with that found in similar rat tissues (lungs studied in place of gills). Soluble puromycin-sensitive aminopeptidase showed the highest values in the pituitary gland of the sea bream, whereas the membrane-bound form was found to be more active in the trout kidney. Very high levels of activity of aminopeptidase N were detected in trout and sea bream plasma. In contrast, the highest levels of activity of aminopeptidase B were found in rat tissues, with the exception of the gills of the trout. Aminopeptidase N levels tended to be higher in sea bream tissues with respect to those of trout. In contrast, the level of activity of aminopeptidase B was found to be consistently much higher in trout tissues than in those of the sea bream. Prolyl endopeptidase activity was principally detected in the pituitary gland and in the brain areas of teleosts. These differences between species could be related to different mechanisms of osmoregulation in saltwater- and in freshwater-adapted fish.

Stanniocalcin in the euryhaline flounder ( Platichthys flesus): Primary structure, tissue distribution, and response to altered salinity

Stanniocalcin (STC) is a homodimeric glycoprotein hormone that was first discovered in fish, where it is largely produced by a unique endocrine gland, the corpuscles of Stannius (CS). In bony fish, it is thought to be an important regulator of calcium and phosphate uptake from the aquatic environment. This report describes the molecular cloning of STC from euryhaline flounder ( Platichthys flesus) CS cDNA and genomic DNA. The flounder STC encodes a prehormone of 251 amino acids (aa) with a signal peptide of 17 aa, followed by another 15 aa sequence before the mature protein of 219 aa. The deduced aa sequence of flounder STC shows 62.9–89.0% similarity and 50.4–83.1% identity with other known fish STC sequences, but only 42.3% identity with mouse STC1, 24.4% identity with fugu and zebrafish STC2, and 22.3% identity with mouse STC2. Primary structural analysis demonstrated that flounder STC gene contains five exons in contrast to the four exons present in mammalian STC gene. RT-PCR revealed the expression of flounder STC mRNA to be widely spread in many tissues and organs, similar to the situation in mammals and other fish. Quantitative PCR (Q-PCR) was conducted to measure relative STC expression levels in the CS, which showed STC mRNA expression levels in seawater-adapted fish CS were about 3-fold higher than in freshwater-adapted fish CS.

Electrical activity of caudal neurosecretory neurons in seawater and freshwater-adapted Platichthys flesus, in vivo

This study examined the electrical firing activity of neuroendocrine Dahlgren cells in the caudal neurosecretory system (CNSS) of the euryhaline flounder in vivo. Intracellular recordings revealed generally similar activity patterns and membrane properties to those previously reported in vitro. To investigate the potential role of the CNSS in osmoregulatory adaptation, extracellular, multiunit, recordings compared the activity patterns of Dahlgren cells in fully seawater- and freshwater-adapted fish. The proportion of cells showing bursting (as opposed to phasic or tonic) activity was greater in seawater-than in freshwater-adapted fish, as was the Correlation Index, a measure of the degree of correlation between firing activities of cells recorded simultaneously from the same preparation. Acute transfer of fish from seawater to freshwater gill perfusion led to recruitment of previously silent Dahlgren cells and a reduction in Correlation Index; freshwater to seawater transfer increased the Correlation Index. Severing the spinal cord anterior to the CNSS led to an increase in overall Dahlgren cell activity. Electrical stimulation of branchial nerve branches providing input to the brainstem, or tactile (pinch) stimulation of lips or fins, led to a reduction in CNSS activity lasting up to 500 s, indicating the presence of descending modulatory pathways from the brain. These results are consistent with a role for CNSS neuropeptides, urotensins, in supporting survival in a hypertonic, seawater, environment.

Effects of Low Salinities on Oxygen Consumption of Selected Euryhaline and Stenohaline Freshwater Fish

The amount of energy required for osmoregulation depends on the difference between internal and external concentrations of ions (Rao 1968; Farmer and Beamish 1969), changes in corticosteroid hormone levels (Morgan and Iwama 1996), glomerular filtration rates (Furspan et al. 1984), gill and kidney Na+, K+‐ATPase activity (McCormick et al. 1989; Morgan and Iwama 1998), tissue permeability to water and ions, and gill ventilation, perfusion, and functional surface area (Rankin and Bolis 1984). Differences in the energetic cost of osmoregulation play a significant role in the difference in growth rate between seawater‐and freshwater‐adapted fish (Morgan and Iwama 1991; Ron et al. 1995; Wang et al. 1997). Oxygen consumption is an indirect indicator of metabolic rate in fish (Cech 1990) and can be used to determine effects of salinity changes on energy costs.

Environmental levels of sediment-associated tri- n -butyltin chloride (TBTCl) and ionic regulation in flounders during seawater adaptation

The effects of exposure to sediment-associated tri-n-butyltin chloride (TBTCl) were examined in the euryhaline European flounder, Platichthys flesus (L.). The effects were quantified by measuring the changes in sodium efflux; Na+/K+-ATPase activity; and the numbers, areas, and distribution of chloride cells in the gills of freshwater-adapted fish, following a rapid transfer to seawater. Following the transfer to seawater, the Na+/K+-ATPase activity and the sodium efflux were significantly increased in the control group but remained unchanged in the TBTCl-exposed group. The normal morphological changes to the gill epithelium associated with seawater adaptation, which involve chloride cell distribution, took place in the control group but were significantly inhibited or delayed in the TBTCl group. The results presented in this study lead to the conclusion that environmental concentrations of tri-n-butyltin chloride in sediments are capable of significantly disrupting both the physiological and the morphological components of iono-regulatory functions of an estuarine flatfish.

The effects of sediment-associated triorganotin compounds on the gills of the European flounder, Platichthys flesus (L.)

The effects of exposure to sediment-associated tri- n-butyltin chloride (TBT) and triphenyltin chloride (TPhT) were examined in the euryhaline European flounder, Platichthys flesus (L.). The effects were quantified by measuring the changes in sodium efflux, Na +/K +-ATPase activity and the numbers, areas and distribution of chloride cells in the gills of freshwater-adapted fish, following a rapid transfer to seawater. After transfer, the Na +/K +-ATPase activity and the sodium efflux significantly increased in both the TPhT and control groups but not in the TBT group. However, Na +/K +-ATPase activity and the sodium efflux in the TPhT group had returned to pre-salinity transfer levels by day 15 after the initial exposure to TPhT. Morphological changes in the numbers and areas of chloride cells, known to be associated with seawater adaptation, took place in the control group, i.e. there was a significant reduction in the number of lamellar chloride cells accompanied by an increase in the number of interlamellar chloride cells. There was a reduction in the numbers of lamellar chloride cells in the TBT-exposed group following transfer to seawater but the mean number was significantly higher than the control group by the end of the experiment. In the TPhT-exposed group, the reduction was not significantly different to that seen in the control group. By the end of the experiment, both organotin-exposed groups had significantly lower mean numbers of interlamellar chloride cells than the control group. Before transfer to seawater, the mean areas of lamellar and interlamellar chloride cells of all three groups were not significantly different. On transfer, the mean areas of lamellar chloride cells in the control group became significantly smaller than the mean areas of the organotin groups. There was no significant difference in the mean areas of interlamellar chloride cells in the control and TBT groups between the start and finish of the experiment but there was a significant increase in the mean area of TPhT-treated animals at the end of the experiment when compared to the control group. The results presented in this study lead to the conclusion that tri- n-butyltin chloride and triphenyltin chloride in sediments are capable of significantly disrupting both the physiological as well as morphological components of ionic regulatory functions of an estuarine fish, at concentrations currently found in estuarine sediments.

Morphology and function of gill mitochondria-rich cells in fish acclimated to different environments.

The objective of this study is to test the hypothesis that morphologically different mitochondria-rich (MR) cells may be responsible for the uptake of different ions in freshwater-adapted fish. Tilapia (Oreochromis mossambicus) were acclimated to high-Ca, mid-Ca, low-Ca, and low-NaCl artificial freshwater, respectively, for 2 wk. Cell densities of wavy-convex, shallow-basin, and deep-hole types of gill MR cells as well as whole-body Ca(2+), Na(+), and Cl(-) influxes were measured. Low-Ca fish developed more shallow-basin MR cells in the gills and a higher Ca(2+) influx than those acclimated to other media. However, fish acclimated to low-NaCl artificial freshwater predominantly developed wavy-convex cells, and this was accompanied by the highest Na(+) and Cl(-) influxes. Relative abundance of shallow-basin and wavy-convex MR cells appear to be associated with changes in Ca(2+) and Na(+)/Cl(-) influxes, suggesting that shallow-basin and wavy-convex MR cells are mainly responsible for the uptake of Ca(2+) and Na(+)/Cl(-), respectively.

Haloplasticity of black seabream (Mylio macrocephalus): Hypersaline to freshwater acclimation

Black seabream (Mylio macrocephalus) were acclimated to various salinities (50, 33, 12 and 6‰) for eight months. Acclimation of fish to 6‰ for eight months allowed successful adaptation to freshwater (0‰) for a further 21 days without mortality. This is the first report of freshwater acclimation of a “true” marine fish for an acceptable experimental duration. Osmoregulatory and metabolic strategies were characterized via alterations in branchial chloride cell (CC) numbers and surface ultrastructural morphometrics along with changes in serum chemistry, muscle moisture, liver glycogen and branchial, renal, hepatic and intestinal enzyme activities. Branchial CC numbers were elevated in 50 and 6‰ environments; however, freshwater acclimation resulted in return to low numbers. Branchial Na+-K+-ATPase activity was generally higher in 50 and 33‰ environments and exhibited a declining trend in 12 and 6‰ environments. Freshwater acclimation resulted in a marked elevation in branchial Na+-K+-ATPase activity. Elevated CC exposure areas were typically found at salinity extremes. Serum Na+, Cl– and muscle moisture content did not vary between groups acclimated from 50 to 6‰. Freshwater acclimation resulted in significant hyponatremia, hypochloremia and muscle hydration. Branchial ICDH activity was lowest in a 12‰ environment and highest at salinity extremes. Renal Na+-K+-ATPase exhibited lower activity in 12 and 6‰ and was markedly elevated in 0‰. Enzyme activities of both liver and kidney indicated elevated gluconeogenic activity in freshwater-adapted fish. Total intestinal Na+-K+-ATPase activity tended to decline in lower salinities; however, lowest activity was found in fish adapted to 12‰. Na+-K+-ATPase activities in different segments of the intestine may reflect the osmoregulatory role of this organ in varying salinities. The data indicated efficient homeostatic control in Mylio macrocephalus acclimated from hypersaline to freshwater environments and clearly demonstrates the haloplasticity of this marine fish species. J. Exp. Zool. 283:226–241, 1999. © 1999 Wiley-Liss, Inc.

Diel changes in plasma arginine vasotocin, isotocin, and melatonin in rainbow trout (Oncorhynchus mykiss)

The diel changes in plasma AVT, IT and Mel in rainbow trout (Oncorhynchus mykiss) were studied to assess potential relationships. Blood was sampled at 05:00, 11:00, 16:00, 22:30 and 05:00 in freshwater-adapted fish and at 22:30 in brackish water-adapted fish maintained under natural photoperiod. A few of the FW-acclimated fish were assigned to one of two experimental groups and adapted to DD or LL lighting regimes. Blood samples were taken at 11:00 and 22:30. Hormones were extracted from plasma by solid phase extraction and determined by high-performance liquid chromatography. Marked diel variations in AVT and Mel were detected in fish maintained under natural photoperiod. Plasma AVT (fmol ml−1) increased during the light to reach the maximal level at the end of that phase (261.7±23.1). Thereafter, AVT concentration decreased and became minimal at 05:00 (68.9±11.5) 3 h before the sunrise. Plasma Mel (pmol ml−1) increased between 16:00 and 22:30 when a peak value was reached (1204.0±55.5). Thereafter, Mel levels decreased and were minimal after the onset of the light phase (242.8±37.0). IT levels displayed no significant diel changes. Linear regression analysis indicated the negative correlation between plasma Mel and AVT for five collecting times of the daily 24 h cycle in freshwater fish and at 22:30 in brackish water fish. A similar correlation occurred at 11:00 in the DD group and at 22:30 in the LL group. To elucidate the character of the Mel-AVT relationships further studies are required.

Response of circulating arginine vasotocin and isotocin to rapid osmotic challenge in rainbow trout

The plasma levels of arginine vasotocin (AVT) and isotocin (IT) in rainbow trout ( Oncorhynchus mykiss) were studied to demonstrate changes in neurohypophysial nonapeptides following rapid osmotic challenge. Freshwater-adapted fish were transferred to brackish Baltic water (BW) and brackishwater-adapted fish were transferred to fresh water (FW). In both experiments blood samples for AVT, IT and osmolality were taken 30 min, 1, 2, 24 hr, and 10 days after transfer. Hormones were determined by gradient high-performance liquid chromatography preceded by solid-phase extraction. The pattern of AVT and IT response to external salinity changes was similar during the first hours and showed transient increase or decrease after transfer to brackish or fresh water, respectively. The AVT plasma concentration after 10 days in FW was significantly higher than that in BW, whereas the IT plasma concentrations in both salinities did not differ. These data suggest that the synthesis and or release of AVT and IT are controlled independently, and the roles of hormones in long-term osmotic adaptation are different.

Diurnal Changes in Plasma Arginine Vasotocin and Isotocin in Rainbow Trout Adapted to Fresh Water and Brackish Baltic Water

The plasma levels of arginine vasotocin (AVT) and isotocin (IT) in rainbow trout (Oncorhynchus mykiss) were studied to assess possible diurnal variations in neurohypophysial nonapeptides. Fish were kept under natural photoperiod and adapted to fresh and brackish Baltic water. Blood was sampled at 5:00, 11:00, 16:00, 22:30, and again at 5:00. Hormones were determined by gradient high-performance liquid chromatography preceded by solid-phase extraction. The marked diurnal changes of AVT were detected in plasma of fish adapted to both fresh and brackish waters. AVT levels (fmol/ml) were maximal at 16:00 and minimal at 5:00 (253.4±35.7 and 45.5±17.3, respectively). Unlike AVT, isotocin levels displayed no diurnal changes. AVT concentrations at 11:00, 16:00, and 22:30 were significantly higher than IT values measured throughout the day. Plasma AVT concentrations determined in brackish water-adapted fish at 16:00 were significantly lower than those of freshwater-adapted fish at the same time. These data suggest that synthesis and/or release of AVT and IT are controlled independently, so that these nonapeptides have different physiological roles in teleost fish. AVT might participate in circadian time-keeping system in fish.

Calcium pump activities in the kidneys of Oreochromis mossambicus

The mechanism that underlies transcellular Ca2+ reabsorption in the kidney of the euryhaline teleost Oreochromis mossambicus was studied. Preparations of membrane vesicles made from the kidneys of freshwater- and seawater-adapted fish were more than sevenfold enriched in the basolateral plasma membrane marker Na+/K+-ATPase. Significant recovery of NADH­ cytochrome c reductase enzyme activity and of oxalate-stimulated Ca2+ pump activities in the membrane preparations indicated that the membrane fraction was of endoplasmic reticular origin. Indeed, thapsigargin specifically inhibited Ca2+ pump activity that could be attributed to oxalate-permeable endoplasmic reticular fragments. Kinetic analysis of thapsigargin-insensitive Ca2+ pump activity indicated the existence of a homogeneous, high-affinity, ATP-driven Ca2+ pump. No Na+-driven Ca2+ transport mechanism could be demonstrated. Plasma membrane Ca2+ pump activity was 56 % lower in preparations from seawater-adapted fish than in preparations from freshwater-adapted fish, suggesting a physiological role for this Ca2+ pump activity in renal Ca2+ handling by euryhaline species, with an involvement in the regulation of Ca2+ reabsorption.

Northern blot analysis of the Na +, K +-ATPase α-subunit in salmonids

A 450-bp long fragment of the gene encoding the Na +, K +-ATPase α-subunit was PCR (polymerase chain reaction)-amplified from the rainbow trout ( Oncorhynchus mykiss) genome. The protein coding sequence of the cloned PCR product shared 85 and 73% identity with Na +, K +-ATPase α-subunit-encoding sequences of the rat (α 2-isoform) and the teleost species of the white sucker ( Catostomus commersoni), respectively. Northern blot analysis demonstrated that the fragment hybridized to a major transcript of about 3.7 kb in several salmonid and non-salmonid species. The observed changes in Na +, K +-ATPase mRNA levels in osmoregulatory organs of seawater- and freshwater-adapted fish further confirm the specificity of the probe.

Evidence that two tilapia (Oreochromis niloticus) prolactins have different osmoregulatory functions during adaptation to a hyperosmotic environment

Two forms of prolactin (tiPRLI and tiPRLII), with only 69% sequence identity, have been previously described in the cichlid fish tilapia (Oreochromis species). In the present study we have attempted to investigate the biological activity of these two prolactin forms during adaptation to a hyperosmotic environment. For this purpose, we have developed two highly sensitive (sensitivity: 0.05 ng/ml) and specific (cross-reactivity < 0.04%) radioimmunoassays for tiPRLI and tiPRLII, using recombinant hormones. When fish were directly transferred from fresh to brackish water, the measured levels of plasma tiPRLI and tiPRLII dropped abruptly until 12 h after transfer. Thereafter, plasma tiPRLII remained stable (around 0.5 ng/ml) until the end of the experiment, whereas plasma tiPRLI continued to decrease to undetectable levels. These different patterns of change are reflected in the calculated ratio of plasma tiPRLII to tiPRLI, which increased from 2-3 in fresh water-adapted fish to over 10 in fish which had spent 3 days or more in brackish water. The pituitary contents of tiPRLI and tiPRLII varied in a qualitatively similar fashion after transfer to brackish water. The tiPRLI content dropped continuously after 12 h, reaching one-twelfth of its initial level after 2 weeks. The pituitary tiPRLII content, on the other hand, did not decrease significantly until day 7, and after a 2-week exposure to brackish water it had only decreased by 50%. When injected into tilapia adapted to brackish water, both ovine prolactin and recombinant tiPRLI induced a clear dose-dependent ion-retaining effect. In contrast, the effect induced by tiPRLII treatment was markedly smaller and not dose-dependent. Northern blot analysis of tiPRL mRNAs using either a tiPRLI or a tiPRLII cDNA probe indicated the presence of two mRNAs differing in size: a 1.7 kb mRNA coding for tiPRLI and a 1.3 kb mRNA coding for tiPRLII. After transfer to brackish water, levels of the two mRNAs decreased similarly. The present study indicates that, in O. niloticus, the two forms of prolactin have different osmoregulatory roles during adaptation to brackish water. Accordingly, their synthesis are differentially regulated after transfer to a hyperosmotic environment, presumably at a post-transcriptional level.

Difference between summer and winter in gill NaK-ATPase activity and hypoosmoregulatory ability of seafarmed anadromous arctic char ( Salvelinus alpinus)

1. 1. In July, both fish kept in seawater of 30–32‰ and 20‰ salinity (temperature 10–12°C) showed good hypoosmoregulatory ability and their gill NaK-ATPase activity was high. 2. 2. In January (30–32‰ salinity, 5°C), mean plasma Cl − concentration was 30 mM higher than that in July, and gill NaK-ATPase activity was only a half of that recorded in July. 3. 3. This difference in hypoosmoregulatory abilities recorded in summer and in winter is suggested to be due to a reversion during late summer and early autumn in the state of osmo-ionoregulatory system towards that of a freshwater-adapted fish, thus reflecting the migratory pattern of this species in nature.

Effects of changes in environmental calcium on prolactin secretion in Japanese eel, Anguilla japonica

Effects of changes in environmental Ca2+ on the secretion of prolactin, a possible hypercalcemic hormone, were examined both in vivo and in vitro in the Japanese ecl, Anguilla japonica. Transfer of seawater- or freshwater-adapted fish to fresh water, fresh water containing 10 mmol Ca2+ · 1-1 sea water, Ca2+-free sea water, or deionized water was accompanied by significant changes in plasma Ca2+ levels after 7 days, except for the fish transferred from fresh water to fresh water and from sea water to sea water. Changes in external Ca2+ concentrations did not affect plasma prolactin levels, although plasma prolactin levels as well as pituitary prolactin contents were significantly greater in fish in a hypotonic environment than those in a hypertonic environment, regardless of the external Ca2+ concentration. Hypercalcemia, induced by removal of the corpuscles of Stannius, did not alter plasma prolactin levles. Incubation of the pituitary in the medium with different Ca2+ concentrations (up to 2.9 mmol·l-1) did not affect the basal release of prolactin, except at an extremely low Ca2+ concentration (less than 0.1 mmol·l-1) where prolactin release was inhibited. Addition of Ca2+ ionophore (A23187) to the medium led to a marked and significant increase in prolactin release, indicating that an increase in intracellular Ca2+ stimulates prolactin release. However, the effect was not specific to prolactin cells; a similar increase was seen in growth hormone release. These results indicate that changes in environmental Ca2+ concentration may not be the primary factor influencing prolactin secretion in the eel; changes in environmental osmolality or Na+ levels seem to be more critical for the regulation of prolactin secretion.

Secretory renal proximal tubules in seawater- and freshwater-adapted killifish

A population of proximal tubules when isolated from the glomerular kidneys of seawater-adapted (SW) and freshwater-adapted (FW) killifish (Fundulus heteroclitus) spontaneously secrete fluid. Regardless of SW or FW adaptation, Na and Cl are the dominant electrolytes in secreted fluid. Mg concentrations in fluid secreted by both tubules are significantly greater than those in the peritubular bath, and Mg concentrations are inversely related to Na concentrations. Proximal tubules from either SW or FW fish exhibit low transepithelial voltage (-1 to -2 mV) and low transepithelial resistances (20-30 omega.cm2) typical of other vertebrate proximal tubules. Transepithelial diffusion potentials for Na, Cl, Mg, and SO4 suggest that the paracellular pathway is Na selective and impermeable to divalent ions. Consideration of transepithelial electrochemical potential differences for Na, Cl, Mg, and SO4 suggests active transport of Mg, SO4, and Cl in proximal tubules isolated from SW- and FW-adapted fish. The similarities in the functional properties of secretory proximal tubules isolated from SW- and FW-adapted killifish are striking and raise questions about the in vivo role of these tubules in the renal adaptations to seawater and freshwater.

Histochemical demonstration of carbonic anhydrase in gills and opercular epithelium of seawater- and freshwater-adapted killyfish (Fundulus heteroclitus)

Gills and operculum of seawater- and freshwater-adapted killyfish (Fundulus heteroclitus) were stained histochemically for carbonic anhydrase (CA). In the seawater-acclimatized specimens, CA was found predominantly in the chloride cells which were considerably larger than in the freshwater-adapted ones. Within these cells, the reaction products were concentrated in the apical parts of the cytoplasm. In contrast, chloride cells of freshwater-adapted fish were not, or only faintly, stained both in gills and opercular epithelium. Reaction products for CA were seen additionally in the cytoplasm of the outer respiratory cells lining the lamellae of gills both in seawater- and freshwater-adapted fish.