Effect of transfer to sea water and back to fresh water on the histological structure of the eel kidney

Summary A cytometrical study of the various segments of kidney tubules was performed on silver or silvering male eels during acclimation to sea water (SW) for 2 to 160 days and their return to fresh water (FW). Tubular epithelial cell height and nuclear area are markedly reduced during the first two days, and further reduced in the following period to reach a stable level around the 20th day. The glomerular size is slightly decreased. Phospholipids are less abundant and the brush borders become thinner. In a single eel kept for 9 months in pure SW slowly concentrated by evaporation (NaCl 45 g/l), cell and nuclear values were similar to those obtained in full strength SW after 160 days. Distal and collecting tubules exhibit maximal reactivity, that of the first proximal tubule being minimal.When eels kept for 20 days in SW are returned to FW, there is a rapid increase (48 h), sometimes with an overshoot, of all measured parameters, followed by a rapid decrease (5 days) and a normalization of the values in the distal and collecting tubules.The rapid response of eel kidney to salinity changes does not appear to be linked to cellular hydration as the various segments of the kidney tubules react differently. The data are discussed with respect to osmotic acclimation and in relation to variations of prolactin secretion. Prolactin (PRL) release during transfer to FW stimulates the renal tubules less strongly than mammalian PRL: while mitotic activity and differentiation of new nephrons are induced by ovine PRL treatment in intact or hypophysectomized FW eels (Olivereau and Lemoine, 1969b), no such events were detected during acclimation to this hypotonic medium, at least during the first ten days. This rather reflects insufficient PRL release than a different biological activity, despite immunological differences between fish and ovine prolactin.

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Résumé L'étude cytométrique (hauteur épithéliale et aire nucléaire) des divers segments du tube rénal, réalisée chez l'Anguille mâle argentée ou en cours d'argenture lors de l'adaptation à l'eau de mer (SW) montre une réduction marquée dès les premières 48 h en SW qui s'accuse légèrement pour atteindre un niveau stable vers le 20^e jour; la taille des glomérules est réduite. Les phospholipides sont moins abondants, la bordure en brosse est plus mince. Chez une Anguille conservée 9 mois en SW initialement normale et lentement concentrée par évaporation (NaCl 45 g/l), les valeurs obtenues sont peu différentes de celles après 160 jours en SW pure. La réactivité maximale s'observe au niveau du tube distal et du collecteur, celle du tube proximal l étant minimale.Le retour en eau douce (FW) provoque une rapide augmentation (2 jours), parfois excessive, de ces paramètres, suivie d'une baisse rapide (5 jours) et de la normalisation des valeurs du tube distal et collecteur (10 jours).Les réponses du rein aux changements de salinité, très rapides, ne paraissent pas liées à des processus de déshydratation ou hydratation cellulaire car elles affectent inégalement les divers segments du néphron. Elles sont discutées en fonction des variations des électrolytes plasmatiques et de la sécrétion hypophysaire de prolactine (PRL): sa décharge qui accompagne le retour en FW stimule moins intensément le tube rénal que l'injection de PRL ovine, elle ne provoque pas une intense activité mitotique ni une différenciation de nouveaux néphrons, au moins pendant les 10 premiers jours. Ce fait représente probablement plus une sécrétion insuffisante de PRL lors du retour en milieu hypotonique qu'une différence d'activité biologique, malgré des différences immunologiques entre prolactines ovine et de Poisson.

     

The effects of calcium and magnesium on water and ionic permeabilities in the sea water adapted eel,Anguilla anguilla L.

Summary The permeability characteristics to water and ions, of the seawater adapted eel, have been studied in the absence and presence of external calcium and magnesium.The absence of these divalent ions from the external medium produces an increase in the unidirectional and net water fluxes as well as a decrease in temperature coefficients. Similarly, an increase in the effluxes of sodium and chloride and a large increase in the passive loss of these ions was observed when fish were transferred to deionized water. The stimulating effect of externally added (10 mM) potassium on the sodium and chloride effluxes, when fish are transferred to fresh water is abolished in the absence of calcium and magnesium (Fig. 1). These kinetic changes are accompanied by ultrastructural changes concerned principally with the chloride cells (Figs. 2 and 3).The results are discussed in terms of the possible role of calcium and magnesium in the maintenance of the hydro-mineral equilibrium of the eel.     

Ca2+ transport by opercular epithelium of the fresh water adapted euryhaline teleost,Fundulus heteroclitus

We examined transepithelial transport of Ca²⁺ across the isolated opercular epithelium of the euryhaline killifish adapted to fresh water. The opercular epithelium, mounted in vitro with saline on the serosal side and fresh water (0.1 mmol·l^–1 Ca²⁺) bathing the mucosal side, actively transported Ca²⁺ in the uptake direction; net flux averaged 20–30 nmol·cm^–2·h^–1. The rate of Ca²⁺ uptake varied linearly with the density of mitochondria-rich cells in the preparations. Ca²⁺ uptake was saturable, apparent K _1/2 of 0.348 mmol·l^–1, indicative of a multistep transcellular pathway. Ca²⁺ uptake was inhibited partially by apically added 0.1 mmol·l^–1 La³⁺ and 1.0 mmol·l^–1 Mg²⁺. Addition of dibutyryl-cyclic adenosine monophosphate (0.5 mmol·l^–1)+0.1 mmol·l^–1 3-isobutyl-l-methylxanthine inhibited Ca²⁺ uptake by 54%, but epinephrine, clonidine and isoproterenol were without effect. Agents that increase intracellular Ca²⁺, thapsigargin (1.0 mol·l^–1, serosal side), ionomycin (1.0 mol·l^–1, serosal side) and the calmodulin blocker trifluoperazine (50 mol·l^–1, mucosal side) all partially inhibited Ca²⁺ uptake. In contrast, apically added ionomycin increased mucosal to serosal unidirectional Ca²⁺ flux, indicating Ca²⁺ entry across the apical membrane is rate limiting in the transport. Verapamil (10–100 mol·l^–1, mucosal side), a Ca²⁺ channel blocker, had no effect. Results are consistent with a model of Ca²⁺ uptake by mitochondria rich cells that involves passive Ca²⁺ entry across the apical membrane via verapamil-insensitive Ca²⁺ channels, intracellular complexing of Ca²⁺ by calmodulin and basolateral exit via an active transport process. Increases in intracellular Ca²⁺ invoke a downregulation of transcellular Ca²⁺ transport, implicating Ca²⁺ as a homeostatic mediator of its own transport.Abbreviations DASPEI 2-(4-dimethylaminostyryl)-N-ethylpyridinium iodide - db-cAMP dibutyryl-cyclic adenosine monophosphate - FW fresh water - G _t transepithelial conductance - I _sc short-circuit current - IBMX 3-isobutyl-1-methylxanthine - SW sea water - TFP trifluoperazine - V _t transepithelial potential     

The effects of temperature, river flow, and tidal cycles on the onset of glass eel and elver migration into fresh water in the American eel

Anguilla rostrata elvers were collected in the Annaquatucket R., Rhode Island, and their otoliths extracted. Daily increments beyond the check mark formed upon entry into fresh water were counted and the date of freshwater entry was estimated. The effects of river temperature, difference between seawater and freshwater temperature, river flow and tidal stage on the number of elvers arriving on each date were estimated for six collection dates. At the earliest collection date (23 March), increasing river temperatures and reduced flow increased elver migration. At later dates (16 May-12 June), tidal stage was the most important factor in determining the magnitude of elver migration.     

The ultrastructure of plasma and thylakoid membrane vesicles from the fresh water cyanobacteriumAnacystis nidulans adapted to salinity

Summary Photoautotrophically growing cultures of the fresh water cyanobacteriumAnacystis nidulans adapted to the presence of 0.4–0.5 M NaCl (about sea water level) with a lag phase of two days after which time the growth rate reassumed 80–90% of the control. Plasma and thylakoid membranes were separated from cell-free extracts of French pressure cell treatedAnacystis nidulans by discontinuous sucrose density gradient centrifugation and purified by repeated recentrifugation on fresh gradients. Identity of the plasma and thylakoid membrane fractions was confirmed by labeling of intact cells with impermeant protein markers prior to breakage and membrane isolation. Electron microscopy revealed that each type of membrane was obtained in the form of closed and perfectly spherical vesicles. Major changes in structure and function of the plasma membranes (and, to a much lesser extent, of the thylakoid membranes) were found to accompany the adaptation process. On the average, diameters of plasma membrane vesicles from salt adapted cells were only one-third of the diameters of corresponding vesicles from control cells. By contrast, the diameters of thylakoid membrane vesicles were the same in both cases.Freeze-etching the cells and counting the number of membrane-intercalating particles on both protoplasmic and exoplasmic fracture faces of plasma and thylakoid membranes indicated a roughly 50% increase of the particle density in plasma membranes during the adaptation process while that in thylakoid membranes was unaffected. Comparison between particle densities on isolated membranes and those on corresponding whole cell membranes permitted an estimate as to the percentage of inside-out and right-side-out vesicles. Stereometric measurement of particle sizes suggested that two distinct sub-populations of the particles in the plasma membranes increased during the adaptation process, tentatively correlated to the cytochrome oxidase and sodium-proton antiporter, respectively. The effects of salt adaptation described in this paper were fully reversed upon withdrawal of the additional NaCl from the growth medium (deadaptation). Moreover, they were not observed when the NaCl was replaced by KCl.Abbreviations CM cytoplasmic or plasma membrane - ICM intracytoplasmic or thylakoid membrane - EF exoplasmic fracture face - PF protoplasmic fracture face - DABS diazobenzosulfonate; Hepes N-2-hydroxyethylpiperazine-N-2-ethane-sulfonate - PMSF phenylmethylsulfonylfluoride Dedicated to the memory of Professor Oswald Kiermayer     

Surfactant-induced lipid peroxidation in a tropical euryhaline teleost Oreochromis mossambicus (Tilapia) adapted to fresh water

Exposure to anionic (sodium dodecyl sulfate, SDS), cationic (cetyl trimethyl ammonium bromide CTAB) and non ionic (Triton X-100) surfactants at a sub lethal concentration of 1 ppm resulted in severe oxidative stress in the hepatic, renal and cardiac tissues of fresh water adapted Oreochromis mossambicus. Hepatic catalase showed significant increase (P<0.001) in all the surfactant exposed fish, but the renal enzyme was significantly increased only in CTAB dosed fish (P<0.001) and the cardiac enzyme showed significant increase in Triton (P<0.05) and CTAB dosed fish (P<0.001). SOD levels were significantly increased (P<0.001) in hepatic, renal and cardiac tissues of all the surfactant-treated fish. Glutathione reductase also was significantly increased (P<0.001) in the hepatic and renal tissues of surfactant dosed fish except cardiac tissues of CTAB exposed animals. Glutathione levels in the tissues studied were significantly higher in the surfactant treated animals (P<0.001) whereas malondialdehyde levels were significantly elevated only in the hepatic tissues of animals exposed to Triton (P<0.001). The surfactants based on their charge, antioxidant profile and in vivo metabolism may be arranged in the order of decreasing toxicity as CTAB > Triton > SDS. Thus it may be inferred from the present study that the antioxidant defenses and the in vivo metabolism of the surfactants are key factors in deciding the surfactant toxicity.     

Gossypol affects ion transport in the isolated intestine of the seawater adapted eel, Anguilla anguilla

Cottonseed (Gossypium sp.) meals are protein rich and inexpensive, but the presence of the polyphenolic dialdehyde, gossypol, is responsible of many toxic effects in animals including fishes. Recently an effect on the transepithelial ion transport in rat colon has been demonstrated. In this study we investigated the effect of gossypol on the transepithelial electrical parameters of the isolated intestine of seawater adapted eel, Anguilla anguilla, by employing a Ussing chamber technique. We showed that the addition of gossypol to the perfusion media reduced short circuit current (I(sc)), a measure of Cl- active absorption in this tissue, and increased tissue conductance (g(t)). The observation that the effect of gossypol on both I(sc) and g(t) was modified by the pretreatment with TFP, a calmodulin inhibitor, suggests that the substance acts via a Ca2+ calmodulin pathway and excludes the possibility that the observed effects were due to a cytotoxic action. In addition, experiments performed in the presence of verapamil suggest that the polyphenolic pigment increases Ca2+ influx. It is likely that gossypol stimulates a basolateral quinine sensitive K+ conductance producing a K+ flux in absorptive direction that explains the reduction of I(sc). In addition dilution potential experiments showed that the polyphenolic aldehyde increases the anion conductance of the paracellular pathway. In conclusion our study suggests that gossypol alters ion transport in eel intestine by acting on both transcellular and paracellular pathways. Since the intestine is an important organ for maintaining the water and ion balance in seawater adapted fish, it is conceivable that gossypol could impair the ability of the animals to adapt to the environment.     

Renal effects of fresh water-induced hypo-osmolality in a marine adapted seal

With few exceptions, marine mammals are not exposed to fresh water; however quantifying the endocrine and renal responses of a marine-adapted mammal to the infusion of fresh water could provide insight on the evolutionary adaptation of kidney function and on the renal capabilities of these mammals. Therefore, renal function and hormonal changes associated with fresh water-induced diuresis were examined in four, fasting northern elephant seal ( Mirounga angustirostris) (NES) pups. A series of plasma samples and 24-h urine voids were collected prior to (control) and after the infusion of water. Water infusion resulted in an osmotic diuresis associated with an increase in glomerular filtration rate (GFR), but not an increase in free water clearance. The increase in excreted urea accounted for 96% of the increase in osmotic excretion. Following infusion of fresh water, plasma osmolality and renin activity decreased, while plasma aldosterone increased. Although primary regulators of aldosterone release (Na(+), K(+) and angiotensin II) were not significantly altered in the appropriate directions to individually stimulate aldosterone secretion, increased aldosterone may have resulted from multiple, non-significant changes acting in concert. Aldosterone release may also be hypersensitive to slight reductions in plasma Na(+), which may be an adaptive mechanism in a species not known to drink seawater. Excreted aldosterone and urea were correlated suggesting aldosterone may regulate urea excretion during hypo-osmotic conditions in NES pups. Urea excretion appears to be a significant mechanism by which NES pups sustain electrolyte resorption during conditions that can negatively affect ionic homeostasis such as prolonged fasting.     

The nature and properties of the inducible sodium-plus-potassium ion-dependent adenosine triphosphatase in the gills of eels (Anguilla anguilla) adapted to fresh water and sea water

1. Gill tissue from eels adapted to fresh water or to sea water was disrupted in 0.32m-sucrose containing 0.1% (w/v) sodium deoxycholate and the subcellular distribution of (Na(+)+K(+))-dependent adenosine triphosphatase was determined. 2. About 70% of the recovered enzyme was in a fraction sedimenting between 225000g(av.)-min and 6000000g(av.)-min; the specific activities of enzymes from tissues of freshwater and seawater eels were 16 and 51 mumol of phosphate/h per mg of protein respectively. 3. The enzymes from gills of freshwater and seawater eels were indistinguishable on the basis of a number of parameters. These included phosphorylation by [gamma-(32)P]ATP, the binding of [(3)H]ouabain, the extent to which bound [(3)H]ouabain was displaced by increasing concentrations of KCl and pH optima. 4. Electrophoresis on polyacrylamide gels in sodium dodecyl sulphate showed that enzyme preparations from both sources had an identical number of protein components. 5. The higher specific activity of (Na(+)+K(+))-dependent adenosine triphosphatase from tissue of seawater eels was accompanied by increased amounts of two protein components. One of these proteins retained (32)P after treatment of the enzyme with [gamma-(32)P]ATP and had mol.wt. 97000; the other component was a glycoprotein with mol.wt. approx. 46000. 6. The results are discussed in terms of the nature of the transepithelial NaCl pumps in the gills of freshwater and seawater fish.     

The biochemical nature of the cell periphery of the salt gland secretory cells of fresh and salt water adapted mallard ducks

Summary Both ruthenium red and dialyzed iron techniques indicated that anionic protein-carbohydrates were associated with the plasmalemma of secretory cells in mallard salt glands. Digestion of the tissue with neuraminidase prior to dialyzed iron staining provided evidence that much of the anionic nature of this surface was due to sialic acid. These results were confirmed by biochemical assays showing that the salt-gland tissue contained a relatively high sialic-acid content and that the amount of sialic acid increased with salt-water adaptation. Possible roles of these anionic sites are discussed in relation to currently accepted hypotheses of electrolyte transport.Research supported by Public Health Service Grant AM-13705     

Actinomycete virus in fresh water

An ecological study was made of the freshwater distribution of Actinomycete virus. One component of this, the virus of Actinoplanes, is highly specific and its pattern of occurrence mirrors that of its host. The occurrence of Streptomyces virus is more difficult to explain in ecological terms, but this is attempted. Micromonospora virus was not obtained and the significance of this is discussed. The Actinoplanes virus is characterized at the fine-structure level by a polyhedral-shaped head with a tail bearing a contractile sheath, the latter feature being previously undescribed for actinophage.     

Chloride-dependent sodium and water transport in the seawater eel intestine

Summary Simultaneous measurements of transepithelial potential difference (PD) and net water flux were made in the stripped seawater eel intestine, and the effects of removal of Ca²⁺ and replacement of Cl^– with other anions on these two parameters were examined. Removal of Ca²⁺ from normal (NaCl) Ringer solution on both mucosal and serosal sides reduced the serosa-negative PD and the net water flux. Since SO ₄ ^2– binds Ca²⁺ strongly, the effects of substitution of SO ₄ ^2– for Cl^– could be due to deficiency in both Cl^– and Ca²⁺. Among five anions used in this study, CH₃SO ₄ ^– (with low affinity to Ca²⁺) seems to be the most suitable substitute for Cl^–. When both mucosal and serosal Cl^– were replaced with CH₃SO ₄ ^– , both the PD and the net water flux decreased to approximately zero. When mucosal Cl^– was replaced progressively with other anions, the serosa-negative PD and the net water flux decreased in association with the decrease in Cl^– concentration, and a linear regression was observed between the decrease in the net water flux and that in the PD. These results indicate that Na⁺ and water transport depend closely on Cl^– transport.     

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.     

Renal water and solute excretion in the Atlantic stingray in fresh water

Freshwater, male Atlantic stingrays Dasyatis sabina , from Lake Jesup, Florida, U·S·A·, excreted a dilute urine similar in composition to freshwater teleosts and lampreys with the exception that urea was the primary osmolyte. Urine flow rate was 2·5 to 10 fold higher than that reported for freshwater teleosts resulting in high free-water clearance. Mass-specific free-water clearance values from euryhaline elasmobranchs inhabiting freshwater environments greatly exceed those for freshwater teleosts and are nearly equivalent to those of freshwater lampreys.