Morphological changes in gill mitochondria-rich cells in cultured Japanese eel <Emphasis Type="Italic">Anguilla japonica</Emphasis> acclimated to a wide range of environmental salinity

Morphological changes in gill mitochondria-rich (MR) cells were examined in cultured Japanese eel acclimated to deionized freshwater (DFW), freshwater (FW), 30%-diluted seawater (DSW), and seawater (SW). The gill Na⁺/K⁺-ATPase activity was higher in SW-acclimated eel than in those acclimated to DFW, FW, and DSW. Immunocytochemical observations revealed that MR cells in the gill filaments were most developed in SW, whereas MR cells in the lamellae were preferentially observed in DFW, suggesting that filament and lamellar MR cells are responsible for ion secretion and absorption, respectively. In scanning electron microscopic observations, the apical membrane of lamellar MR cells appeared as a flat or slightly projecting disk with a mesh-like structure on its surface. In contrast, the apical membrane of filament MR cells showed a slightly concave surface. Whole-mount immunocytochemistry revealed that most MR cells showed cystic fibrosis transmembrane conductance regulator immunoreaction in their apical region in fish in DSW and SW, but not in those in DFW and FW, indicating that MR cells developed in DSW and SW function as an ion-secreting site. In addition to MR cells, distinct Na⁺/K⁺-ATPase immunoreaction was observed in the outermost layer of gill epithelia, suggesting that pavement cells are an additional site of ion uptake in the gills.     

花鲈ncc、nkcc基因在海、淡水适应中鳃组织的表达与定位分析

为探究ncc、nkcc基因在花鲈渗透调节中发挥的作用,实验通过全基因组鉴定、多重序列比对、系统进化树构建以及蛋白结构预测对花鲈ncc进行了鉴定及序列分析,利用实时荧光定量PCR (qRT-PCR)检测ncc和nkcc在海水、淡水花鲈鳃组织中的表达水平,利用原位杂交技术确定ncc2和nkcc1a在海水及淡水花鲈鳃中的表达位置。结果显示,从花鲈中鉴定出2个ncc基因,即ncc1和ncc2,其编码序列(CDS)长度分别为2 691和3 120bp,编码896和1 039个氨基酸,在进化上具有保守性。ncc2在淡水花鲈鳃组织中的表达量显著高于海水,而nkcc1a在海水花鲈鳃组织中的表达量显著高于淡水,ncc1、nkcc1b、nkcc2在海淡水中的表达量则无显著差异。淡水适应过程中花鲈鳃组织中的ncc2的表达量逐渐上调,而nkcc1a的表达量逐渐下调;海水适应过程则呈现相反的表达趋势。此外,原位杂交结果显示,ncc2和nkcc1a基因分别位于淡水与海水中鳃组织的相邻鳃小片间的鳃丝上皮。以上结果表明,ncc2和nkcc1a基因分别编码淡水及海水花鲈鳃中重要的Na⁺及Cl     

Excretion of cesium and rubidium via the branchial potassium-transporting pathway in Mozambique tilapia

Using an insolubilization technique in combination with element analysis, we examined the possible excretion of Cs⁺ and Rb⁺, both known to be biochemical analogs of K⁺, through the K⁺-transporting pathway in the gills of seawater-adapted Mozambique tilapia. To detect ion excretion from gills infused with Rb⁺ or Cs⁺, the isolated gills were treated with sodium tetraphenylborate, which reacts not only with K⁺ but also with Rb⁺ and Cs⁺ to form insoluble precipitates. The precipitates were formed specifically at the apical openings of mitochondrion-rich (MR) cells, which are responsible for K⁺ excretion in marine teleosts. Elemental analysis showed that Cs and Rb were distributed at the area of precipitates on the gill surface, and that these elements were colocalized with K. Our findings indicate that the gills eliminate unnecessary Cs⁺ and Rb⁺ in the body fluid, presumably through the same pathway as the K⁺ excretion in MR cells.     

Guanylyl cyclase stimulation by homologous and heterologous natriuretic peptides in salmonids

A range of homologous (trout ANP, trout CNP, trout VNP) and heterologous (eel ANP, eel ANP-NH₂, rat ANP, porcine CNP) NPs were tested for their effect on guanylyl cyclase in gill and kidney membrane preparations from freshwater and seawater-acclimated rainbow trout and Atlantic salmon. All NPs stimulated guanylyl cyclase at 1 μmol l⁻¹in all preparations. ANP was the most potent stimulator of kidney guanylyl cyclase and CNP the most potent stimulator of guanylyl cyclase in gills. Some differences were apparent between the potencies of homologous and heterologous peptides at 1 μmol l⁻¹: tANP was more potent than rANP in the SW trout kidney and tCNP was more potent than pCNP in FW salmon tissues. While eANP was more potent than tANP in trout gills, it was less potent than tANP in FW salmon gills. However, there was no significant difference between the potencies of eANP and eANP-NH₂ in trout or salmon gills. Salinity did not affect guanylyl cyclase activity with the exception that trout ANP at 1 μmol l⁻¹was more potent in SW trout kidneys than in FW trout kidneys. These results suggest a predomination of NPR-A in the kidney and NPR-B in the gill. It appears that salmonid NPR-A and NPR-B are relatively promiscuous in their ligand affinity, with few differences in the potencies of trout and mammalian NPs and only small differences in cGMP production where these differences do occur. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effects of growth hormone on plasma ionic regulation,respiration and extracellular acid-base status in trout (Oncorhynchus mykiss) transferred to seawater

The effects of trout recombinant growth hormone (rtGH) treatment (0.25 g g^–1 by intraperitoneal implant) on plasma ionic regulation, extracellular acid-base status and respiration were investigated in freshwater rainbow trout and during a 4-day period after direct transfer into seawater (35 g 1^–1).In freshwater, rtGH treatment resulted in a significant increase in gill (Na⁺, K⁺) ATPase activity and in standard metabolism (MO₂). The latter would mainly result from a higher rate of protein synthesis. Direct transfer from freshwater to seawater induced a decrease in arterial blood pH, far more pronounced in controls than in treated fish. This effect could be regarded in both groups mainly as a metabolic acidosis resulting from extracellular ion composition changes (i.e., an increase higher in chloride than in sodium, more marked in controls than in treated fish). As the rise in PaCO₂, in spite of an increase in ventilatory activity, is more significant in controls than in treated fish, it can be assumed that rtGH treatment lightened the decrease in the gas diffusing capacity of gills induced by transfer to seawater. The initial increase in MO₂ in both controls and treated fish could be the consequence of an increase in energetic cost of ventilation and osmoregulation. Then, in treated fish, the persistent high level of M may indicate a stimulation of intermediary metabolism by rtGH. In addition, the absence in treated fish of an increase in plasma lactate concentration, as observed in controls, would indicate that rtGH attenuated the decrease in O₂ affinity of haemoglobin foreseeable from the metabolic acidosis.This article is dedicated to Professor Claude Peyraud, whose recent death has deeply saddened us. We respectfully pay a tribute to his memory.     

Proton pumps in the fish gill and kidney

The proton pump or vacuolar type H⁺-ATPase is an oligomeric protein responsible for electrogenic H⁺ secretion in a variety of acid-secreting epithelia. Recently, the proton pump was identified in both the gill and kidney of freshwater-adapted rainbow trout (Oncorhynchus mykiss). Using immunocytochemistry, H⁺-ATPase has been localized in the pavement cells and chloride cells of the lamellar epithelium. During periods of internal acidosis, there is a marked increase in the expression of the branchial proton pump as identified by Western analysis, immunocytochemistry and in situ hybridization. This augmented expression of proton pumps occurs concomitantly with a marked increase in branchial acid excretion and Na⁺ uptake. Immunocytochemical studies suggest that the pavement cell, rather than the chloride cell, is the predominant site of acid excretion during periods of acidosis. These findings are consistent with the notion that in freshwater teleosts, Na⁺ uptake and H⁺ excretion are linked via the coupling of the electrogenic proton pump to apical membrane Na⁺ channels. This mechanism may be controlled by hormones including cortisol and/or growth hormone. The fish kidney plays an important role in regulating acidosis via the re-absorption of filtered HCO₃ ^-. Recently, we have demonstrated using Western analysis and immunocytochemistry, the presence of proton pump in rainbow trout kidney and observed increased H⁺-ATPase expression during respiratory acidosis. These new findings suggest a role for the renal proton pump in acid-base regulation.     

Osmoregulatory ability of chum salmon,Oncorhynchus keta,reared in fresh water for prolonged periods

The osmoregulatory ability of chum salmon (Oncorhynchus keta), reared in fresh water for a prolonged period, was examined by transferring them directly to seawater and then back to fresh water. When fry and juveniles weighing 0.3–125g, reared in fresh water for 1.5–13 months, were transferred directly to seawater, they adjusted their plasma Na⁺ concentration to the seawater-adapted level within 12–24h. When they were transferred back to fresh water after having been adapted to seawater for 2 weeks, the plasma Na⁺ level gradually decreased during the first 12–24h, and then increased to reattain the initial freshwater level after 5–7 days. No mortality was observed during the experiment except among the smallest fry weighing about 0.3g after transfer to seawater (2.1%). The maintenance of good osmoregulatory ability of the chum salmon for a prolonged period in fresh water seems to be unique among Pacific salmon, with the possible exception of the pink salmon.Changes in plasma levels of hormones during the transfer experiments were recorded in juveniles reared in fresh water for 13 months. Prolactin levels increased maximally 3 days after transfer from seawater to fresh water, as would be expected from its well-established role in freshwater adaptation in several euryhaline teleosts. In addition, an increase in plasma growth hormone was observed during the first 12h after seawater transfer, along with a tendency towards a decrease during freshwater transfer, suggesting an important role for this hormone in seawater adaptation. There were no consistent changes in plasma levels of thyroxine and cortisol during freshwater to seawater or seawater to freshwater transfer.     

Acclimation of brackish water pearl spot (Etroplus suratensis) to various salinities: relative changes in abundance of branchial Na+/K+-ATPase and Na+/K+/2Cl− co-transporter in relation to osmoregulatory parameters

The present study was conducted to elucidate the osmoregulatory ability of the fish pearl spot (Etroplus suratensis) to know the scope of this species for aquaculture under various salinities. Juvenile pearl spot were divided into three groups and acclimated to freshwater (FW), brackish water (BW) or seawater (SW) for 15 days. The fish exhibited effective salinity tolerance under osmotic challenges. Although the plasma osmolality and Na⁺, K⁺ and Cl^? levels increased with the increasing salinities, the parameters remained within the physiological range. The muscle water contents were constant among FW-, BW- and SW-acclimated fish. Two Na+/K+-ATPase α-isoforms (NKA α) were expressed in gills during acclimation in FW, BW and SW. Abundance of one isoform was up-regulated in response to seawater acclimation, suggesting its role in ion secretion similar to NKA α1b, while expression of another isoform was simultaneously up-regulated in response to both FW and SW acclimation, suggesting the presence of isoforms switching phenomenon during acclimation to different salinities. Nevertheless, NKA enzyme activities in the gills of the SW and FW individuals were higher (p < 0.05) than in BW counterparts. Immunohistochemistry revealed that Na⁺/K⁺-ATPase immunoreactive (NKA-IR) cells were mainly distributed in the interlamellar region of the gill filaments in FW groups and in the apical portion of the filaments in BW and SW groups. The number of NKA-IR cells in the gills of the FW-acclimated fish was almost similar to that of SW individuals, which exceeded that of the BW individuals. The NKA-IR cells of BW and SW were bigger in size than their FW counterparts. Besides, the relative abundance of branchial Na⁺/K⁺/2Cl^? co-transporter showed stronger evidence in favor of involvement of this protein in hypo-osmoregulation, requiring ion secretion by the chloride cells. To the best of our knowledge, this is the first study reporting the wide salinity tolerance of E. suratensis involving differential activation of ion transporters and thereby suggesting its potential as candidate for fish farming under different external salinities.     

Lead accumulation and its effects on the branchial physiology of Prochilodus lineatus

The purpose of this work was to determine the tissue accumulation of lead (Pb) and its effects on osmoregulatory processes of the freshwater fish Prochilodus lineatus. Juvenile fish were exposed to Pb (from 1.7 to 0.7 mg of dissolved Pb L^?1) for 6, 24 and 96 h and Pb accumulation was analyzed in the gills, liver, kidneys, blood cells and muscle. The following parameters were also analyzed: hematologic (hemoglobin content, hematocrit and number of red blood cells), metabolic (blood glucose), endocrine (blood cortisol), osmo ionic (plasma osmolality and Na⁺, K⁺, Cl^? and Ca⁺² concentrations), gill enzymes (Na⁺/K⁺-ATPase and carbonic anhydrase), chloride cell (CC) density and CC location in the gills. Pb accumulated in all the analyzed tissues, with the kidneys showing the highest concentration, followed by the gills and liver. The lowest Pb concentrations were found in blood cells and muscle. Pb promoted an increase in blood glucose after 6 and 24 h exposure. Gill Na⁺/K⁺-ATPase was inhibited after 24 h of exposure, but its activity was restored at 96 h, probably due to the increase in CC in gill lamellae. Plasma Na⁺ was reduced after 6 and 96 h, while K⁺ concentrations increased at all the experimental times. Fish exposed to Pb showed reduced plasma Ca⁺² at all experimental periods. Hematologic parameters remained unchanged. Overall, this study demonstrated that Pb interferes in osmoregulatory processes of P. lineatus and the proliferation of CC in the gills is a response in order to reestablish adequate ion concentrations.     

Gill organ culture of rainbow trout, Salmo gairdneri Richardson: an experimental model for the study of pathogenicity of Mycoplasma mobile 163 K

Abstract. A method has been developed to maintain gills from rainbow trout, Salmo gairdneri Richardson, in vitro. The gill organ cultures were used to examine the cytotoxic capacities of Mycoplasma mobile 163 K, isolated from the gills of a tench, Tinea tinea L. Histological investigations using light and electron microscopy showed that M. mobile causes heavy damage to the gill epithelium characterized by cytoplasmic vacuolization, peripheral orientation of the condensed nuclear chromatin, separation of the epithelial cells from each other and from the basal membrane, desquamation of the epithelial layer, shrinkage of the cytoplasmic net structure, and a swollen and bleb–like appearance of the apical area of the secondary lamella. The gill organ culture appears to be a suitable model to investigate the cell injuring capacity of M. mobile and may also be applicable to the study of the pathogenic properties of other microorganisms or viruses of fish.     

Response of rainbow trout gill (Na++K+)-ATPase and chloride cells to T3 and NaCl administration

With the aim of comparing the effects of oral T₃ and NaCl administration on trout hypoosmoregulatory mechanisms, three groups of rainbow trout (Oncorhynchus mykiss Walbaum) held in freshwater (FW) were fed a basal diet (C), the same diet containing 8.83 ppm of 3,5,3-triiodo-L-thyronine (T₃) (T) or 10% (w/w) NaCl (N) respectively for 30 d. They were then transferred to brackish water (BW) for 22 d and fed on diet C. Gill (Na⁺+K⁺)-ATPase activity and its dependence on ATP, Na⁺ and pH, number of gill chloride cells (CC), serum T₃ level as well as fish growth, condition factor (K) and mortality were evaluated. During the FW phase, as compared to C trout, T trout showed a two fold higher serum T₃ level, had unchanged gill (Na⁺+K⁺)-ATPase activity and increased CC number, whereas N trout showed higher gill (Na⁺+K⁺)-ATPase activity and CC number. At the end of the experiment the enzyme activity was in the order T>N>C groups and all groups showed similar CC number. Both treatments changed the enzyme activation kinetics by ATP and Na⁺. A transient increase in K value occurred in N group during the period of salt administration. In BW, T and N groups had higher and lower survival than C group respectively. Other parameters were unaffected by the treatments. This trial suggests that T₃ administration promotes the development of hypoosmoregulatory mechanisms of trout but it leaves the (Na⁺+K⁺)-ATPase activity unaltered till the transfer to a hyperosmotic environment.     

Enhanced hypoosmoregulatory response to growth hormone after cortisol treatment in immature rainbow trout,Salmo gairdneri

The growth-independent effect of ovine growth hormone (oGH) and oGH + cortisol treatment on seawater (SW) adaptation in immature rainbow trout, Salmo gairdneri was investigated. Fish were injected every second day with saline, 2.0 μg oGH/g or 2.0 μg oGH + 8.0 μg cortisol/g for a maximum of 8 injections in freshwater (FW). Subgroups were transferred to 28‰ SW after 4 or 8 injections, and changes in plasma Na⁺ and Cl⁻, muscle water content and gill Na⁺/K⁺-ATPase activity were measured. In both of the hormone-treated groups retained in FW, gill Na⁺/K⁺-ATPase activity and interlamellar chloride cell density increased. The effects were most pronounced in the oGH + cortisol group after 2 weeks of treatment. After transfer to SW most of the control fish died due to the osmotic stress, whereas in the hormone-treated groups, mortality was low and there was a positive correlation between pretransfer gill Na⁺/K⁺-ATPase and the ability to maintain ionic-osmotic homeostasis after SW transfer. After two weeks of oGH + cortisol treatment, gill Na⁺/K⁺-ATPase activity was maximal. In contrast, after SW transfer, Na⁺/K⁺-ATPase activity increased further in the oGH-treated group. This group regulated ionic-osmotic parameters less effectively than the oGH + cortisol-treated group. The data indicate that GH and cortisol are important hormones in the regulation of hypoosmoregulatory mechanisms in S. gairdneri.     

Importance of river migration to the development of seawater tolerance in Columbia River anadromous salmonids

Gradually increasing levels of gill Na⁺K⁺ ATPase activity were observed in juvenile chinook, Oncorhynchus tshawytscha, and coho, Oncorhynchus kisutch, salmon and steelhead trout, Salmo gairdneri, undergoing parr-smolt transformation in artificial rearing facilities on the Columbia River. Portions of the same populations released to migrate seaward, however, generally showed much greater increases in enzyme activity with time and distance from the release point. After migrating 714 km to the Columbia River estuary, spring chinook salmon had a mean gill Na⁺K⁺ ATPase activity 2.5 times greater than fish retained at the hatchery and 1.9 times greater than fish adapted to 28 ppt seawater for 208 days. Similar observations were made on coho salmon.     

Environment-induced gill disease of cultured rainbow trout (Salmo gairdneri)

Rainbow trout from eight North German freshwater culture facilities were investigated for chronic health damage resulting from captivity. The fishes were raised under a wide variety of conditions in net cages, circulation tanks, raceways, and earthen ponds.The most obvious non-contagious health problem was gill injury. Hyperplasia of the gill filaments, leading to lamellar fusion or the development of apical caps, as well as necrotic or advanced degenerative processes were observed. In addition, the gill filaments were frequently shortened, significantly reducing the functional surface of the organ.The lesions occurred in almost the same form in all of the culture units. It is likely that they are part of a syndrome induced by the environmental conditions typical of freshwater aquaculture facilities. These are characterized by an accumulation of nitrogenous excretion and decomposition products, extreme diurnal fluctuations in the concentration of dissolved gases, and intensive feeding involving the sudden addition of large amounts of artificial products. Under such conditions even small quantities of un-ionized ammonia can be toxic for epithelial tissues and disturb the normal elimination of protein metabolites across the gills. The pathological processes may be accelerated by secondary bacterial infections.     

Seawater tolerance in Atlantic salmon, Salmo salar L., brown trout, Salmo trutta L., and S. salar?×?S. trutta hybrids smolt

High levels of hybridization between Atlantic salmon (Salmo salar) and brown trout (Salmo trutta) have been reported in the Gyrodactylus salaris infected Rivers Vefsna and Driva in Norway. The survival and behaviour during the sea phase of such hybrids is unknown. The reported work documents ionoregulatory status after 24 h seawater challenge tests (24hSW) and gill Na⁺/K⁺-ATPase (NKA) activity of migrating wild smolts of Atlantic salmon, brown trout and hybrids at two sampling dates during the 2006 smolt run in River Driva. Salmon, trout and hybrids contributed to 27, 52 and 21% of the catches, respectively. The large contribution of hybrids suggests both a high hybridization rate and a high survival rate from fry to smolt. Both salmon and hybrids had a well-developed seawater tolerance at the time of downstream migration, revealed by small ionoregulatory effects and no or low mortality rates during the 24hSW tests. The trout were not fully adapted to seawater, and high mortality rates were observed (71 and 92%) during the 24hSW tests. The NKA activity was not significantly different between salmon and hybrids. Most of the hybrids were physiologically capable of direct entry to full strength seawater. The incomplete seawater tolerance in trout compared to salmon corresponds well with differences in life-history patterns between these two species. The life history strategy of the hybrids during the sea phase is not known, and further investigations on the marine behaviour and survival is needed to evaluate the role of hybrids in the risk of spreading G. salaris to nearby river systems.     

Osmoregulatory balance in Murray cod, Maccullochella peelii peelii (Mitchell), affected with chronic ulcerative dermatopathy

This study examined the osmoregulatory capability of Murray cod, Maccullochella peelii peelii, affected by chronic ulcerative dermatopathy (CUD) in intensive aquaculture. This condition appears to arise only in facilities utilizing groundwater, with the causative agent suggested to be a water-borne factor. Healthy Murray cod (~ 700 g) were transferred to a CUD-affected farm to monitor the progression of the syndrome and began to show signs of CUD after approximately five months. In order to evaluate possible effects of CUD on osmoregulation; plasma electrolyte concentrations, osmolality, and Na⁺,K⁺-ATPase activities were measured, and gill histology and immunohistochemistry were analyzed. Plasma electrolyte concentrations and osmolality of CUD-affected Murray cod were consistent with reference values determined for non CUD-affected fish. A greater number of gill mucous cells were observed in Murray cod cultured at the CUD-affected farm compared to non CUD-affected fish. We also found an un-identified cell type that was present solely in the gills of CUD-affected Murray cod. Gill Na⁺,K⁺-ATPase activity was significantly higher in severely CUD-affected Murray cod compared to individuals transferred to the CUD-affected farm. While there appeared to be some minor changes in the gills of CUD-affected fish, this study demonstrated that Murray cod were able to effectively osmoregulate, although, perhaps at an energetic cost.     

Cortisol treatment improves the development of hypoosmoregulatory mechanisms in the euryhaline rainbow trout,Salmo gairdneri

The effect of cortisol on osmoregulatory parameters was studied in rainbow trout, (Salmo gairdneri), kept in freshwater (FW) and/or transferred to seawater (SW). Repeated injections of 20 μg cortisol/g fish stimulated gill and gut Na⁺/K⁺-ATPase activity and reduced plasma Na⁺ and Cl⁻ levels after 2 weeks of treatment in FW-adapted fish. Cortisol doses of 0.05 and 1.0 μg/g were without effect. Repeated injections of 10 μg cortisol/g stimulated gill Na⁺/K⁺-ATPase activity and reduced plasma Na+ and Cl⁻ levels in fish in FW, and significantly improved ion regulation after their transfer to 28SW. Higher doses of cortisol (10 and 20 μg/g) induced hyperglycemia, whereas low doses (0.05 and 1.0 μg/g were without effect or induced hypoglycemia. Plasma glucose levels decreased in cortisol-treated fish transferred to SW, whereas transient hyperglycemia was seen in the control fish.     

Transepithelial ion exchange in smolting atlantic salmon (Salmo Salar L.)

Whole-body (but predominantly gill) Na⁺ exchange, gill Na⁺/K⁺/ATPase activity and seawater tolerance were examined in juvenile Atlantic salmon during the smolting period. Transepithelial net Na⁺ gain decreased steadily from late February showing a net loss in April and early May, returning to approximate equilibrium in mid-May. This seasonal net loss of Na⁺ to the environment occurred slightly after maximal gill epithelial Na⁺/K⁺/ATPase activity and preceded maximal seawater tolerance. The results are discussed in relation to changes in gill permeability and salt intake via the diet.