Changes in osmotic water permeability of the eel gills during seawater and freshwater adaptation

Summary Changes in osmotic water permeability of the isolated gills of the Japanese eel,Anguilla japonica were studied during transfer to seawater or to fresh water. The water permeability increased gradually during the course of seawater transfer and attained a maximal level after 2 weeks. The water permeability of the freshwater eel gills was reduced when calcium ions were added to the incubation medium at a concentration of 1 mM, where-as no effect of the ion was observed on the gills of the seawater-adapted eel even at a higher concentration (10 mM). In contrast to seawater transfer, the water permeability decreased to a low level almost immediately (3 h) after transfer from seawater to fresh water. The acute reduction of the water permeability was also seen in the gills of the hypophysectomized eel after transfer to fresh water.The gradual increase in the gill water permeability during seawater transfer is correlated with an increase in the number of chloride cells. In scanning electron microscopy, chloride cells of seawater-adapted eel gills exhibit a pit-like structure, which was larger than in the freshwater eel. On transfer from seawater to fresh water, the pit diameter became smaller within 6 h. Hypophysectomy did not affect the change in gill surface structures during transfer to fresh water. The junctions between the chloride cells of seawater eel gills are reported to be of the leaky type. The parallel change in osmotic water permeability and in pit size of the chloride cells during seawater or freshwater transfer or after hypophysectomy suggests that these cells could provide a major route of water as well as ion movement.This paper is a portion of a thesis presented to Hokkaido University by t. Ogasawara in partial fulfilment of the requirements for Doctor of Fisheries     

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.     

Effect of environmental salinity change on osmotic permeability of the isolated gill of the eel, Anguilla anguilla L.

Net water fluxes in the isolated gills of Anguilla anguilla were studied during incubation in fresh water (FW) and in sea water (SW). When incubated in FW, water entry was greater in SW-adapted eels than in FW-adapted eels. In contrast, water loss in SW was less in SW-adapted eels than in FW-adapted eels. Rectification of osmotic water fluxes was observed for both FW and SW-adapted eels, net water fluxes in the mucosal-serosal (m-s) direction being greater than those in the opposite (s-m) direction. These results indicate that adaptation to a given external medium brings about a decrease in the osmotic permeability so that water gain in FW or water loss in SW is minimal.     

Salt excretion by the perfused head of trout adapted to sea water and its inhibition by adrenaline

Summary Isolated heads of trout (Salmo gairdneri) were used to study the unidirectional flux of sodium and chloride across the gills in salt water.Two perfusion techniques were employed. Under constant pressure perfusion, the addition of adrenaline during the perfusion causes an increase in the flow-rate. Under constant flow-rate conditions, adrenaline provokes a decrease in pressure. A comparison of influx determination made with these two techniques of perfusion shows that variations in flow-rate of perfusion do not affect the assessment of these fluxes.A net efflux of sodium, but not of chloride, is demonstrated in sea water. The effluxes of sodium and chloride observed in sea water are decreasedd about 50% during a rapid transfer to fresh water. The addition of potassium to this medium stimulates the effluxes of sodium and chloride, suggesting a Na/K exchange participating in the chloride excretion.Adrenaline causes an inhibition of sodium and chloride efflux in sea water which persists after transfer to fresh water and the addition of potassium. Only the influx of chloride is inhibited at a concentration of 10^–5 M whereas the sodium influx is unaffected. The presence of adrenaline results in a net influx of both sodium and chloride.The differential action of adrenaline on the influxes of sodium and chloride suggests that the hemodynamic modifications provoked by this catecholamine occur independently of its aforementioned ion exchange effects.     

The heart-gill preparation of the eel and its perfusion for the study of a natural membrane in situ

Summary The isolated heart-gill preparation of the eel is presented. Essentially it is a double perfusion system in which both internal and external mediums are circulated separately. The applicability of this preparation to other species of fish besides the eel is indicated.The apparatus arrangement, operative technique, and the procedure for making the most carefully controlled studies of the permeability and chemical activity of the gills are described in detail.The composition and preparation of various concentrations of a perfusion medium developed for use in this preparation are given.It is shown that the preparation retains its vital properties for a period of hours and is physiologically acceptable.The utilization of the preparation as a potent research tool in the investigation of the physiology 6f the gills and heart, as well as its use from the aspect of the properties of membranes, are discussed.Fellow of the National Research Council of America.     

RING finger, B-box, and coiled-coil (RBCC) protein expression in branchial epithelial cells of Japanese eel, Anguilla japonica.

An RBCC (RING finger, B-box, and coiled-coil) protein was identified that belongs to the superfamily of zinc-binding proteins and is specifically expressed in the gill of eel, Anguilla japonica. Euryhaline fishes such as eels can migrate between freshwater and seawater, which is considered to be accomplished by efficient remodeling of the architecture and function of the gill, a major osmoregulatory organ. To identify molecules involved in such adaptive changes, we performed differential display using mRNA preparations from freshwater and seawater eel gills and obtained an RBCC clone among several differentially expressed clones. The clone encoded a protein of 514 amino acid residues with structural features characteristic of the RBCC protein; we therefore named it eRBCC (e for eel). eRBCC mRNA was specifically expressed in the gills with a greater extent in the gills of freshwater eels. Immunohistochemistry revealed that the expression of eRBCC is confined to particular epithelial cells of the gills including freshwater-specific lamellar chloride cells. The RING finger of eRBCC was found to have a ubiquitin ligase activity, suggesting an important regulatory role of eRBCC in the remodeling of branchial cells.     

Enhanced expression and release of C-type natriuretic peptide in freshwater eels

C-type natriuretic peptide (CNP) is recognized as a paracrine factor acting locally in the brain and periphery. To assess the role of CNP in teleost fish, a cDNA encoding a CNP precursor was initially cloned from the eel brain. CNP message subsequently detected by ribonuclease protection assay, using the cDNA as probe, was most abundant in the brain followed by liver, gut, gills, and heart. Expression was generally higher in freshwater (FW) than in seawater (SW) eels, but not in the brain. Plasma CNP concentration measured by a newly developed homologous radioimmunoassay for eel CNP was higher in FW than in SW eels. The CNP concentration was also higher in the heart of FW eels but not in the brain. These results show that CNP is abundantly synthesized in peripheral tissues of FW eels and secreted constitutively into the circulation. Therefore, CNP is a circulating hormone as well as a paracrine factor in eels. Together with our previous demonstration that CNP-specific receptor expression is enhanced in FW eels, it appears that CNP is a hormone important for FW adaptation. Because atrial NP (ANP) promotes SW adaptation in eels, CNP and ANP, despite high sequence identity, appear to have opposite effects on environmental adaptation of the euryhaline fish.     

Colonization, larval survival and epidemiology of the nematode Anguillicola crassus, parasitic in the eel, Anguilla anguilla, in Britain

In late 1987, immature Anguillicola crassus were reported for the first time in Britain from eels from two river systems. By late 1988, gravid adults were present in a number of rivers in the east of England in two discrete centres of distribution: one in East Anglia correlated with the route taken by lorries exporting eels to the continent, and one in the R. Thames correlated with the import of eels to London. The parasite was firmly established in the R. Trent, where prevalence levels reached 100% in some places. Laboratory investigations showed that adult parasites and their eggs remained viable even after infected eels had been maintained for 4 weeks in 100% sea water. Hatching of eggs declined with increasing salinity, but was not totally inhibited by sea water. Survival and infectivity of freeliving second stage larvae were maximal in natural fresh water (95% survival for 4 months, and 50% still infective to copepod intermediate hosts after 70 days), but declined in alkaline water and with increased salinity. Nevertheless, in 100% sea water, 50% of larvae were still infective after 8 days. Specificity to the intermediate host was low, and eels of all sizes could be infected. These characteristics, plus a high reproductive potential, give the parasite exceptional colonization potential and ability, enabling it to survive natural movements of eels from catchment to catchment and to increase rapidly within a new locality. The ability of free-living larvae to adhere to the substratum and survive in sea water enables them to survive in eel-transport lorries from which they will not readily be removed by flushing, the normal cleansing procedure. It is concluded that there were two separate introductions of the parasite to Britain; via the eel import trade through London, and, totally unexpectedly, via the eel export trade in lorries traversing East Anglia. The parasite is now firmly established in Britain and will continue to spread by natural movements of eels but especially by human-assisted movements of infected eels for stocking and market. This latter practice is recognized as a major factor in introducing and disseminating fish parasites.     

The effects of 11-ketotestosterone on occupation of downstream location and seawater in the New Zealand shortfinned eel, Anguilla australis

The androgen 11-ketotestosterone (11KT) is associated with the physiological and morphological changes that occur during the transformation of sedentary ('yellow') freshwater eels (Anguilla spp.) into their migratory form ('silver') prior to their spawning migration in the ocean. In this study, we investigate the possible role of 11KT in modulating behaviors consistent with downstream migration; i.e., downstream and salinity preference in the New Zealand shortfinned eel (A. australis). Unlike silvering, 11KT did not induce preference for downstream locations, scored as presence at the downstream ends of 35 m raceways. Likewise, there was no evidence for increased salinity preference in 11KT-treated yellow eels, scored as preference for sea water over fresh water in a choice experiment. However, the 11KT treatment induced higher frequency of movements between fresh water and sea water, which may indicate restlessness.     

Environmental cues and extended estuarine residence in seaward migrating eels (Anguilla australis)

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Ouabain inhibition of gill Na-K-ATPase: relationship to active chloride transport.

Ouabain circulating in blood inhibits Na-K-ATPase in the gills of seawater eels at a concentration similar to that necessary for inhibition in vitro. By contrast, a much higher concentration is required when ouabain is applied to the exterior of the gill. Inhibition by external ouabain occurs only when the drug gains access to the circulation of the fish, as evidenced by simultaneous inhibition of Na-K-ATPase in the kidney. These results suggest that the Na-K-ATPase of gill chloride cells faces inward, lining intracytoplasmic tubular channels continuous with the extracellular fluid. Inhibition of gill Na-K-ATPase by ouabain in intact salt water eels results in almost complete inhibition of the efflux of both Na+ and Cl-. The efflux is tritiated water was much less reduced, to 60% of normal. Since chloride is actively transported outward across the gill of seawater teleosts, it is suggested that active chloride transport is coupled to Na-K-ATPase. A neutral sodium chloride carrier is postulated that is energized by the movement of sodium from extracellular fluid down its electrochemical gradient into the chloride cell.     

Differences in the Trace Element Deposition in Otoliths Between Marine- and Freshwater-resident Japanese Eels, Anguilla japonica, as Determined by Laser Ablation ICPMS

Synopsis In order to determine whether the trace element composition in otolith of the Japanese eel Anguilla japonica could be used to determine its habitat use, we used laser ablation inductivity coupled plasma mass spectrometry (LA-ICPMS) to assay sectioned otoliths of both marine-resident (sea eels) and freshwater-resident (river eels) eels. A close linear relationship in the Sr:Ca ratios between EPMA (X-ray analysis with an electron microprobe) and LA-ICPMS analyses was found, suggesting that the latter technique could be used to separate the marine and freshwater life phases. Elemental signatures in the otolith outside the elver mark showed significant differences in Cr:Ca, Mn:Ca, and Ba:Ca ratios as well as Sr:Ca ratios between sea and river eels. These results indicate that the elemental compositions may reflect environmental variability between marine and fresh water masses. Thus, those elemental ratios determined by LA-ICPMS analysis seem to have the potential to help distinguish the habitat of the eel.     

Densities and swimbladder development of juvenile American eels, Anguilla rostrata (Lesueur) as related to energetics of migration

Glass eels and elvers of the American eel were negatively buoyant. Those adapted to sea water were more dense (1.072 ± 0.001 g cm⁻³) than those adapted to fresh water (1.061 ± 0.001 g cm⁻³). Adaptation to fresh water increased relative body water content, but did not account for the observed decrease in total body density. Histological examination revealed the presence of a potentially functional swimbladder in the glass eels, although this hydrostatic organ did not becomegas-filled until after freshwater residency had occurred. Calculation of lift as used in the selective tidal transport mechanism suggests that hydrodynamic compensation for horizontal swimming during the estuarine phase of migration is energetically adaptive.     

The role of the transbranchial potential difference in hyperosmotic regulation of the shore crabCarcinus maenas

When isolated gills of the shore crabCarcinus maenas were bathed and perfused with identical solutions on both sides (50 % sea water), a spontaneous transepithelial potential difference (PD) of some millivolts (hemolymph side negative) was established. This PD is of active nature and requires the metabolism of the living cell, since it uses its own sources of energy in addition to organic nutrients offered in the flow of artificial hemolymph. Addition of sodium cyanide and dinitrophenole to bathing and perfusion medium resulted in reversible breakdown of PDs in a concentration-dependent mode. In posterior gills ofC. maenas, the potential differences were more negative compared to data measured in anterior gills of the same individuals. These results are correlated with higher specific activities of Na-K-ATPase in posterior gills. Experiments with triamterene indicate that sodium uptake inC. maenas is sensitive to this diuretic drug, when applied on the apical side of the epithelial cell. The results obtained show that active uptake of sodium from medium to blood across the gills is performed by a complex mechanism including participation of several basal and apical transport steps.     

The demonstration of chloride ions in the “chloride cells” of the gills of eels (Anguilla anguilla L.) adapted to sea water

Summary The ultrastructure of chloride cells in the gills of eels kept in artificial sea water and of a control animal kept in fresh water was studied. In addition to glutaraldehydeosmium tetroxide and simple osmium tetroxide fixation, a special method for the demonstration of chloride ions was used (Komnick, 1962, 1963). Based on the principle of silver chloride precipitation in the presence of chloride ions, the procedure showed positive results in the chloride cells of eels adapted to sea water. Smooth-surfaced tubules of the endoplasmic reticulum contained a material of medium to strong electron density, that was often in communication with the plasma membrane. The same material, always of very high density, was present in the intercellular spaces, thus forming conspicuous lines around cells. The silver precipitate was found very often in large quantities in the pits of chloride cells, having thereby the aspect of a secretory product. However, a direct communication between the system of endoplasmic reticulum tubules containing the silver reaction product with the above-mentioned masses of silver chloride was not demonstrated. Schultz (1958) first published an electron micrograph of these cells. Two features, numerous mitochondria and highly developed agranular endoplasmic reticulum, affirmed by all later investigators, have remained the most important criteria for the identification of chloride cells (Kessel and Beams, 1962; Philpott, 1962; Philpott and Copeland, 1963; Rhodin, 1964; Henrikson and Matoltsy, 1968). Kessel and Beams (1962) and Philpott and Copeland (1963) demonstrated pits, or apical cavities (Oberg, 1967) filled with an amorphous granular substance of medium electron density. (Threadgold and Houston, 1964). These cavities are found in animals adapted to sea water only, and correspond to the excretory vesicles described earlier by Copeland (1948). Philpott and Copeland demonstrated numerous vesicles and tubules in the cytoplasm surrounding the apical cavities and opening into them, possibly contributing to their granular material. Several authors attempted to demonstrate a chloride excretory function of chloride cells with the aid of some histochemical reactions. Copeland (1948), Datta Munshi (1964) and Philpott (1966) used silver techniques     

A comprehensive hypothesis on the migration of European glass eels (Anguilla anguilla)

The European eel (Anguilla anguilla) is a catadromous fish that spawns in the Sargasso Sea. As larvae, eels cross the Atlantic Ocean and reach the continental slope of Europe, where they metamorphose into post‐larval glass eels. These reach the continent, where some enter fresh water, some remain in marine waters, and others move between fresh and marine waters. After 5–25 years, as adult silver eels, they migrate back from fresh water to the Sargasso Sea to spawn and die. The glass eel stage is a critical step during which the eels cross the continental shelf and recruit to estuaries, where they facultatively transition to fresh water. Extensive research has been conducted to understand the behavioural mechanisms and environmental cues that aid and guide glass eels' migration. Glass eels follow odours and salinity gradients, they avoid light, and they change orientation and depth according to the tides. Recent work revealed that European glass eels also use Earth's magnetic field and lunar cues to orient. However, while we understand many aspects of their orientation behaviour, a unifying theory describing how glass eels migrate from the continental slope to fresh water is lacking. The goal of this review is to develop a comprehensive hypothesis on the migration of European glass eels, integrating previous knowledge on their orientation behaviour with recent findings on magnetic and celestial orientation. This review follows the journey of a hypothetical glass eel, describing the nature and the role of orientation cues involved at each step. I propose that, although glass eels have the sensory capacity to use multiple cues at any given time, their migration is based on a hierarchical succession of orientation mechanisms dictated by the physical properties of the environments that they occupy: (i) lunar and magnetic cues in pelagic water; (ii) chemical and magnetic cues in coastal areas; and (iii) odours, salinity, water current and magnetic cues in estuaries.     

A description of rodlet cells from the alimentary canal of Anguilla anguilla and their relationship with parasitic helminths

Rodlet cells in intestinal epithelia of infected and uninfected European eels Anguilla anguilla from brackish and fresh water were studied by light and electron microscopy. Deropristis inflata (Trematoda) was found in eels from brackish water, whereas eels from fresh water were infected with Acanthocephalus clavula (Acanthocephala). In a comparison between uninfected and infected eels from brackish water, a higher number of rodlet cells was recorded in the intestinal epithelia of infected fish. Evidence is presented that rodlet cells secrete their contents in a holocrine manner into the lumen of the eel intestine. The occurrence of organelles within the mature rodlet cell was rare. ? 1998 The Fisheries Society of the British Isles     

Timing and pattern of annual silver eel migration in two European watersheds are determined by similar cues

Many animals perform long‐distance migrations in order to maximize lifetime reproductive success. The European eel migrates several thousand kilometers between their feeding habitats in continental waters (fresh‐, brackish, and sea water) and their spawning area in the Sargasso Sea. Eels residing in freshwaters usually initiate their spawning migration as silver eels during autumn, triggered by diverse environmental cues. We analyzed the time series of silver eel downstream migration in Burrishoole, Ireland (1971–2015), and Imsa, Norway (1975–2015), to examine factors regulating the silver eel migration from freshwater to the sea. The migration season (90% of the run) generally lasted from 1 August to 30 November. Environmental factors acting in the months before migration impacted timing and duration of migration, likely through influencing the internal processes preparing the fish for migration. Once the migration had started, environmental factors impacted the day‐to‐day variation in number of migrants, apparently stimulating migration among those eels ready for migration. Both the day‐to‐day variation in the number of migrants and the onset of migration were described by nearly identical models in the two rivers. Variables explaining day‐to‐day variation were all associated with conditions that may minimize predation risk; number of migrants was reduced under a strong moon and short nights and increased during high and increasing water levels. Presence of other migrants stimulated migration, which further indicates that silver eel migration has evolved to minimize predation risk. The onset of migration was explained mainly by water levels in August. The models for duration of the migration season were less similar between the sites. Thus, the overall migration season seems governed by the need to reach the spawning areas in a synchronized manner, while during the actual seaward migration, antipredator behavior seems of overriding importance.     

Role of the metalloprotease Vvp and the virulence plasmid pR99 of Vibrio vulnificus serovar E in surface colonization and fish virulence

The virulence for eels of Vibrio vulnificus biotype 2 serovar E (VSE) is conferred by a plasmid that codifies ability to survive in eel serum and cause septicaemia. To find out whether the plasmid and the selected chromosomal gene vvp plays a role in the initial steps of infection, the VSE strain CECT4999, the cured strain CT218 and the Vvp-deficient mutant CT201 (obtained in this work by allelic exchange) were used in colonization and virulence experiments. The eel avirulent biotype 1 (BT1) strain YJ016, whose genome has been sequenced, was used for comparative purposes. The global results demonstrate that the plasmid does not play a significant role in surface colonization because (i) CECT4999 and CT218 were equally chemoattracted towards and adherent to eel mucus and gills, and (ii) CT218 persisted in gills from bath-infected eels 2 weeks post infection. In contrast, mutation in vvp gene reduced significantly chemoattraction and attachment to eel mucus and gills, as well as virulence degree by immersion challenge. Co-infection experiments by bath with CECT4999 and CT201 confirmed that Vvp was involved in eel colonization and persistence in gills, because CECT4999 was recovered at higher numbers compared with CT201 from both internal organs of moribund fish (ratio 4:1) and gills from survivors (ratio 50:1). Interestingly, YJ016 also showed chemoattraction and attachment to mucus, and complementation of CT201 with BT1- vvp gene restored both activities together with virulence degree by immersion challenge. Additional experiments with algae mucus and purified mucin gave similar results. In conclusion, the protease Vvp of V. vulnificus seems to play an essential role in colonization of mucosal surfaces present in aquatic environments. Among the V. vulnificus strains colonizing fish mucus, only those harbouring the plasmid could survive in blood and cause septicaemia.     

A novel guanylin family (guanylin,uroguanylin, and renoguanylin) in eels: possible osmoregulatory hormones in intestine and kidney

As the intestine is an essential organ for fish osmoregulation, the intestinal hormone guanylins may perform major functions, especially in euryhaline fish such as eels and salmonids. From the intestine of an eel, we identified cDNAs encoding three distinct guanylin-like peptides. Based on the sequence of mature peptide and sites of production, we named them guanylin, uroguanylin, and renoguanylin. Renoguanylin is a novel peptide that possesses the characteristics of both guanylin and uroguanylin and was abundantly expressed in the kidney. By immunohistochemistry, guanylin was localized exclusively in goblet cells, but not enterochromaffin cells, of the intestine. After transfer of eels from fresh water to seawater, mRNA expression of guanylin and uroguanylin did not change for 3 h, but it increased after 24 h. The increase was profound (2-6-fold) after adaptation to seawater. The expression of uroguanylin was also up-regulated in the kidney of seawater-adapted eels, but that of renoguanylin was not so prominent as other guanylins in both intestine and kidney. Collectively, the novel eel guanylin family appears to have important functions for seawater adaptation, particularly long-term adaptation. Eel guanylin may be secreted from goblet cells into the lumen with mucus in response to increased luminal osmolality and act on the epithelium to regulate water and salt absorption.