Embryonic occurrence of ionocytes in the sea bass Dicentrarchus labrax

Because of the permeability of the chorion, sea bass embryos are exposed to seawater before hatching and hence require precocious osmoregulatory processes. Several studies of other species have demonstrated the existence of ion-transporting cells located on the yolk sac membrane of embryos. In these cells, called ionocytes, ion movements are controlled by a pool of transmembrane proteins. Among them, the Na⁺/K⁺-ATPase, an abundant driving enzyme, has been used to reveal the presence or absence of ionocytes. We have immunostained the Na⁺/K⁺-ATPase in sea-bass embryos and shown the presence of the first ionocytes on the yolk sac membrane at stage 12 somites and the occurrence of ionocytes at other sites before hatching. Ionocytes located on the first gill slits have been identified at stage 14 somites. Primitive enteric ionocytes have also been detected at stage 14 somites in the mid and posterior gut. The presence of these cells might be related to the early opening of the gut to perivitelline fluids, both anteriorly by the gill slits and posteriorly by the anus. The role of embryonic ionocytes in osmoregulation before hatching is discussed.     

Environmental Regulation of Mitochondria-Rich Cells in Chalcalburnus tarichi (Pallas, 1811) During Reproductive Migration

Chalcalburnus tarichi is an anadromous cyprinid fish that has adapted to extreme conditions (salinity 22 ‰, pH 9.8 and alkalinity 153 mEq × l^?1) in Lake Van in eastern Turkey. Changes in immunoreactivity of Na⁺/K⁺-ATPase in gill tissue and osmolarity and ion levels in plasma were investigated in C. tarichi during reproductive migration. Physicochemical characteristics and ion levels in Lake Van were high compared freshwater. Plasma osmolality and plasma ion concentrations ([Na⁺], [K⁺] and [Cl^?]) increased after transfer from freshwater to Lake Van. The mitochondria-rich (MR) cells of the gill were stained in both filament and lamellar epithelia of C. tarichi by immunocytochemistry with a specific antiserum for Na⁺/K⁺-ATPase in river fish samples. Density and area of MR cells were decreased in lake-adapted fishes. These results indicated that freshwater acclimation capacity is correlated with the size and distribution of MR cells in C. tarichi, in contrast to many teleost fishes.     

Na+-dependent Ca2+ uptake in isolated opercular epithelium of Fundulus heteroclitus

It is concluded that Ca²⁺ transport across the basolateral membranes of the ionocytes in killifish skin is mediated for the major part by a Na⁺/Ca²⁺-exchange mechanism that is driven by the (transmembrane) Na⁺ gradient established by Na⁺/K⁺-ATPase. The conclusion is based, firstly, on the biochemical evidence for the presence of a Na⁺/Ca²⁺-exchanger next to the Ca²⁺-ATPase in the basolateral membranes of killifish gill cells. Secondly, the transcellular Ca²⁺ uptake measured in an Ussing chamber setup was 85% and 80% reduced in freshwater (FW) and SW (SW) opercular membranes, respectively, as the Na⁺ gradient across the basolateral membrane was directly or indirectly (by ouabain) reduced. Thapsigargin or dibutyryl-cAMP/IBMX in SW opercular membranes reduced Ca²⁺ influx to 46%, comparable to the effects seen in FW membranes [reduction to 56%; Marshall et al. 1995a]. Basal Ca²⁺ influx across the opercular membrane was 48% lower in membranes from fish adapted to SW than in membranes from fish adaptated to FW. Branchial Na⁺/K⁺-ATPase activity was two times higher in SW adapted fish. Accepted: 29 October 1996     

Ion-poor water and dietary salt deprivation upregulate the ghrelinergic system in the goldfish (Carassius auratus)

Because the ghrelinergic system in teleost fishes is broadly expressed in organs that regulate appetite as well as those that contribute to the regulation of salt and water balance, we hypothesized that manipulating salt and water balance in goldfish (Carassius auratus) would modulate the ghrelinergic system. Goldfish were acclimated to either freshwater (FW) or ion-poor FW (IPW) and were fed either a control diet containing 1% NaCl or low-salt diet containing 0.1% NaCl. Endpoints of salt and water balance, i.e., serum Na⁺ and Cl⁻ levels, muscle moisture content and organ-specific Na⁺-K⁺-ATPase (NKA) activity, were examined in conjunction with brain, gill and gut mRNA abundance of preproghrelin and its receptor, growth hormone secretagogue receptor (ghs-r). Acclimation of fish to IPW reduced serum osmolality and Cl⁻ levels and elevated kidney NKA activity, while FW fish fed a low NaCl diet exhibited a modest reduction in muscle moisture content but otherwise no apparent osmoregulatory disturbance. In contrast, a combined treatment of IPW acclimation and low dietary NaCl content reduced serum osmolality and Cl⁻ levels, elevated muscle moisture content and increased gill, kidney and intestinal NKA activity. This intensified response to the combined effects of water and dietary ion deprivation is consistent with an increased effort to enhance ion acquisition. In association with these latter observations, a significant upregulation of preproghrelin mRNA expression in brain and gut was observed. A significant increase in ghs-r mRNAs was also observed in the gill of goldfish acclimated to IPW alone but a reduction in dietary NaCl content did not impact the ghrelinergic system of goldfish in FW. The results support the hypothesis that the ghrelinergic system is modulated in response to manipulated salt and water balance. Whether the central and peripheral ghrelinergic system contributes to ionic homeostasis in goldfish currently remains unclear and warrants further research.     

Sulfatide and Na<Superscript>+</Superscript>-K<Superscript>+</Superscript>-ATPase: A Salinity-sensitive Relationship in the Gill Basolateral Membrane of Rainbow Trout

We investigated the effect of salinity on the relationship between Na⁺-K⁺-ATPase and sulfogalactosyl ceramide (SGC) in the basolateral membrane of rainbow trout (Oncorhynchus mykiss) gill epithelium. SGC has been implicated as a cofactor in Na⁺-K⁺-ATPase activity, especially in Na⁺-K⁺-ATPase rich tissues. However, whole-tissue studies have questioned this role in the fish gill. We re-examined SGC cofactor function from a gill basolateral membrane perspective. Nine SGC fatty acid species were quantified by tandem mass spectrometry (MS/MS) and related to Na⁺-K⁺-ATPase activity in trout acclimated to freshwater or brackish water (20 ppt). While Na⁺-K⁺-ATPase activity increased, the total concentration and relative proportion of SGC isoforms remained constant between salinities. However, we noted a negative correlation between SGC concentration and Na⁺-K⁺-ATPase activity in fish exposed to brackish water, whereas no correlation existed in fish acclimated to freshwater. Differential Na⁺-K⁺-ATPase/SGC sensitivity is discussed in relation to enzyme isoform switching, the SGC cofactor site model and saltwater adaptation.This revised version was published online in June 2005 with a corrected cover date.     

Effects of cytochalasin B on water, Na+ and Cl? exchanges in the gill of seawater adapted mulletMugil capito

Summary Electron microscopy study shows that cytochalasin treatment of the mullet damages the microfilaments system in the apex of gill ionocytes: the microfilaments are reduced in number and shortened. Cytochalasin causes a reduction of transgill potential difference and an increase of the Na⁺ and Cl^– blood concentration, of the diffusional water permeability of the gill, of the Na⁺ branchial influx and of Cl^– efflux. The increase of the Na⁺ influx may result in a reduction of the Na⁺ net excretion flux compared to the control. The increased permeability in cytochalasin treated fish facilitates the Cl^– entry probably leading to a reduction of the net Cl^– excretion. The partial inhibition of the K⁺ dependent components of Na⁺ and Cl^– effluxes also contributes to the reduction of Na⁺ and Cl^– excretion. The role of microfilaments in the mechanisms of ionic excretion by the gill is discussed.     

A Time Differential Staining Technique Coupled with Full Bilateral Gill Denervation to Study Ionocytes in Fish

Branchial ionocytes (ICs) are the functional units for ionic regulation in fish. In adults, they are found on the filamental and lamellar epithelia of the gill where they transport ions such as Na⁺, Cl^- and Ca²⁺ via a variety of ion channels, pumps and exchangers. The teleost gill is extrinsically innervated by the facial (VI), glossopharyngeal (IX) and vagus (X) nerves. The IX and X nerves are also the extrinsic source of branchial IC innervation. Here, two techniques used to study the innervation, proliferation and distribution of ICs are described: a time differential staining technique and a full bilateral gill denervation technique. Briefly, goldfish are exposed to a vital mitochondrion-specific dye (e.g., MitoTracker Red) which labels (red fluorescence) pre-existing ICs. Fish were either allowed to recover for 3 - 5 days or immediately underwent a full bilateral gill denervation. After 3 - 5 days of recovery, the gills are harvested and fixed for immunohistochemistry. The tissue is then stained with an α-5 primary antibody (targets Na⁺/K⁺ ATPase containing cells) in conjunction with a secondary antibody that labels all (both new and pre-existing) ICs green. Using confocal imaging, it was demonstrated that pre-existing ICs appear yellow (labelled with both a viable mitochondrion-specific dye and α-5) and new ICs appear green (labelled with α-5 only). Both techniques used in tandem can be applied to study the innervation, proliferation and distribution of ICs on the gill filament when fish are exposed to environmental challenges.     

Changes in branchial and intestinal osmoregulatory mechanisms and growth hormone levels during smolting in hatchery-reared and wild brown trout

Evidence of smolting was studied in Danish hatchery-reared brown trout Salmo trutta L. Twenty-four hour seawater (SW) challenge tests (28‰, 10°C) at regular intervals showed that maximal hypo-osmoregulatory ability developed within a 3–4-week period in March and April. The improved ability to regulate plasma osmolality, muscle water content and plasma total [Mg] developed asynchronously, indicating that developmental changes in the gill, the gastrointestinal system and the kidney may not necessarily concur during smolting. Gill Na⁺, K⁺-ATPase activity peaked in April at the time of optimal hypo-osmoregulatory ability. Na⁺, K⁺-ATPase a -subunit mRNA level in gills was unchanged from January until April, but decreased in May in parallel with a decrease in the activity of the enzyme. In the middle region of the intestine, Na⁺, K⁺-ATPase activity increased in February and remained high until April. In the posterior region of the intestine, the activity was stable from January until April after which it decreased. In vitro fluid transport capacitity, J_v, in the middle intestine fluctuated throughout the spring. In the posterior intestine, J_v was low until late March, when it increased fivefold until early May. Drinking rate in fish transferred to SW for 24 h surged during spring. Na⁺, K⁺-ATPase activity in the pyloric caeca was elevated from March until May, and increased in response to SW transfer in June, suggesting a hypo-osmoregulatory function of the pyloric caeca. Plasma GH levels surged in FW trout during spring, concurring with the increase in gill Na⁺, K⁺-ATPase activity and SW tolerance, but peaked in May when gill Na⁺, K⁺-ATPase activity and SW tolerance were regressing. GH levels were generally low in SW-challenged fish, and there was no consistent effect of 24-h SW exposure on GH levels. In wild anadromous trout, gill Na⁺, K⁺-ATPase activity varied seasonally as in hatchery-reared fish, but peaked at higher levels suggesting a more intense smolting in fish living in their natural environment.     

Embryonic ionocytes in the European sea bass (Dicentrarchus labrax): Structure and functionality

Early ionocytes have been studied in the European sea bass (Dicentrarchus labrax) embryos. Structural and functional aspects were analyzed and compared with those observed in the same conditions (38 ppt) in post hatching stages. Immunolocalization of Na⁺/K⁺‐ATPase (NKA) in embryos revealed the presence of ionocytes on the yolk sac membrane from a stage 12 pair of somites (S), and an original cluster around the first gill slits from stage 14S. Histological investigations suggested that from these cells, close to the future gill chambers, originate the ionocytes observed on gill arches and gill filaments after hatching. Triple immunocytochemical staining, including NKA, various Na⁺/K⁺/2Cl^? cotransporters (NKCCs) and the chloride channel “cystic fibrosis transmembrane regulator” (CFTR), point to the occurrence of immature and mature ionocytes in early and late embryonic stages at different sites. These observations were completed with transmission electronic microscopy. The degree of functionality of ionocytes is discussed according to these results. Yolk sac membrane ionocytes and enteric ionocytes seem to have an early role in embryonic osmoregulation, whereas gill slits tegumentary ionocytes are presumed to be fully efficient after hatching.     

Effects of lanthanum on sodium and chloride fluxes in the goldfishCarassius auratus

Summary The effect of lanthanum (La³⁺) on Na⁺ and Cl^– fluxes and on gill potentials was investigated in the goldfish,Carassius auratus. Initially La³⁺ caused large increases in both influx and efflux but after 15 min these decreased with influx returning to near normal levels while effluxes remained elevated, leading to a significant net loss of ions. Both La³⁺ and Ca²⁺ influenced gill potentials in an identical way. Possible physiological effects of La³⁺ on gill ion exchange mechanisms are discussed.     

Physiological and molecular analysis of the interactive effects of feeding and high environmental ammonia on branchial ammonia excretion and Na+ uptake in freshwater rainbow trout

Recently, a “Na⁺/NH₄ ⁺ exchange complex” model has been proposed for ammonia excretion in freshwater fish. The model suggests that ammonia transport occurs via Rhesus (Rh) glycoproteins and is facilitated by gill boundary layer acidification attributable to the hydration of CO₂ and H⁺ efflux by Na⁺/H⁺ exchanger (NHE-2) and H⁺-ATPase. The latter two mechanisms of boundary layer acidification would occur in conjunction with Na⁺ influx (through a Na⁺ channel energized by H⁺-ATPase and directly via NHE-2). Here, we show that natural ammonia loading via feeding increases branchial mRNA expression of Rh genes, NHE-2, and H⁺-ATPase, as well as H⁺-ATPase activity in juvenile trout, similar to previous findings with ammonium salt infusions and high environmental ammonia (HEA) exposure. The associated increase in ammonia excretion occurs in conjunction with a fourfold increase in Na⁺ influx after a meal. When exposed to HEA (1.5 mmol/l NH₄HCO₃ at pH 8.0), both unfed and fed trout showed differential increases in mRNA expression of Rhcg2, NHE-2, and H⁺-ATPase, but H⁺-ATPase activity remained at control levels. Unfed fish exposed to HEA displayed a characteristic reversal of ammonia excretion, initially uptaking ammonia, whereas fed fish (4 h after the meal) did not show this reversal, being able to immediately excrete ammonia against the gradient imposed by HEA. Exposure to HEA also led to a depression of Na⁺ influx, demonstrating that ammonia excretion can be uncoupled from Na⁺ influx. We suggest that the efflux of H⁺, rather than Na⁺ influx itself, is critical to the facilitation of ammonia excretion.     

Physiological, Endocrine, and Genetic Bases of Anadromy in the Brook Charr, Salvelinus Fontinalis, of the Laval River (Québec, Canada)

Brook charr, Salvelinus fontinalis, often display alternate life history styles in coastal areas. In the Laval River, some brook charr remain freshwater residents, while others undergo seasonal migrations between freshwater and saltwater environments. In the present paper, we examined physiological (electrolyte concentrations, gill Na⁺, K⁺-ATPase activity, and thyroid hormone levels) as well as genetic differences (neutral genetic markers) between anadromous and river-resident fish from the Laval River. We also examined how artificial rearing conditions affected seasonal variations in the osmoregulatory physiology of a domestic strain derived from wild anadromous fish. Sympatric anadromous and resident forms of brook charr of the Laval River exhibited differences in gill Na⁺, K⁺-ATPase activity, plasma thyroxine (T₄), and triidothyronine (T₃) concentrations. In domestic anadromous charr, rearing conditions during development had no negative impact on osmoregulatory ability or on gill Na⁺, K⁺-ATPase activity. These results argued for an important hereditary component of gill Na⁺, K⁺-ATPase activity. However, the spring increase in T₄ was present only in wild fish. Significant differences observed at microsatellite loci further suggested that at least some level of reproductive isolation may have occurred between anadromous and resident charr in the Laval River.     

A model system using confocal fluorescence microscopy for examining real-time intracellular sodium ion regulation

The gills of euryhaline fish are the ultimate ionoregulatory tissue, achieving ion homeostasis despite rapid and significant changes in external salinity. Cellular handling of sodium is not only critical for salt and water balance but is also directly linked to other essential functions such as acid–base homeostasis and nitrogen excretion. However, although measurement of intracellular sodium ([Na⁺]_i) is important for an understanding of gill transport function, it is challenging and subject to methodological artifacts. Using gill filaments from a model euryhaline fish, inanga (Galaxias maculatus), the suitability of the fluorescent dye CoroNa Green as a probe for measuring [Na⁺]_i in intact ionocytes was confirmed via confocal microscopy. Cell viability was verified, optimal dye loading parameters were determined, and the dye–ion dissociation constant was measured. Application of the technique to freshwater- and 100% seawater-acclimated inanga showed salinity-dependent changes in branchial [Na⁺]_i, whereas no significant differences in branchial [Na⁺]_i were determined in 50% seawater-acclimated fish. This technique facilitates the examination of real-time changes in gill [Na⁺]_i in response to environmental factors and may offer significant insight into key homeostatic functions associated with the fish gill and the principles of sodium ion transport in other tissues and organisms.     

A new model for fish ion regulation: identification of ionocytes in freshwater- and seawater-acclimated medaka (Oryzias latipes)

The ion regulation mechanisms of fishes have been recently studied in zebrafish (Danio rerio), a stenohaline species. However, recent advances using this organism are not necessarily applicable to euryhaline fishes. The euryhaline species medaka (Oryzias latipes), which, like zebrafish, is genetically well categorized and amenable to molecular manipulation, was proposed as an alternative model for studying osmoregulation during acclimation to different salinities. To establish its suitability as an alternative, the present study was conducted to (1) identify different types of ionocytes in the embryonic skin and (2) analyze gene expressions of the transporters during seawater acclimation. Double/triple in situ hybridization and/or immunocytochemistry revealed that freshwater (FW) medaka contain three types of ionocyte: (1) Na⁺/H⁺ exchanger 3 (NHE3) cells with apical NHE3 and basolateral Na⁺-K⁺-2Cl^? cotransporter (NKCC), Na⁺-K⁺-ATPase (NKA) and anion exchanger (AE); (2) Na⁺-Cl^? cotransporter (NCC) cells with apical NCC and basolateral H⁺-ATPase; and (3) epithelial Ca²⁺ channel (ECaC) cells [presumed accessory (AC) cells] with apical ECaC. On the other hand, seawater (SW) medaka has a single predominant ionocyte type, which possesses apical cystic fibrosis transmembrane conductance regulator (CFTR) and NHE3 and basolateral NKCC and NKA and is accompanied by smaller AC cells that express lower levels of basolateral NKA. Reciprocal gene expressions of decreased NHE3, AE, NCC and ECaC and increased CFTR and NKCC in medaka gills during SW were revealed by quantative PCR analysis.     

Dietary lipid composition affects the gene expression of gill Na+/K+-ATPase α1b but not the α1a isoform in juvenile fall chinook salmon (Oncorhynchus tshawytscha)

We assessed the effects of dietary fatty acid composition on sodium–potassium ATPase (Na⁺/K⁺-ATPase) activity and isoform expression in the gills of juvenile fall chinook salmon, Oncorhynchus tshawytscha by supplementing diets with either anchovy oil (AO) or AO blended with canola oil (CO) so that CO comprised 0% (0CO), 11% (11CO), 22% (22CO), 33% (33CO), 43% (43CO), or 54% (54CO) of the measured dietary lipid content. The effects of diet were assessed in freshwater (FW) following 104 days of diet manipulation, in response to 24-h seawater (SW) transfer at this time, and following an additional 35 days of SW acclimation. Gill Na⁺/K⁺-ATPase activity was not significantly affected by diet at any sampling time, and there were no consistent effects of diet on the expression of the Na⁺/K⁺-ATPase α1a isoform. As dietary CO increased, Na⁺/K⁺-ATPase α1b mRNA decreased in fish held in FW, with the 43CO and 54CO diet groups having significantly lower levels than fish fed the 0CO and 11CO diets. Twenty-four-hour SW challenge did not affect the expression of the Na⁺/K⁺-ATPase α1a isoform in any diet group, but this isoform was down-regulated in all diet groups following 35 days of SW acclimation. Na⁺/K⁺-ATPase α1b expression levels increased in response to 24-h SW transfer and SW acclimation only in fish fed the 54CO diet. The effects of the two extreme diets (0CO and 54CO) were also assessed at various time points during 104 days of rearing in FW. Na⁺/K⁺-ATPase α1b mRNA levels were greater in fish fed diet 0CO versus those fed diet 54CO at all times during the FW culture period. These data demonstrate that dietary fatty acid composition can influence the gill Na⁺/K⁺-ATPase isoform physiology of juvenile fall-run chinook salmon prior to SW transfer.     

Expression of Na+/K+-ATPase α-subunit mRNA during embryonic development of the crayfish Astacus leptodactylus

Astacus leptodactylus is a decapod crustacean fully adapted to freshwater where it spends its entire life cycle after hatching under huge osmoconcentration differences between the hemolymph and surrounding freshwater. We investigated the expression of mRNA encoding one ion transport-related protein, Na⁺/K⁺-ATPase α-subunit, and one putative housekeeping gene, β-actin, during crayfish ontogenesis using quantitative real-time PCR. A 216-amino acid part of the open reading frame region of the cDNA coding for the Na⁺/K⁺-ATPase α-subunit was sequenced from total embryo, juvenile and adult gill tissues. The predicted amino acid sequence showed a high percentage similarity to those of other invertebrates (up to 95%) and vertebrates (up to 69%). β-actin expression exhibited modest changes through embryonic development and early post-embryonic stage. The Na⁺/K⁺-ATPase α-subunit gene was expressed in all studied stages from metanauplius to juvenile. Two peaks of expression were observed: one in young embryos at 25% of embryonic development (EI = 100 μm), and one in embryos just before hatching (at EI = 420 μm), continuing in the freshly hatched juveniles. The Na⁺/K⁺-ATPase expression profile during embryonic development is time-correlated with the occurrence of other features, including ontogenesis of excretory antennal glands and differentiation of gill ionocytes linked to hyperosmoregulation processes and therefore involved in freshwater adaptation.     

Mechanisms of seawater acclimation in a primitive,anadromous fish,the green sturgeon

Relatively little is known about salinity acclimation in the primitive groups of fishes. To test whether physiological preparative changes occur and to investigate the mechanisms of salinity acclimation, anadromous green sturgeon, Acipenser medirostris (Chondrostei) of three different ages (100, 170, and 533 dph) were acclimated for 7 weeks to three different salinities (<3, 10, and 33 ppt). Gill, kidney, pyloric caeca, and spiral intestine tissues were assayed for Na⁺, K⁺-ATPase activity; and gills were analyzed for mitochondria-rich cell (MRC) size, abundance, localization and Na⁺, K⁺-ATPase content. Kidneys were analyzed for Na⁺, K⁺-ATPase localization and the gastro-intestinal tract (GIT) was assessed for changes in ion and base content. Na⁺, K⁺-ATPase activities increased in the gills and decreased in the kidneys with increasing salinity. Gill MRCs increased in size and decreased in relative abundance with fish size/age. Gill MRC Na⁺, K⁺-ATPase content (e.g., ion-pumping capacity) was proportional to MRC size, indicating greater abilities to regulate ions with size/age. Developmental/ontogenetic changes were seen in the rapid increases in gill MRC size and lamellar length between 100 and 170 dph. Na⁺, K⁺-ATPase activities increased fourfold in the pyloric caeca in 33 ppt, presumably due to increased salt and water absorption as indicated by GIT fluids, solids, and ion concentrations. In contrast to teleosts, a greater proportion of base (HCO₃ ⁻ and 2CO₃ ²⁻) was found in intestinal precipitates than fluids. Green sturgeon osmo- and ionoregulate with similar mechanisms to more-derived teleosts, indicating the importance of these mechanisms during the evolution of fishes, although salinity acclimation may be more dependent on body size.     

Seaward migrating Atlantic salmon smolts with low levels of gill Na+, K+ -ATPase activity; is sea entry delayed?

Two groups of migrating wild Atlantic salmon (Salmo salar) smolts caught within a 1 week interval in the River Alta, northern Norway, were tagged with acoustic transmitters and measured for gill Na⁺, K⁺ -ATPase activity in order to compare their smolt status with timing of sea entry. The first group of smolts had low levels of gill Na⁺, K⁺ -ATPase activity and resided in the lower part of the river twice as long as the second group that had high levels of gill Na⁺, K⁺ -ATPase activity. This indicates that early migrating smolts may not be completely physiologically adapted for salt water and delay their sea entry, thereby also synchronizing their seaward migration with the later migrating smolts.     

The interactive effects of hypoxemia, hyperoxia, and temperature on the gill morphology of goldfish (Carassius auratus)

Acclimation of crucian carp and goldfish to temperatures below 15°C causes covering of the gill lamellae by a mass of cells termed the interlamellar cell mass (ILCM). Here we explore the cues underlying gill remodeling (removal or growth of an ILCM) and specifically test the hypotheses that 1) depletion of internal O(2) stores in the absence of any change in external O(2) status can trigger the removal of the ILCM in goldfish acclimated to 7°C, 2) exposing fish acclimated to 25°C to an abundance of O(2) (hyperoxia) can reverse the gill remodeling (i.e., cause the covering of lamellae by an expansion of the ILCM), and 3) neuroepithelial cells (NECs) are involved in signaling the shedding of the ILCM. Hypoxemia induced by phenylhydrazine (anemia) or 5% CO caused a decrease in the ILCM from 80% to 23% and 35%, respectively. Hyperoxia exposure at 25°C caused an increase to 67% of total ILCM and a smaller decrease in the size of the ILCM when fish were transferred from 7 to 25°C. Daily sodium cyanide injections were used to stimulate NECs; this treatment led to a significant decrease in the ILCM. Thus, the three major conclusions of this study are 1) that gill remodeling can occur during periods of internal hypoxemia, 2) that O(2) supply and demand may be a significant driving force shaping gill remodeling in goldfish, and 3) the NECs may play a role in triggering the shedding of the ILCM during hypoxia.     

Cl− influx across posterior gills of the Chinese crab (Eriocheir sinensis): potential energization by a V-type H+-ATPase

Cl⁻ absorption across isolated, perfused gills of freshwater adapted Chinese crabs (Eriocheir sinensis) was analysed by measuring transepithelial potential differences (PD_te) and radioactive tracer fluxes across isolated, perfused posterior gills. Applying hemolymph-like NaCl salines on both sides of the epithelium PD_te amounted to −30±1 mV (n=14). Undirectional Cl⁻ influxes of 470±38 and effluxes of 245±27 μmol·hr⁻¹·g⁻¹ wet weight (ww) (n=14) resulted in a Cl⁻ net influx of 226±31 μmol·hr⁻¹·g⁻¹ ww. Symmetrical substitution of Na⁺ by choline resulted in a substantial hyperpolarisation of the gill. Cl⁻ influx was unchanged under these conditions. However, net influx of Cl⁻ decreased by 40%, due to an increase of the Cl⁻ efflux.Nevertheless, a significant Cl⁻ net influx remained which was independent of the presence of Na⁺. When 2 mmol/l ouabain were added to the internal perfusion medium, PD_te increased, although the fluxes remained unchanged. Following external application of 1μmol/l of the V-type H⁺-ATPase inhibitor bafilomycin, A_l PD_te and Cl⁻ effluxes were not significantly affected. However, Cl⁻ influxes decreased. These findings suggest that Cl⁻ can be taken up independently of Na⁺ and that active Na⁺ independent Cl⁻ uptake across the posterior gill of Eriocheir sinensis is probably driven by a V-type H⁺-ATPase localized in the apical membrane.