Gills Of Freshwater Fish Research Articles

Arsenic induced toxicity and histopathological changes in gill and liver tissue of freshwater fish, tilapia (Oreochromis mossambicus)

Acute toxicity of arsenic to tilapia (Oreochromis mossambicus) and its histopathological impacts on gill and liver tissue were evaluated. The median lethal concentration (96 h; LC50) of arsenic (NaAsO₂) was calculated as 28.22 ppm in repeated semi static test method. Fish were exposed to 3 ppm, 28 ppm and 56 ppm concentrations of NaAsO₂ and gill and liver samples were collected after 48 h, 96 h and 192 h of exposure. The changes in gill were characterized by epithelial hyperplasia, epithelial lifting and oedema, lamellar fusion, aneurism, desquamation and necrosis, whereas, the liver tissue showed focal lymphocytic and macrophage infiltration, congestion, vacuolization and shrinkage of hepatocytes, dilation of sinusoids, cloudy swelling, vacuolar degeneration, focal necrosis and nuclear hypertrophy. The result showed that acute arsenic toxicity severely affects the normal behavior and vital organs which is deleterious for the exposed fish.

Molecular Structure of Isolated MvspI, a Variable Surface Protein of the Fish Pathogen Mycoplasma mobile

Mycoplasma mobile is a parasitic bacterium that causes necrosis in the gills of freshwater fishes. This study examines the molecular structure of its variable surface protein, MvspI, whose open reading frame encodes 2,002 amino acids. MvspI was isolated from mycoplasma cells by a biochemical procedure to 92% homogeneity. Gel filtration and analytical ultracentrifugation suggested that this protein is a cylinder-shaped monomer with axes of 66 and 2.7 nm. Rotary shadowing transmission electron microscopy of MvspI showed that the molecule is composed of two rods 30 and 45 nm long; the latter rod occasionally features a bulge. Immuno-electron microscopy and epitope mapping showed that the bulge end of the molecular image corresponds to the C terminus of the amino acid sequence. Partial digestion by various proteases suggested that the N-terminal part, comprised of 697 amino acids, is flexible. Analysis of the predicted amino acid sequence showed that the molecule features a lipoprotein and 16 repeats of about 90 residues; 15 positions exist between residues 88 and 1479, and the other position is between residues 1725 and 1807. The amino acid sequence of MvspI was mapped onto a molecular image obtained by electron microscopy. The present study is the first to elucidate the molecular shape of a variable surface protein of mycoplasma.

Ammonia excretion in the freshwater planarianSchmidtea mediterranea

In aquatic invertebrates, metabolic nitrogenous waste is excreted predominately as ammonia. Very little is known, however, of the underlying mechanisms of ammonia excretion, particularly in freshwater species. Our results indicate that in the non-parasitic freshwater planarian Schmidtea mediterranea, ammonia excretion depends on acidification of the apical unstirred layer of the body surface and consequent ammonia trapping. Buffering of the environment to a pH of 7 or higher decreased the excretion rate. Inhibitor experiments suggested further that the excretion mechanism involves the participation of the V-type H(+)-ATPase and carbonic anhydrase and possibly also the Na(+)/K(+)-ATPase and Na(+)/H(+) exchangers. Alkalinization (pH 8.5, 2 days) of the environment led to a 1.9-fold increase in body ammonia levels and to a downregulation of V-ATPase (subunit A) and Rh-protein mRNA. Further, a 2 day exposure to non-lethal ammonia concentrations (1 mmol l(-1)) caused a doubling of body ammonia levels and led to an increase in Rh-protein and Na(+)/K(+)-ATPase (α-subunit) mRNA expression levels. In situ hybridization studies indicated a strong mRNA expression of the Rh-protein in the epidermal epithelium. The ammonia excretion mechanism proposed for S. mediterranea reveals striking similarities to the current model suggested to function in the gills of freshwater fish.

Ammonia excretion via Rhcg1 facilitates Na+uptake in larval zebrafish, Danio rerio, in acidic water

The involvement of a Na(+)/H(+) exchanger (NHE) in mediating Na(+) uptake by freshwater fish is currently debated. Although supported indirectly by empirical molecular and pharmacological data, theoretically its operation should be constrained thermodynamically, owing to unfavorable chemical gradients. Recently, there has been an increasing focus on ammonia channels (Rh proteins) as potentially contributing to Na(+) uptake across the freshwater fish gill. In this study, we tested the hypothesis that Rhcg1, a specific apical isoform of Rh protein, is critically important in facilitating Na(+) uptake in zebrafish larvae via its interaction with NHE. Treating larvae (4 days postfertilization) with 5-(N-ethyl-N-isopropyl) amiloride (EIPA), an inhibitor of NHE, caused a significant reduction in Na(+) uptake in fish reared in acidic water (pH ∼ 4.0). A role for NHE in Na(+) uptake was further confirmed by translational knockdown of NHE3b, an isoform of NHE thought to be responsible for Na(+)/H(+) exchange in zebrafish larvae. Exposing the larvae reared in acidic water to 5 mM external ammonium sulfate or increasing the buffering capacity of the water with 10 mM HEPES caused concurrent reductions in ammonia excretion and Na(+) uptake. Furthermore, translational knockdown of Rhcg1 significantly reduced ammonia excretion and Na(+) uptake in larvae chronically (4 days) or acutely (24 h) exposed to acidic water. Unlike in sham-injected larvae, EIPA did not affect Na(+) uptake in fish experiencing Rhcg1 knockdown. Additionally, exposure of larvae to bafilomycin A1 (an inhibitor of H(+)-ATPase) significantly reduced Na(+) uptake in fish reared in acidic water. These observations suggest the existence of multiple mechanisms of Na(+) uptake in larval zebrafish in acidic water: one in which Na(+) uptake via NHE3b is linked to ammonia excretion via Rhcg1, and another facilitated by H(+)-ATPase.

Effect of cortisol on permeability and tight junction protein transcript abundance in primary cultured gill epithelia from stenohaline goldfish and euryhaline trout

Primary cultured gill epithelia from goldfish and rainbow trout were used to investigate a role for cortisol in the regulation of paracellular permeability and tight junction (TJ) protein transcript abundance in representative stenohaline versus euryhaline freshwater (FW) fish gills. Glucocorticoid and mineralocorticoid receptors are expressed in cultured goldfish gill preparations and cortisol treatment (100, 500 and 1000ng/mL) dose-dependently elevated transepithelial resistance (TER) and reduced paracellular [3H]PEG-4000 flux across cultured goldfish gill epithelia. Despite these dose-dependent ‘tightening’ effects of cortisol, the response of goldfish TJ protein transcripts (i.e. occludin, claudin b, c, d, e, h, 7, 8d and 12, and ZO-1) were surprisingly small, with only claudin c and h, and ZO-1 transcript levels significantly decreasing at a dose of 1000ng/mL. Extending the duration of cortisol exposure from 24 to 48 or 96h (at 500ng/mL) did little to alter this phenomenon. By comparison, exposing primary cultured trout gill epithelia (i.e. a euryhaline fish gill model) to 500ng/mL cortisol resulted in a qualitatively similar, but quantitatively stronger epithelial ‘tightening’ response. Furthermore, transcript abundance of orthologous trout TJ proteins (i.e. occludin, and claudin 30, 28b, 3a, 7, 8d and 12) significantly elevated as would be expected in a ‘tighter’ epithelium. Taken together, data suggest a conservative role for cortisol in the endocrine regulation of paracellular permeability across the goldfish gill that may relate to stenohalinity.

Freshwater Fish Gill Ion Transport: August Krogh to morpholinos and microprobes

August Krogh proposed that freshwater fishes (and other freshwater animals) maintain body NaCl homoeostasis by extracting these ions from the environment via separate Na(+) /NH(4)(+) and Cl(-) /HCO(3)(-) exchangers in the gill epithelium. Subsequent data from other laboratories suggested that Na(+) uptake was more probably coupled to H(+) secretion via a vesicular proton pump (V-ATPase) electrically coupled to a Na(+) channel. However, despite uncertainty about electrochemical gradients, evidence has accrued that epithelial Na(+) /H(+) exchange indeed may be an alternative pathway for Na(+) uptake. The specific pathways for Na(+) uptake may be species and environment specific. An apical Cl(-) /HCO(3)(-) exchanger is generally accepted for most species (some species do not extract Cl(-) from freshwater), but the relative roles of anion exchanger-like (SLC4A1) vs. pendrin-like (SLC26Z4) exchangers are unknown, and also may be species specific. Most recently, data have supported the presence of an apical Na(+) + Cl(-) cotransporter (NCC-type), despite thermodynamic uncertainty. Ammonia extrusion may be via NH(3) diffusing through the paracellular junctions or NH(4) (+) substitution on both basolateral and apical ionic exchangers (Na(+) + K(+) -ATPase; Na(+) + K(+) + Cl(-) - cotransporter; and Na(+) /H(+) exchanger), but recent evidence suggests that Rhesus-glycoproteins mediate both basolateral and apical movement of ammonia.

Fluoride induced alterations in the arginase activity of freshwater catfish, Clarias batrachus (Linn)

Fluoride induced alterations in arginase activity in liver, muscle, kidney, gill and brain of freshwater cat fish, Clarias batrachus (Linn.) were carried out. The effect of fluoride was observed in different concentrations like 1, 10, 20 and 30 ppm in 1, 30 and 60 days of exposure. The arginase activity was found increased in all the tissues, throughout the exposure span. Maximum activity was observed on 60th day of exposure in 30 ppm of fluoride concentration. In most of the cases the elevation of arginase activity significantly differed from the controls. Gill showed maximum activity followed by liver, brain, kidney, and muscle. The increase in arginase activity indicates production of urea or ornithine.

Cloning and regulation of expression of the Na+–Cl––taurine transporter in gill cells of freshwater Japanese eels

Our previous studies have demonstrated the hypertonic-induced expression of osmotic stress transcription factor and the regulatory volume increase (RVI) response in gill cells isolated from freshwater eels. In this study, we aimed to clone one of the organic osmolyte transporters, the Na+-Cl--taurine transporter (TauT), and to characterize its expression in anisosmotic conditions, using both in vivo and in vitro approaches. A cDNA clone encoding TauT was isolated from gill tissues of Japanese eels, Anguilla japonica. The deduced amino acid sequence shows 88-90% identity to other reported piscine TauT sequences. Our data indicated that TauT mRNA was detectable in both freshwater and seawater fish gills. The expression level of TauT mRNA increased in gills of seawater-acclimating fish. A high abundance of TauT protein was found to be localized in seawater gill chloride cells. Using primary gill cell culture, expression of the gene was induced when the ambient osmolarity was raised from 320 to 500 mosmol l(-1). Hypertonic treatment of the culture caused an increase of F-actin distribution in the cell periphery. Treatment of the cells with colchicine or cytochalasin D significantly reduced TauT transcript level following hypertonic exposure. The inhibition of myosin light chain (MLC) kinase by ML-7 had a significant additive effect on hypertonic-induced TauT expression. Collectively, the data of this study reveal, for the first time, the regulation of TauT expression in gill cells of euryhaline fish. We have demonstrated the involvement of ionic strength, the cytoskeleton and MLC kinase in the regulation of TauT expression. The results shed light on the osmosensing and hyperosmotic adaptation in fish gills.

Histopathological changes in the gill and liver tissues of freshwater fish, Cirrhinus mrigala exposed to dichlorvos

The histopathological effects of dichlorvos, an organophosphate pesticide, on the gill and liver tissues in Cirrhinus mrigala were determined by light microscopy. The fishes were exposed to sublethal concentrations (0.91 and 1.82 ppm) of dichlorvos for 10 days with parallel untreated control. No histopathological effects were observed in control group. Hyperplasia, desquamation, and necrosis of epithelial, epithelial lifting, oedema, lamellar fusion, collapsed secondary lamellae, curling of secondary lamellae and aneurism in the secondary lamellae were observed in gill tissues exposed to dichlorvos. Hepatic lesions in the liver tissues of fishes exposed to dichlorvos were characterized by cloudy swelling of hepatocytes, congestion, vacuolar degeneration, karyolysis, karyohexis, dilation of sinusoids and nuclear hypertrophy.

Protective Immunity of Goldfish Against Ichthyophthirius multifiliis Infection Induced by Different Trophont Vaccine Preparations

Abstract Ichthyophthirius multifiliis, commonly called “Ich,” is a protozoan parasite that infects the epidermis and gills of freshwater fish. Here, we used goldfish (Carassius auratus) to determine whether the four vaccine preparations (live trophonts, formalin‐fixed trophonts, freeze‐thawed trophont lysates, and trophont cilial and cell cortical fractions) of I. multifiliis (Fouquet) can elicit resistance of immunized fish against subsequent theront challenge, and whether a relationship exists between in vitro immobilization of theronts in sera or mucus from immunized fish and host protective immunity against the parasite. Experimental goldfish were randomly divided into five groups with five parallel controls, with each group or subgroup consisting of 20 individuals. Each test goldfish was immunized with one of the four Ich vaccine preparations administered by one of the three routes: live trophonts by gavage (Group 1), formalin‐fixed trophonts by injection (Group 2) or by immersion (Group 3), freeze‐thawed trophont lysates by injection (Group 4), and trophont cilial and cell cortical fractions by injection (Group 5). The fish were then challenged by a standard Ich theront challenge on Day 21 postimmunization. For every 10 d following immunization, we tested the in vitro immobilization of Ich theronts by sera or mucus from immunized and control fish. We found that although all control (nonimmunized) and Group 3‐immunized goldfish died following theront challenge, more than half (55–65%) of the immunized fish from Groups 1, 2, 4, and 5 survived. Furthermore, except for the fish from Groups 3 and 4, the number of mean days to death in each test group was significantly higher than that in the respective control. These results indicate that goldfish immunized by injection or by gavage with any of the four vaccine preparations gained resistance to Ich infection. Further analysis indicated that this protective immunity was closely associated with the in vitro immobilization of Ich theronts by fish sera or mucus.

Heavy metal uptake, bioaccumulation and histopathological responses in gills of freshwater fish from the Indus River, Pakistan

The high nutritional value of fish makes it an ideal component of a healthy and balanced diet. Elevated levels of heavy metals in fish represent both an ecological and human health concern. Fish species are often the top consumers in aquatic ecosystems and thus metal concentrations in fish can act as an indicator of the state of the aquatic environment. Fish are known to bio-accumulate metals and are considered as one of the most indicative factors, in freshwater systems, for the estimation of trace metals pollution potential (Rashed, 2001). Histopathology can be an indicator of the effects of various anthropogenic pollutants on organisms and so could be used as a reflection of the overall health of the entire population in an ecosystem. This study aimed to assess the bioaccumulation of heavy metals in gills and muscles and their effects on histopathology of gills of Oreochromis mossambicus from the Indus River, Pakistan which receives potential pollutants from the domestic, municipal and agricultural runoffs.

Ammonia transport in cultured gill epithelium of freshwater rainbow trout: the importance of Rhesus glycoproteins and the presence of an apical Na+/NH4+ exchange complex

The mechanisms of ammonia excretion at fish gills have been studied for decades but details remain unclear, with continuing debate on the relative importance of non-ionic NH(3) or ionic NH(4)(+) permeation by various mechanisms. The presence of an apical Na(+)/NH(4)(+) exchanger has also been controversial. The present study utilized an in vitro cultured gill epithelium (double seeded insert, DSI) of freshwater rainbow trout as a model to investigate these issues. The relationship between basolateral ammonia concentration and efflux to apical freshwater was curvilinear, indicative of a saturable carrier-mediated component (K(m)=66 micromol l(-1)) superimposed on a large diffusive linear component. Pre-exposure to elevated ammonia (2000 micromol l(-1)) and cortisol (1000 ng ml(-1)) had synergistic effects on the ammonia permeability of DSI, with significantly increased Na(+) influx and positive correlations between ammonia efflux and Na(+) uptake. This increase in ammonia permeability was bidirectional. It could not be explained by changes in paracellular permeability as measured by [(3)H]PEG-4000 flux. The mRNA expressions of Rhbg, Rhcg2, H(+)-ATPase and Na(+)/H(+) exchanger-2 (NHE-2) were up-regulated in DSI pre-exposed to ammonia and cortisol, CA-2 mRNA was down-regulated, and transepithelial potential became more negative. Bafilomycin (1 micromol l(-1)), phenamil (10 micromol l(-1)) and 5-(N,N-hexamethylene)amiloride (HMA, 10 micromol l(-1)) applied to the apical solution significantly inhibited ammonia efflux, indicating that H(+)-ATPase, Na(+) channel and NHE-2 pathways on the apical surface were involved in ammonia excretion. Apical amiloride (100 micromol l(-1)) was similarly effective, while basolateral HMA was ineffective. Pre-treatment with apical freshwater low in [Na(+)] caused increases in both Rhcg2 mRNA expression and ammonia efflux without change in paracellular permeability. These data suggest that Rhesus glycoproteins are important for ammonia transport in the freshwater trout gill, and may help to explain in vivo data where plasma ammonia stabilized at 50% below water levels during exposure to high environmental ammonia ( approximately 2300 micromol l(-1)). We propose an apical ;Na(+)/NH(4)(+) exchange complex' consisting of several membrane transporters, while affirming the importance of non-ionic NH(3) diffusion in ammonia excretion across freshwater fish gills.

Enzymes as Biomarkers of Cypermethrin Toxicity: Response of Clarias batrachus Tissues ATPase and Glycogen Phosphorylase as a Function of Exposure and Recovery at Sublethal Level

The present study was conducted to evaluate the sublethal effects of a synthetic pyrethroid, cypermethrin on ATPase, and glycogen phosphorylase in gill, kidney, liver, and muscle tissue of freshwater fish, Clarias batrachus (Linn) during exposure and cessation of exposure. Thirty-six fish were exposed to 1/3 of LC 50 concentration i, e. 0.07 mg/L cypermethrin for 10 days. After 10 days, 18 fish were transferred to freshwater and 10 days recovery response was observed. Thirty-six fish were kept as a control group. The effect of cypermethrin intoxication was studied on total, Mg+2, Na+-K+ ATPase, glycogen, and glycogen phosphorylase (a) and (ab) in various physiological tissues at the end of 1, 5, and 10 days of exposure and recovery period. Cypermethrin intoxication resulted in a significant inhibition in the activities of total, Mg+2, and Na+-K+ ATPase enzyme and glycogen content, whereas it caused a significant induction in the levels of glycogen phosphorylase (a). The activity level of glycogen phosphorylase (ab) showed mixed response. When exposed fish were released into freshwater, they were able to normalize the activities of ATPase enzyme in liver tissue whereas gill, kidney, and muscle tissues showed slight recovery. At the end of 10 days of recovery period, glycogen and glycogen phosphorylase activities in kidneys recover better than muscle. Therefore, the present study clearly suggests that ATPase and glycogen phosphorylase enzymes can be used as biomarkers of exposure to aquatic organisms under cypermethrin intoxication.

Cultured trout gill epithelia enriched in pavement cells or in mitochondria-rich cells provides insights into Na+ and Ca2+ transport

The lack of a suitable flat epithelial preparation isolated directly from the freshwater fish gill has led, in recent years, to the development of cultured gill epithelia on semipermeable supports. To date, their minimal capacity to actively transport ions has limited their utility as ionoregulatory models. The current study describes a new method of culturing gill epithelia consisting either of an enriched population of pavement (PV) cells or a mixed population of PV cells and mitochondria-rich (MR) cells from the gills of adult rainbow trout. Although the cell culture approach is similar to the double-seeded insert (DSI) technique described previously, it makes use of Percoll density centrifugation to first separate populations of PV and MR cells, which are then seeded on cell culture supports in varying proportions on successive days so as to produce preparations enriched in one or the other cell types. Based on rhodamine staining, the MR cell-rich epithelia exhibited a threefold higher enrichment of MR cells compared to traditional DSI preparations. In general, MR cell-rich epithelia developed extremely high transepithelial resistances (TER; >30 kOmega cm(2)) and positive transepithelial potentials (TEP) under symmetrical conditions (i.e., L15 medium on both apical and basolateral sides). Apical exposure of cell cultures to freshwater reduced TER and produced a negative TEP in all the epithelial preparations, although MR cell-rich epithelia maintained relatively high TER and negative TEP for over 2 d under these asymmetrical conditions. Measurement of unidirectional Na(+) fluxes and application of the Ussing flux ratio criterion demonstrated active Na(+) uptake in PV cell-rich and MR cell-rich epithelia under both symmetrical and asymmetrical conditions. In comparison, Ca(2+) uptake and Na(+)/K(+)-ATPase activity were significantly elevated in MR cell-rich preparations relative to the traditional DSI or PV cell-rich cultures under symmetrical conditions. This new methodology enhances our ability to tailor cultured gill epithelia on semipermeable supports with different proportions of PV cells and MR cells, thereby illuminating the ionoregulatory functions of the two cell types.

Na+/H+exchanger isoform 3 expressed in apical membrane of gill mitochondrion-rich cells in Mozambique tilapiaOreochromis mossambicus

A cDNA encoding a homolog of mammalian Na+/H+ exchanger isoform 3 (NHE3) has been cloned from gills of Mozambique tilapia Oreochromis mossambicus using reverse transcription-polymerase chain reaction (RT-PCR). The deduced amino acid sequence shared 48–56% homology with other vertebrate NHE3 homologs. RT-PCR revealed that tilapia NHE3 was expressed in the gill, kidney and intestine in tilapia adapted to fresh water (FW) and sea water (SW), suggesting its involvement in osmoregulation. We also examined NHE3 MRNA expression levels in the gills of FW and SW fish, using real-time quantitative PCR. NHE3 mRNA was highly expressed in the gills of FW fish, compared with those of SW fish. Whole-mount immunohistochemistry of the gill filaments revealed that tilapia NHE3 was localized in the apical region of mitochondrion-rich (MR) cells in FW-and SW-adapted tilapia. Abundance of NHE3 mRNA in FW gills and apical localization of NHE3 in gill MR cells suggest that NHE3 is involved in FW adaptation and acid-base regulation, in which Na+ is absorbed from external environments in exchange for H+ secretion.

Five New Species of Urocleidoides (Monogenoidea) (Mizelle and Price 1964) Kritsky, Thatcher, and Boeger, 1986, Parasitizing the Gills of Panamanian Freshwater Fishes

During an investigation of gill monogenoidean parasites from freshwater fishes in central Panama, 5 new species of Urocleidoides (sensu stricto) were found: Urocleidoides cultellus n. sp., Urocleidoides visiofortatus n. sp., and Urocleidoides advenai n. sp. from the gymnotiform Brachyhypopomus occidentalis (Hypopomidae); Urocleidoides neotropicalis n. sp. and Urocleidoides piriatiu n. sp. from the characiforms Saccodon dariensis (Parodontidae) and Ctenolucius beani (Ctenoluciidae), respectively. Our findings represent the first known species of Urocleidoides from these fishes in Central America and demonstrate that they are morphologically linked to their South American congeners.

Evidence for a protective response by the gill proteome of rainbow trout exposed to X‐ray induced bystander signals

The bystander effect occurs when cells which are not directly exposed to radiation, but which receive signals from irradiated cells, respond as though they were irradiated. An X-ray induced bystander effect has been demonstrated in rainbow trout gills. Therefore, a proteomic comparison was made of gills from X-ray treated trout and trout exposed to X-ray induced bystander signals. 2-D gel analysis revealed X-ray exposure increased the expression of the cancer related protein annexin II. The proteomic changes associated with the bystander effect differed from those associated with direct radiation exposure. Expression of a hemopexin-like protein, Rho GDP dissociation inhibitor (RhoGDI) and pyruvate dehydrogenase (PDH) were increased. These proteins possess protective properties against reactive oxygen damage (a component of the bystander signal), regulate epithelial polarity and prevent lactate acidosis, respectively. There was also evidence for an increase in chromosome 1 SR-like CTD-associated factor (SCAF) protein turnover, which could suggest the protective response is transcriptionally regulated. The freshwater fish gill is a polarised barrier, separating against an external hypotonic environment. Since the maintenance of epithelial polarity is vital to gill function, these bystander effect proteomic changes could collectively protect the structural, functional and intracellular integrity of gill epithelia.

Gene expression of Na+/H+ exchanger in zebrafish H+-ATPase-rich cells during acclimation to low-Na+ and acidic environments

In mammalian nephrons, most of the Na(+) and HCO(3)(-) is reabsorbed by proximal tubular cells in which the Na(+)/H(+) exchanger 3 (NHE3) is the major player. The roles of NHEs in Na(+) uptake/acid-base regulation in freshwater (FW) fish gills are still being debated. In the present study, functional genomic approaches were used to clone and sequence the full-length cDNAs of the nhe family from zebrafish (Danio rerio). A phylogenetic tree analysis of the deduced amino acid sequences showed that zNHE1-8 are homologous to their mammalian counterparts. By RT-PCR analysis and double/triple in situ hybridization/immunocytochemistry, only zebrafish NHE3b was expressed in zebrafish gills and was colocalized with V-H(+)-ATPase but not with Na(+)-K(+)-ATPase, indicating that H(+)-ATPase-rich (HR) cells specifically express NHE3b. A subsequent quantitative RT-PCR analysis demonstrated that acclimation to low-Na(+) FW caused upregulation and downregulation of the expressions of znhe3b and zatp6v0c (H(+)-ATPase C-subunit), respectively, in gill HR cells, whereas acclimation to acidic FW showed reversed effects on the expressions of these two genes. In conclusion, both NHE3b and H(+)-ATPase are probably involved in Na(+) uptake/acid-base regulation in zebrafish gills, like mammalian kidneys, but the partitioning of these two transporters may be differentially regulated depending on the environmental situation in which fish are acclimatized.

Monogeneans on native and introduced freshwater fishes from Cuba with the description of a new species of Salsuginus Beverley-Burton, 1984 from Limia vittata (Poeciliidae)

During a parasitological survey carried out between March and September 2003 in Cuba, the following monogeneans were found on the gills of freshwater fishes: Salsuginus cubensis n. sp. on the Cuban molly Limia vittata Guichenot (Poeciliidae); Cichlidogyrus sclerosus Paperna & Thurston, 1969 and C. tilapiae Paperna, 1960 on the African cichlid Tilapia rendalli Boulenger (Cichlidae); Haplocleidus dispar Mueller, 1936 and Pterocleidus acer Mueller, 1936 (all Dactylogyridae) on the sunfish Lepomis macrochirus Rafinesque (Centrarchidae) (new geographical records); and Gyrodactylus sp. (Gyrodactylidae) on the biajaca Nandopsis tetracanthus Valenciennes (Cichlidae) (new host and geographical record). Salsuginus cubensis differs from all other species of the genus in the size and morphology of the copulatory complex. The occurrence of C. sclerosus, C. tilapiae, H. dispar and P. acer in their respective hosts is due to the introduction of these hosts to Cuba. A review of the species composition of the Monogenea in native and introduced freshwater fish from Cuba is presented and the zoogeographical distribution of the species found is briefly discussed.

Effects of copper on CYP1A activity and epithelial barrier properties in the rainbow trout gill

The effects of copper on β-naphthoflavone (βNF)-induced ethoxyresorufin O-deethylase (EROD) activity were studied in rainbow trout ( Oncorhynchus mykiss) gill filaments (after in vivo exposure) and in gill cells cultured as both primary cultures and as polarised epithelia, i.e. with water in the apical compartment and culture medium in the basolateral compartment. In the in vivo study βNF and copper were added to the water, in primary cultures both chemicals were added to the culture medium and in cultured epithelia copper was added to the apical water whilst βNF was added to the basolateral culture medium. In primary cultures this investigation was repeated with and without foetal bovine serum (FBS) supplementation of the culture media. Gill barrier properties, specifically polyethylene glycol (PEG-4000) permeability ( i.e. paracellular permeability), sodium efflux and transepithelial electrical resistance (TER) were also investigated in cultured gill cell epithelia after apical treatment with copper. Two micromolar copper had no effect on EROD activity in gill filaments in vivo irrespective of whether EROD was induced by 0.01, 0.1 or 1.0 μM βNF. Similarly, 0.5–100 μM copper had no effect on EROD induction in cultured epithelia. In primary cultures copper did reduce EROD induction but the effective concentration was dependent on whether the cells were supplemented with FBS, i.e. EROD activity was reduced by all copper concentrations of 5 and above if FBS was included, but only by 1000 μM if FBS was omitted. In cultured epithelia PEG-4000 permeability increased, whilst sodium efflux and TER were unaffected following treatment with 75 μM copper. Based on these results we conclude that the branchial monooxygenase system is a less sensitive target for copper than the barrier properties of the gill. Indeed, these data suggest the apical membrane of the gill epithelial cells minimises the uptake of waterborne copper and therefore protects the intracellular environment, including the CYP1A system. This could enable the freshwater fish gill to retain their potential of first-pass metabolism of waterborne organic compounds whilst simultaneously being exposed to waterborne copper.