Branchial Chloride Cells Research Articles

Ambient salinity and osmoregulation, energy metabolism and growth in juvenile yellowtail kingfish (Seriola lalandiValenciennes 1833) in a recirculating aquaculture system

The effects of salinity on plasma osmolality, branchial chloride cell density, feed consumption and conversion and growth performance of yellowtail kingfish (Seriola lalandi) were evaluated. Fish (11.6 ± 0.6 g) were kept for 29 days at 14, 18, 22, 26 (experimental) and 30 g L−1 (control) salinity in independent, pilot-scale recirculation aquaculture systems. No differences in plasma osmolality or chloride cell numbers in gills were observed, pointing to a strong osmoregulatory capacity in the juveniles. Fish at 14, 18 and 22 g L−1 (7.61 ± 0.19, 7.61 ± 0.01 and 7.61 ± 0.13% day−1, respectively) had higher growth rates than fish at 26 and 30 g L−1 (7.10 ± 0.05 and 6.97 ± 0.06% day−1 respectively). The higher growth rate at lower salinity resulted from increased feed intake; feed conversion was not different. An evaluation of the impact of salinity on growth rate of on-growing stages (till market size) seems warranted to assess whether the profitable effects of low salinity persist in later stages of this important aquaculture species.

Chloride cell morphometrics of Common carp, Cyprinus carpio, in response to different salinities

Salinity plays an important role in the survival, metabolism, and distribution of fish during development. The successful establishment of species in a given habitat depends upon the ability of each developmental stage to cope with salinity via osmoregulation. The present study was conducted to assess the effect of salinity on number and size of chloride cells or mitochondria rich cells and their relation to osmoregulatory ability in Cyprinus carpio, a freshwater (FW) fish. No mortality occurred when the Common carp was gradually transferred into brackish water. Branchial chloride cell numbers decreased after brackish water exposure, whereas a gradual increase was observed in chloride cell size. Chloride cells exist in both FW and brackish water. The number of chloride cell fish in 9 ppt salinity was greatly significant (p < 0.05) when compared with other treatments, while chloride cells with larger size were observed in 12 ppt salinity (p < 0.05). Hence, our data indicate that in culture conditions, adult Common carp can survive successfully in brackish water with salinity of up to 9 ppt.

Differential expression and induction of two carbonic anhydrase isoforms in the gills of the euryhaline green crab, Carcinus maenas, in response to low salinity

Two isoforms of the enzyme carbonic anhydrase (CA) from the gills of the euryhaline green crab were sequenced and identified; these were found to match the cytoplasmic (CAc) and membrane-associated (CAg) isoforms known from other species. The mRNA of the membrane-associated isoform is present in significantly higher levels of abundance in gills of crabs acclimated to 32 ppt, at which the crab is an osmotic and ionic conformer. Upon transfer to low salinity (15 ppt), in which the crab is an osmoregulator, however, the cytoplasmic isoform undergoes a rapid 100-fold increase in abundance in the posterior gills, becoming the dominant isoform. CAg increases 3-fold initially and then remains elevated through 14 days of low salinity acclimation. The induction of CAc mRNA is believed to be the molecular basis for the 20 fold increase in CA protein-specific activity during low salinity acclimation. The initial increase in CAc mRNA takes place at 6 h, and maximal levels of expression are achieved by 24 h; this precedes the induction of CA activity and is within the time in which hemolymph osmotic and ionic concentrations stabilize at new acclimated levels. The increase in expression of the CAg isoform is believed to be more closely related to changes in the population of branchial chloride cells. Changes in the relative abundance of mRNA for the α-subunit of the Na +/K +-ATPase were smaller in magnitude than those for CAc, but the timing was similar. There were no changes in expression of a control gene, arginine kinase (AK) in posterior gills, and there were no significant changes in expression in anterior gills for any of the genes measured here. These results support the use of a control tissue (anterior gills) in addition to a control gene for expression studies.

The effects of changes in salinity on osmoregulation and chloride cell morphology of juvenile Australian snapper, Pagrus auratus

The effect of rapid transfer of juvenile Australian snapper, Pagrus auratus from ambient seawater (30‰) to concentrated hyperosmotic (45‰) and diluted hyperosmotic (15‰) environments on serum osmolality, serum [Na+], [K+], [Cl−], blood haematocrit and branchial chloride cell morphology was assessed during 168 h after transfer. Serum osmolality, [Na+], [K+] and [Cl−] increased after 24 h in 45‰. In contrast, after 24 h in 15‰, [K+] did not change but serum osmolality, [Na+] and [Cl−] decreased. The serum chemistry changes were transient and had returned to near initial levels after 168 h in 45‰ and 15‰. Transfer from 30‰ to 45‰ and 15‰ did not affect blood haematocrit. Branchial chloride cells were identified in both filament and lamellar epithelia of snapper held in all salinity treatments by an immunocytochemical staining technique using an antiserum specific for Na+, K+–ATPase. In 45‰, the number of filament and lamellar chloride cells did not change, but filament chloride cells were more abundant than lamellar chloride cells. In contrast, filament chloride cells had increased in size after 72 h and by 168 h after transfer from 30‰ were 1.4-fold larger than the initial size. In 15‰, the number of filament chloride cells and the size of both filament and lamellar chloride cells had decreased after 72 h. Our results demonstrate that snapper can osmoregulate in a wide range of salinity and provide indirect evidence that both filament and lamellar chloride cells are responsible for excretion of excess salt from snapper in hyperosmotic environments. The ability for snapper to adapt rapidly and maintain homeostasis in a wide range of salinities supports the fact that snapper are a suitable species for land-based aquaculture in ponds, where rapid fluctuation in salinity can occur.

Immunolocalization of metallothioneins in different tissues of turbot (Scophthalmus maximus) exposed to Cd.

Metallothioneins (MT) were localized by immunochemistry in different organs and cell compartments of turbot exposed to sublethal concentrations (100 ppb) of Cd for 7 days. The polyclonal rabbit anti-cod MT antibody (NIVA, Norway) applied herein exhibited positive cross-reactivity with turbot MTs. Immunoreactive MTs were localized in the branchial epithelium, in the liver and in the kidney of turbot. In Cd exposed fishes MTs were demonstrated mainly in branchial chloride cells (CC) and to a lesser extend in the area where progenitor cells are located and in the cells of the respiratory epithelium (secondary lamellae). A higher staining intensity for MTs was observed in CC of the interlamellar space of the main branchial epithelium in comparison with control CC. MT-staining was also observed in the chondroblasts of the cartilage and in the erythrocytes within blood vessels both in control and Cd-exposed specimens. MT immunoreaction was high in the liver hepatocytes and weak in the epithelium of the proximal portion of the kidney in exposed turbot. The tegument, spleen and muscle were devoid of any immunolabelling in both treatments. Ultrastructural studies at the transmission electron microscope revealed that Cd-induced MTs were mainly located in the cytoplasm of gill CC, the lysosomes and the cytoplasm of hepatocytes and in the basal labyrinth of kidney proximal nephrocytes. The differential localization/induction of MTs in different cell types described hereby suggests that the quantification of the specific expression of MT may be used in biomonitoring programs as a biomarker of Cd exposure in aquatic environments.

Prolonged air-breathing and recovery modify the thyroid and interrenal axes in climbing perch (Anabas testudineus)

To examine the response of thyroid and intrenal axes to hydromineral and metabolic responses in air-breathing fish during their territorial migration, we quantified several physiological markers in the tropical obligate air-breathing perch (Anabas testudineus) during air breathing on land and in water. Plasma T3 and T4 levels remained unaffected after 60 and 120 min of air breathing though T3 had a tendency to increase. Plasma cortisol levels showed a significant 4-fold increase. Plasma cortisol showed a 1.5-fold increase in these recovered fish. Air breathing on land significantly increased plasma glucose (2.8-fold) and plasma urea (5.2-fold) while plasma lactate showed a significant 3.2-fold decrease. Na+,K+-ATPase activity, reflecting the sodium pump activity, showed a 4.4-fold decrease in the gills but produced a 1.8-fold increase in the kidneys after air exposure for 120 min. Significant reversal of the plasma metabolite values and the gill Na+,K+-ATPase activity (15-fold) was obtained during recovery. The immunoreactivity of Na+,K+-ATPase in the branchial chloride cells (CCs) showed little changes. The density of the CCs remained unaffected. A marked increase of the glucocorticoid receptor staining was observed in air-exposed gills. Analyses of the ultrastructure of the branchial epithelia revealed CCs with low Na+, K+-ATPase immunoreactivity, more macrophages and increased intercellular spaces in the air-exposed fish. Our data for the first time provide evidence that the thyroid and interrenals jointly direct adaptive strategies to recover from the stress during prolonged air-breathing thus supporting the hypothesis of a role for thyroid in stress adaptation in fish.

Modulation of branchial ion transport protein expression by salinity in glass eels (Anguilla anguilla L.)

The Anguillid juvenile glass eel must deal with the osmoregulatory consequences of highly variable environmental salinities on its recruitment migration from coastal to fresh waters. Changes in ionoregulatory parameters and branchial ion transport protein [Na+/K+-ATPase, Na+:K+:2Cl− cotransporter (NKCC), cystic fibrosis transmembrane regulator (CFTR) anion channel, V-type proton ATPase] expression (activities, protein and/or mRNA level expression and/or cellular localization) in response to acclimation to a broad range of ionic strengths [distilled water (DW) to hypersaline water (HSW; 150%) sea water (SW 32‰)] was studied. The estuarine glass eels were very euryhaline and successfully acclimated to acute changes in environmental ionic strength from 50% SW, with high mortality only observed in HSW (51%) and sublethal osmoregulatory indicators (whole body water content and sodium levels) disturbed at the extremes (DW and HSW). Central to a high salinity acclimation were elevated branchial Na+/K+-ATPase, NKCC and CFTR expression. At lower salinity, Na+/K+-ATPase expression was maintained and NKCC and CFTR expressions were reduced. Branchial chloride cells increased in size up to SW but decreased in HSW. During hypotonic disturbance (DW), no compensatory elevation in V-ATPase or Na+/K+-ATPase expression was observed.

The Na+/K+/2Cl- cotransporter in the sea bassDicentrarchus labraxduring ontogeny: involvement in osmoregulation

This study combines a cellular and molecular analysis of the Na(+)/K(+)/2Cl(-) cotransporter (NKCC) to determine the osmoregulatory role of this protein in different tissues during the ontogeny of the sea bass. We have characterized the complete sequence of the NKCC1 isoform isolated from the sea bass gills and have identified, by immunofluorescence, NKCC1, and other isoforms, within the epithelium of the major osmoregulatory organs. Different (absorptive and secretory) functions have been attributed to this protein according to the tissue and salinity. The effects of short- (1-4 days), medium- (7-21 days) and long (6 months)-term freshwater (FW) adaptations were investigated, in comparison with seawater (SW)-maintained sea bass. In adult sea bass after long-term adaptation to FW and SW, the gills had the highest expression of NKCC mRNA compared with the median/posterior kidney and to the posterior intestine. Expression of NKCC mRNA in the kidney was 95% (SW) and 63% (FW) lower, and in the intestine 98% (SW) and 77% (FW) lower. Compared to SW-maintained sea bass, long-term FW adaptation induced a significant 5.6-fold decrease in the branchial NKCC gene expression whereas the intestinal and renal expressions did not vary significantly. The cells of the intestine and collecting ducts as well as a part of the epithelium lining the urinary bladder expressed NKCC apically. Within the gill chloride cells, NKCC was found basolaterally in SW-acclimated fish; some apically stained cells were detected after 7 days of FW exposure and their relative number increased progressively following FW acclimation. The appearance of FW-type chloride cells induces a functional shift of the gills from a secretory to an absorptive epithelium, which was only completed after long-term exposure to FW. Short- and medium-term exposure to FW induced a progressive decrease in total NKCC content and an increase in functionally different branchial chloride cells. During development, the cotransporter was already expressed in tegumentary ionocytes and along the digestive tract of late embryos. NKCC was recorded in the branchial chamber and along the renal collecting ducts in prelarvae and also in the dorsal part of the urinary bladder in larvae. The expression of NKCC along the osmoregulatory epithelial cells and the presence of Na(+)/K(+)-ATPase within these cells contribute to the increase of the osmoregulatory capacity during sea bass ontogeny.

The ichthyotoxic alga Chattonella marina induces Na+, K+-ATPase, and CFTR proteins expression in fish gill chloride cells in vivo

Our previous studies demonstrated that the ichthyotoxic Chattonella marina stimulated proliferation of branchial chloride cell (CC) and induced osmotic distress akin to hyperactive elimination of ions in fish (Rhabdosargus sarba). To ascertain the in vivo effects of C. marina on key CC ion transporters, the localization and expression of Na(+), K(+)-ATPase (NKA) and cystic fibrosis transmembrane conductance regulator (CFTR) proteins in response to C. marina exposure were investigated, using a quantitative immunocytochemical approach. The polarized distributions of NKA (alpha subunit) and CFTR proteins in branchial CCs of R. sarba remained unchanged under C. marina exposure. However, significant inductions of these two ion-transporters were detected in CCs of fish after 6h exposure. By real-time PCR, no significant changes in gill NKA and CFTR mRNA expressions were detected, suggesting a post-transcriptional pathway is likely involved in regulating the ion transporters abundance. This study is the first to demonstrate the in vivo effects of harmful algal toxin on NKA and CFTR protein expressions in gill transepithelial cells. Taken together, an augmentation of branchial CCs together with hyper-stimulation of NKA and CFTR in CCs attribute to the rapid development of osmotic distress in C. marina susceptible fish.

Changes to chloride and rodlet cells in gills, kidney and intestine of Dicentrarchus labrax (L.) exposed to reduced salinities

The effect of osmotic shock was investigated mainly in the chloride cells (CCs) and rodlet cells (RCs) of gills, and RCs of intestine and kidney of the European sea bass Dicentrarchus labrax obtained from a farm in the northern Adriatic Sea. During the experiment, fish were abruptly transferred from sea water to a salinity of 15 (15 SW) or to fresh water (FW). Numeric variation and ultrastructural changes of both cell types were evaluated at 24, 48 and 96 h after the transfer to lower salinity levels, using light and transmission electron microscopy (TEM). Exposure to FW produced a significant increase (P &lt; 0·05) in the number of branchial CCs and RCs within 96 and 24 h, respectively. Following osmotic challenge (either transfer to 15 SW or FW), kidney and intestine showed an evident increase in RC numbers. The cellular damage detected by TEM was the same for each sampling time (24, 48 and 96 h), but appeared more severe in fish exposed to FW (higher osmotic shock) than in those exposed to 15 SW. In RCs cytoplasmic vacuolizations, autophagosomes and autophagolysosomes with myelinoid bodies, dissolution and shrinkage of the typical inclusions were documented. Nevertheless, CCs showed vacuolization of endoplasmic reticulum and cytoplasmic dissolution and maintained the apical crypt typical of seawater acclimated fish. Renal tubular cells and intestinal epithelial cells showed similar changes to those reported for CCs and RCs.

Ontogenic change of gill chloride cells in leptocephalus and glass eel stages of the Japanese eel, Anguilla japonica

The development of gill chloride cells was examined in premetamorphic larvae (leptocephali) and juveniles (glass eels) of the Japanese eel, Anguilla japonica. Branchial chloride cells were detected by immunocytochemistry using an antiserum specific for Na+,K+-ATPase. The specificity and availability of the antiserum for the detection of Japanese eel chloride cells were confirmed by Western blot analysis. The chloride cells first appeared on the developing gill filaments in a mid larval stage of leptocephalus (32.2 mm). Both immunoreactivity and the number of chloride cells gradually increased as the fish grew to a late stage of leptocephalus over 54 mm. In glass eels just after metamorphosis, gill lamellae developed from the gill filaments, and a rich population of chloride cells was observed in the gill filaments. In glass eels collected at a coastal area, chloride cells were extensively distributed in the gill filaments. The chloride cell size decreased progressively in glass eels transferred from seawater (SW) to freshwater (FW), whereas there was no difference in cell number. In contrast, some Na+,K+-ATPase immunoreaction distinct from typical chloride cells was observed in the gill lamellae throughout FW-transferred fish, but disappeared in control fish maintained in SW for 14 days. These findings indicate that the gill and gill chloride cells developed slowly during the extremely long larval stage, followed by rapid differentiation during a short period of metamorphosis. The excellent euryhalinity of glass eels may be due to the presence of the filament chloride cells and lamellar Na+,K+-ATPase-immunoreaction, presumably being responsible for SW and FW adaptation, respectively.

Prolactin and prolactin receptor expressions in a marine teleost, pufferfish Takifugu rubripes

To investigate the physiological significance of prolactin (PRL) in a marine teleost, pufferfish (or fugu), Takifugu rubripes, we cloned and characterized cDNAs encoding its PRL and PRL receptor (PRLR) from the pituitary and gills, respectively. The fugu PRL cDNA consisted of 995 bp and encoded a protein of 213 amino acids. The PRLR, consisting of 510 amino acids, contained a putative signal peptide, an extracellular domain with two pairs of cysteines, a WSXWS motif, a single transmembrane domain, and a cytoplasmic (intracellular) domain with box 1 and box 2 regions, all of which are characteristic of the cytokine receptor superfamily. Reverse transcription-PCR showed the expression of PRLR mRNA in osmoregulatory organs, such as gills, kidney, and intestine, whereas pufferfish PRL mRNA was detected only in the pituitary. Furthermore, in situ hybridization revealed the expression of pufferfish PRLR in branchial chloride cells, kidney tubule cells, and intestinal epithelia. The PRL-gene expression levels in the pituitary were about five times higher in 25%-diluted seawater than in full-strength seawater. These results suggest that fugu PRL regulates water and electrolyte balances through PRLR expressed in the osmoregulatory organs, as is the case with freshwater-adapted euryhaline species.

Cadmium tolerance in the Nile tilapia (Oreochromis niloticus) following acute exposure: Assessment of some ionoregulatory parameters

The Nile tilapia (Oreochromis niloticus) can tolerate very high levels of waterborne cadmium. It has one of the highest 96 h LC50 recorded for a freshwater teleost fish (14.8 mg/L Cd; hardness 50 mg/L CaCO(3)). Cadmium is known to perturb ion balance in teleost fishes. However, in an acute time course experiment, plasma Na(+) concentrations were unaffected, and plasma Ca(2+) values only decreased after 96 h exposure in a dose-independent manner. Branchial Na(+)/K(+)-ATPase activity and alpha-subunit protein level expression in crude gill homogenates were not affected by Cd exposure during this period. Branchial chloride cell numbers, identified as Na(+)/K(+)-ATPase immunoreactive cells using immunohistochemistry, decreased 24 h after exposure but recovered thereafter. Histopathological changes did not follow a consistent pattern of variation with exposure time, and the alterations noted in gill epithelium were basically nonspecific to cadmium. Because of its tolerance, it can be concluded that the tilapia O. niloticus would not be a suitable test organism to evaluate sublethal toxicity of cadmium and the realistic impact of this pollutant in the environment. However, it certainly could contribute significantly to our understanding of the toxic mechanism of cadmium exposure in aquatic organisms. This is the first work to investigate the effect of waterborne pollutants on Na(+)/K(+)-ATPase alpha-subunit protein expression in fish gills.

Quantitative changes in metallothionein expression in target cell-types in the gills of turbot ( Scophthalmus maximus) exposed to Cd, Cu, Zn and after a depuration treatment

Turbot ( Scophthalmus maximus) were exposed to two sublethal concentrations (1 and 10 mg metal/l) of cadmium (8.9 and 89 μM Cd), copper (15.26 and 152.6 μM Cu) and zinc (15.3 and 153 μM Zn) for 7 days, and afterwards were maintained depurating for 14 days. Immunoreactive metallothioneins ( irMTs) and metal ions were localized in the branchial epithelium by immunohistochemistry (using an anti-Cod MT antibody) and autometallography (AMG), respectively. Metal ions were demonstrated by AMG as black silver deposits (BSD), mainly in mucocytes (MC) and to a lesser extent in the other branchial cell-types (respiratory cells (RC), chloride cells (CC) and basal layer cells (BLC)). Irrespective of the metal supplied, BSD were rapidly visualized in MC after 1 h of exposure. This accumulation did not increase with increasing exposure time and concentration. Metallothionein expression was mainly observed in mature CC in the interlamellar space for all exposure conditions and it was shown that all mature cells express the same amount of irMT. The number of CC exhibiting irMT in metal-exposed turbots increased following short exposure times (1 h–1 day) in the filament epithelium and following longer exposure times (1–7 days) in the secondary lamellae. Total levels of irMT in the gills (quantified by image analysis and densitometry) increased significantly in metal-exposed turbot and were related to increased exposure times. It can be concluded that the total content of irMT in the gills of metal-exposed turbot is governed by changes in the number of mature CC expressing the protein. The quantification of total irMT in branchial CC can be considered as a reliable biomarker of metal exposure since reflects changes in metal bioavailability. This approach based on cell-selective immunohistochemistry can be simplified by only quantifying the number of mature CC. In addition, the dramatic increase of CC in the gills that produces epithelial thickening of the FE enhances migration of CC up to the edge of the SL and provokes the hypertrophy and fusion of secondary lamellae can be considered as unspecific biomarkers of effect indicating disturbed health in turbot.

Effects of a low-Ca2+environment on branchial chloride cell morphology in chum salmon fry and immunolocalization of Ca2+-ATPase in chloride cells

To clarify the involvement of branchial chloride cells in Ca2+uptake in fresh water (FW), chloride-cell morphology was compared in chum salmon (Oncorhynchus keta) fry acclimated to defined FWs with different Ca2+concentrations (0, 0.1, and 0.5 mM). Using immunocytochemical staining with an antiserum specific for Na+,K+-ATPase, chloride cells were detected in both filament and lamellar epithelia. The numbers and sizes of chloride cells in the lamellar epithelia were greater in the low-Ca2+groups (0 and 0.1 mM Ca2+) than in the normal-Ca2+groups (0.5 mM Ca2+and normal FW), whereas filament chloride cells were not affected in number or size by the environmental Ca2+concentration. Electron-microscope observations also revealed that enlarged lamellar chloride cells were more frequently observed in the 0 mM Ca2+group than in the 0.5 mM Ca2+group. To obtain morphological evidence for Ca2+uptake through the branchial epithelia, cellular localization of Ca2+-ATPase was examined with a monoclonal antibody specific for human erythrocyte Ca2+-ATPase. Ca2+-ATPase immunoreactivity was detected in Na+,K+-ATPase-immunoreactive chloride cells in both filament and lamellar epithelia. Using electron-microscope immunocytochemistry, Ca2+-ATPase was found to be localized in the tubular system, which is continuous with the basolateral membrane of chloride cells. These findings indicate that chloride cells in the lamellar epithelia activated by a low Ca2+concentration may constitute the extra Ca2+and NaCl uptake capacity required to maintain homeostasis in soft water.

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.

Effects of environmental Ca2+ levels on branchial chloride cell morphology in freshwater-adapted killifish Fundulus heteroclitus

: To examine the involvement of chloride cells in the uptake of Ca2+ in freshwater (FW) killifish, chloride cell morphology was compared in fish acclimated to different defined FW environments with Ca2+ concentrations of either 0.1 mM, 0.5 mM, or 2.5 mM. Numerous chloride cells were detected in whole-mount preparations of the gill filaments, which were stained with an antiserum specific for Na+, K+-ATPase. Chloride cells, located mostly on the afferent–vascular edge of the filaments, were larger at lower Ca2+ concentrations. Electron microscopic observations showed that in the 0.1 mM and 0.5 mM Ca2+ experimental groups, the apical membrane of chloride cells were flat or slightly projecting and equipped with numerous microvilli. In the 2.5 mM Ca2+ group, some chloride cells formed an apical pit, whereas other cells were similar to those observed in the 0.1 mM and 0.5 mM Ca2+ groups. Plasma osmolality decreased with decreasing ambient Ca2+ concentration, suggesting that environmental Ca2+ affects the permeability of the body surfaces. Gill Na+, K+-ATPase activity in the 0.1 mM and 0.5 mM Ca2+ groups were significantly higher than that in the 2.5 mM Ca2+ group, implying the involvement of the Na+–Ca2+ exchanger in Ca2+ uptake in the gills. These findings suggest that chloride cells function as the site for Ca2+ uptake in killifish acclimated to low Ca2+ environments.

Evidence for a mineralocorticoid-like receptor linked to branchial chloride cell proliferation in freshwater rainbow trout.

Fish acclimated to ion-deficient water exhibit proliferation of branchial chloride cells. The objective of the present study was to investigate the role of cortisol in this response using the corticosteroid receptor antagonists RU486 and spironolactone. RU486 is a potent antagonist of the glucocorticoid actions of cortisol, whereas spironolactone exhibits high-affinity binding to mineralocorticoid receptors, with a resulting blockade of mineralocorticoid properties in mammals. Untreated rainbow trout, as well as rainbow trout given a single intraperitoneal implant of coconut oil alone, coconut oil containing RU486 (0.5 mg g(-1)) or coconut oil containing spironolactone (0.1 mg g(-1)), were exposed to either dechlorinated city-of-Ottawa tapwater or artificial softwater for 7 days. Neither corticosteroid antagonist nor acclimation condition affected circulating plasma cortisol levels, plasma ion concentrations or gill Na(+)-K(+)-ATPase activity. Kidney Na(+)-K(+)-ATPase activity was significantly higher in softwater-acclimated fish than in fish held in dechlorinated tapwater. In addition, whereas RU486 treatment was found to be without effect on gill morphometrics, treatment with spironolactone inhibited the proliferation of chloride cells normally associated with acclimation to ion-deficient water. The results of the present study provide further evidence for the mineralocorticoid actions of cortisol in freshwater fish, specifically in eliciting chloride cell proliferation. Furthermore, these results support the hypothesis that distinct glucocorticoid and mineralocorticoid receptor populations are present in teleost fish, despite the apparent absence of the classic mineralocorticoid hormone, aldosterone.

Ionic Effects of Infection ofIchthyophthirius multifiliisin Goldfish

Abstract Ichthyophthiriasis is a common parasitic disease of freshwater food and ornamental fish. This study examined the ionic effects of an induced infection of ichthyophthiriasis in goldfish Carassius auratus. The whole-animal net Na+ and Cl– fluxes were measured from 12 goldfish each day for 5 d. The numbers of branchial mucous and chloride cells were also determined histologically. Two days of postexposure to 200 theronts/mL resulted in significant net losses of Na+ and Cl–. Whole-body ionic effluxes were then reduced over the subsequent duration of the experiment. There was a general increase in the numbers of branchial mucous cells as reinfection progressed. Branchial chloride cells were elevated in infected fish, indicating some degree of compensation in response to infection. This suggested that acute ionic disturbances occurred in the host fish as a consequence of different stages of the life cycle of the parasite; the host exhibited a suite of physiological and morphological changes to compensa...

Cloning, Characterization, and Tissue Distribution of Prolactin Receptor in the Sea Bream (Sparus aurata)

The prolactin receptor (PRLR) was cloned and its tissue distribution characterized in adults of the protandrous hermaphrodite marine teleost, the sea bream (Sparus aurata). An homologous cDNA probe for sea bream PRLR (sbPRLR) was obtained by RT-PCR using gill mRNA. This probe was used to screen intestine and kidney cDNA libraries from which two overlapping clones (1100 and 2425 bp, respectively) were obtained. These clones had 100% sequence identity in the overlapping region (893 bp) and were used to deduce the complete amino acid sequence of sbPRLR. The receptor spans 2640 bp and encodes a protein of 537 amino acids. Features characteristic of PRLR, two pairs of cysteines, WS box, hydrophobic transmembrane domain, box 1, and box 2, were identified and showed a high degree of sequence identity to PRLRs from other vertebrate species. SbPRLR is 29 and 32% identical to tilapia (Oreochromis niloticus) and goldfish (Carassius auratus) PRLRs, respectively. In the sea bream two PRLR transcripts of 2.8 and 3.2 kb were detected in the intestine, kidney, and gills and a single transcript of 2.8 kb was detected in skin and pituitary by Northern blot. Spermiating gonads (more than 95% male tissue; gonado–somatic index of 0.6) contained, in addition to the 2.8-kb transcript, three more transcripts of 1.9, 1.3, and 1.1 kb. RT-PCR, which is a far more sensitive method than Northern blot, detected PRLR mRNA in gills, intestine, brain, pituitary, kidney, liver, gonads, spleen, head-kidney, heart, muscle, and bone. Immunohistochemistry using specific polyclonal antibodies raised against an oligopeptide from the extracellular domain of sbPRLR detected PRLR in several epithelial tissues of juvenile sea bream, including the anterior gut, renal tubule, choroid membrane of the third ventricle, saccus vasculosus, branchial chloride cells, and branchial cartilage.