Branchial expression patterns of claudin isoforms in Atlantic salmon during seawater acclimation and smoltification

In euryhaline teleosts, permeability changes in gill epithelia are essential during acclimation to changed salinity. This study examined expression patterns of branchial tight junction proteins called claudins, which are important determinants of ion selectivity and general permeability in epithelia. We identified Atlantic salmon genes belonging to the claudin family by screening expressed sequence tag libraries available at NCBI, and classification was performed with the aid of maximum likelihood analysis. In gill libraries, five isoforms (10e, 27a, 28a, 28b, and 30) were present, and quantitative PCR analysis confirmed tissue-specific expression in gill when compared with kidney, intestine, heart, muscle, brain, and liver. Expression patterns during acclimation of freshwater salmon to seawater (SW) and during the smoltification process were examined. Acclimation to SW reduced the expression of claudin 27a and claudin 30 but had no overall effect on claudin 28a and claudin 28b. In contrast, SW induced a fourfold increase in expression of claudin 10e. In accord, a peak in branchial claudin 10e was observed during smoltification in May, coinciding with optimal SW tolerance. Smoltification induced no significant changes in expression of the other isoforms. This study demonstrates the expression of an array of salmon claudin isoforms and shows that SW acclimation involves inverse regulation, in the gill, of claudin 10e vs. claudin 27a and 30. It is possible that claudin 10e is an important component of cation selective channels, whereas reduction in claudin 27a and 30 may change permeability conditions in favor of the ion secretory mode of the SW gill.     

Growth hormone and seawater adaptation in coho salmon, Oncorhynchus kisutch

1. Effects of growth hormone (GH) were examined on short-term aspects of seawater adaptation in coho salmon smolts. 2. Injection of somatostatin (SRIF) immediately prior to seawater entry suppressed plasma GH levels, but did not have any significant effects at 6 or 12 hr on hematocrits, plasma glucose or plasma Na+ levels. 3. Plasma GH levels increased 250% within 36 hr after seawater exposure. 4. Plasma glucose levels, in contrast, were significantly lower in the seawater fish after 36 hr post-exposure. 5. Plasma Na+ levels increased to 190 mEq/1 by 24 hr but subsequently returned to freshwater levels while hematocrits showed no significant changes over the 72 hr of exposure. 6. The significance of these results is discussed in terms of successful seawater adaptation in coho salmon.     

Purine levels in the skin of juvenile coho salmon (Oncorhynchus kisutch) during parr-smolt transformation and adaptation to seawater.

1. Juvenile coho salmon were examined for changes in guanine and hypoxanthine levels in the skin in relation to common physiological indices of parr-smolt transformation. 2. Guanine levels correlated well with changes in smolt indices, but reached maximum levels up to 1 month earlier than the development of seawater tolerance. 3. Transfer of juveniles into seawater at different times during the smolting cycle did not cause significant alterations in guanine concentration from the cyclic changes found in fish reared in freshwater. 4. Results suggest that guanine deposition in the skin during smolting seems unresponsive to seawater adaptation and therefore may not represent an adaptation in preparation for an oceanic life.     

Rapid changes in the phospholipid composition of gill membranes during thermal acclimation of the rainbow trout,Salmo gairdneri

Summary The phospholipid composition of gill tissue was determined in rainbow trout (Salmo gairdneri) undergoing thermal acclimation between 5°C and 20°C for a period of up to 28 days. Proportions of phosphatidylethanolamine (PE) and cardiolipin (CL) increased during cold acclimation and decreased during warm acclimation; proportions of phosphatidylcholine (PC) changed in the opposite direction (i.e., decreased during cold acclimation). In contrast, levels of phosphatidylserine,-inositol, and sphingomyelin did not vary significantly. Thermal modulation of headgroup composition occurred rapidly as reflected by changes in the ratio of PC-to-PE, which rose significantly from 2.40±0.09 to 2.92±0.09 within 72 h of transfer from 5 to 20°C; adaptation to 5°C was equally rapid. Proportions of PE changed more rapidly than those of PC during cold adaptation, whereas the opposite was true during warm acclimation. Both the time course and the direction of the observed changes in phospholipid composition suggest that such adjustments may contribute to the homeoviscous regulation of membrane properties, particularly during the initial stages of thermal adaptation.     

Chloride cell apical surface changes in gill epithelia of the armoured catfish Hypostomus plecostomus during exposure to distilled water

Armoured catfish Hypostomus plecostomus were exposed to distilled water for 15 days. High chloride cell proliferation occurred on the filament and lamellar gill epithelia. The apical surface of the chloride cells (66% of cells in both epithelia) showed significant reduction in the aquatic environment which was characterized by the development of a sponge-like organization. The chloride cell response suggests that these features could create a microenvironment which may favour either the reduction of ion loss or ion uptake in a environment characterized by an absence of ions.     

Changes of physiological rhythms of N-methyl-d-aspartate receptors in the chum salmon Oncorhynchus keta: effect of seawater acclimation during the parr-smolt transformation

We examined changes on N-methyl-d-aspartate receptors (NRs) in different growth stages (early parr, parr, and early smolt) of chum salmon, Oncorhynchus keta, during parr-smolt transformation from freshwater to seawater. Expression levels of NR genes mRNA and concentration of cortisol, T₃, T₄, dopamine and Na⁺/K⁺-ATPase activity significantly increased at salinity change condition. Moreover, in cultured brain cells, NRs were significantly lower in all groups treated with MK-801 (an antagonist of NRs) than in the early parr stage group in the FW treatment. We confirmed that the reduction in mRNA expression levels of NRs increased from the early parr to the early smolt stage. The information reported here should be taken into account in future studies on the relationship between memory factors of natal streams and homing mechanisms in Salmonidae.     

Cultured gill epithelia as models for the freshwater fish gill

We review recent progress in the development of models for the freshwater teleost gill based on reconstructed flat epithelia grown on permeable filter supports in primary culture. Methods are available for single-seeded insert (SSI) preparations consisting of pavement cells (PVCs) only from trout and tilapia, and double-seeded insert (DSI) preparations from trout, containing both PVCs (85%) and mitochondria-rich cells (MRCs, 15%), as in the intact gill. While there are some quantitative differences, both SSI and DSI epithelia manifest electrical and passive permeability characteristics typical of intact gills and representative of very tight epithelia. Both preparations withstand apical freshwater exposure, exhibiting large increases in transepithelial resistance (TER), negative transepithelial potential (TEP), and low rates of ion loss, but there is only a small active apical-to-basolateral "influx" of Cl(-) (and not of Na(+)). Responses to various hormonal treatments are described (thyroid hormone T3, prolactin, and cortisol). Cortisol has the most marked effects, stimulating Na(+),K(+)-ATPase activity and promoting active Na(+) and Cl(-) influxes in DSI preparations, and raising TER and reducing passive ion effluxes in both epithelia via reductions in paracellular permeability. Experiments using DSI epithelia lacking Na(+) uptake demonstrate that both NH(3) and NH(4)(+) diffusion occur, but are not large enough to account for normal rates of branchial ammonia excretion, suggesting that Na(+)-linked carrier-mediated processes are important for ammonia excretion in vivo. Future research goals are suggested.     

Changes in carbonic anhydrase activity in coho salmon smolts resulting from physical training and transfer into seawater

Carbonic anhydrase (CA) activity was measured in blood and in gill tissue of coho salmon smolts during chronic exercise and subsequent transfer into seawater. The mean level of CA activity was higher in blood than gill tissue in both freshwater and seawater. CA activity in gill tissue increased significantly after the smolts had adapted to seawater. CA activity in blood decreased significantly in the group of fish given the highest exercise level after they had adapted to seawater. There were no significant differences in CA activity in gill tissue between control and exercise groups in either freshwater or seawater.     

A novel betaproteobacterial agent of gill epitheliocystis in seawater farmed Atlantic salmon (Salmo salar)

Epitheliocystis, a disease characterised by cytoplasmic bacterial inclusions (cysts) in the gill and less commonly skin epithelial cells, has been reported in many marine and freshwater fish species and may be associated with mortality. Previously, molecular and ultrastructural analyses have exclusively associated members of the Chlamydiae with such inclusions. Here we investigated a population of farmed Atlantic salmon from the west coast of Norway displaying gill epitheliocystis. Although 'Candidatus Piscichlamydia salmonis', previously reported to be present in such cysts, was detected by PCR in most of the gill samples analysed, this bacterium was found to be a rare member of the gill microbiota, and not associated with the observed cysts as demonstrated by fluorescence in situ hybridization assays. The application of a broad range 16 S rRNA targeted PCR assay instead identified a novel betaproteobacterium as an abundant member of the gill microbiota. Fluorescence in situ hybridization demonstrated that this bacterium, tentatively classified as 'Candidatus Branchiomonas cysticola', was the cyst-forming agent in these samples. While histology and ultrastructure of 'Ca. B. cysticola' cysts revealed forms similar to the reticulate and intermediate bodies described in earlier reports from salmon in seawater, no elementary bodies typical of the chlamydial developmental cycle were observed. In conclusion, this study identified a novel agent of epitheliocystis in sea-farmed Atlantic salmon and demonstrated that these cysts can be caused by bacteria phylogenetically distinct from the Chlamydiae.     

Metabolic changes in coho and chinook salmon resulting from acute insufficiency in pancreatic hormones

Acute deficiency in pancreatic peptides (insulin, somatostatin-25, glucagon, and glucagon-like peptide) was invoked for 9-12 hr in coho, Oncorhynchus kisutch, and chinook, O. tshawytscha, salmon by administration of specific antisera raised against purified salmon hormones. Insulin-deficient fish were hyperglycemic, had diminished glycogen content in the liver (Plisetskaya et al., '88a, elevated liver triacylglycerol lipase activity, and higher concentration of plasma triiodothyronine (T3) compared to a control group of fish injected with nonspecific rabbit serum. After immunoneutralization of somatostatin-25, fish remained normoglycemic, with higher liver glycogen content, decreased lipase activity, and elevated plasma levels of insulin, while the levels of T3 declined. The induced deficiency in glucagon family peptides led to comparatively smaller changes: liver glycogen content was increased after anti-glucagon-like peptide (aGLP) injection and transient hyperglycemia was apparent following anti-glucagon (aGLU) administration. Circulating levels of insulin remained unaffected for at least 9 hr following aGLU and aGLP treatments. The velocity of pyruvate kinase at 2.5 mM phosphoenolpyruvate (V2.5) was depressed, especially after the combined administration of aGLU + aGLP. The effectiveness of immunoneutralization experiments was greatly dependent on the particular stage of the fish life cycle. Antisera against fish pancreatic peptides proved to be a suitable tool in the studies of hormonal regulation of fish metabolism.     

Clearance of plasma corticosteroids during smoltification of coho salmon, Oncorhynchus kisutch

The plasma clearance rate (PCR) of radioactivity after a single intracardial injection of 3H-cortisol was elevated during the spring in yearling coho salmon, Oncorhynchus kisutch. Graphical analysis suggested a seasonal correlation between PCR and gill Na/K-ATPase activity. An explanation for this correlation is suggested. The major metabolite of 3H-cortisol in plasma was 3H-cortisone. It appeared rapidly following injection of the original radiotracer.     

Oxygen uptake of growth hormone transgenic coho salmon during starvation and feeding

Oxygen uptake of growth hormone transgenic coho salmon Oncorhynchus kisutch was measured in individual fish with a closed-system respirometer and was compared with that of similar-sized non-transgenic control coho salmon during starvation and when fed a fixed ration or to satiation. Transgenic and control fish did not differ in their standard oxygen uptake after 4 days of starvation, although control fish had a higher routine oxygen uptake, scope for spontaneous activity and initial acclimation oxygen uptake. During feeding, transgenic fish ate significantly more than control fish, and had an overall oxygen uptake that was 1·7 times greater than control fish. When fish that had eaten the same per cent body mass were compared, transgenic fish had an oxygen uptake that was 1·4 times greater than control fish. Differences in oxygen uptake in growth hormone transgenic coho salmon and non-transgenic fish appear to be due to the effects of feeding, acclimation and activity level, and not to a difference in basal metabolism.     

Morphological changes induced by acclimation to high pressure in the gill epithelium of the freshwater yellow eel

After acclimation either to high pressure (101 ATA) or to low temperature (9°C), the number of mucous cells within gill epithelium of freshwater eel Anguilla anguilla was significantly decreased and the density of chloride cells was significantly increased when compared to control fish (1 ATA, 19°C).     

DNA homologies between the rainbow trout, chum salmon and coho salmon

The thermal stabilities of hybrid duplexes between the DNAs from three salmonid fish species were monitored as measures of DNA homology. The chum salmon, Oncorhynchus keta, and coho salmon, Oncorhynchus kisutch, had more DNA homology with each other than either had with the rainbow trout, Salmo gairdnerii. Morphological, ecological and protein similarities between the coho salmon and the rainbow trout may be due to parallel or convergent evolution.     

Intestinal nutrient transport in coho salmon (Oncorhynchus kisutch) and the effects of development,starvation, and seawater adaptation

Summary Intestinal nutrient transport in yearling coho salmon was characterized and adaptive changes in nutrient transport were described in relation to development, starvation, and environmental salinity. Salmon intestine exhibits a small transepithelial potential difference (TEP: –1.4 to 2.0 mV, mucosa ground) and low resistance (41 to 181 ohms·cm²) that varied with the region along the intestine, with starvation, and with environmental salinity. Addition of glucose or proline to the mucosal side of intestine caused a rapid increase in short-circuit current. Isotopic mucosalto-serosal net fluxes of glucose and proline were achieved across salmon intestine in the absence of transepithelial chemical or electrical gradients. A sleeve technique for measuring proline influx (Karasov and Diamond 1983a) was validated for use in salmon intestine. Comparison of total proline influx in different intestinal regions showed the following order (from highest to lowest rates): pyloric caeca anterior intestine > posterior intestine. Total proline influx was highest in April during the parr-smolt transformation.The kinetics of Na-dependent proline influx were altered by starvation and seawater adaptation. Starved fish exhibited a lowerK _t but similarJ _max in anterior intestine compared with values in fed fish. The effect of seawater adaptation on the kinetics of proline influx varied with the timing of entry into seawater, with length of seawater residence, and with season. Growth-inhibited SW stunts showed a reducedJ _max of proline influx compared with that of normal SW smolts.Abbreviations FW freshwater (-adapted) - SW seawater (-adapted) - TEP transepithelial potential difference - R transepithelial resistance - I _sc short-circuit current - P _a apparent passive permeability coefficient - J _max maximal influx - K _t half-saturation constant     

Intestinal morphology in growth hormone transgenic coho salmon

In two GH transgenic coho salmon Oncorhynchus kisutch , the surface area of the intestine was 2·2 times that of control salmon and the growth rate was about twice that of controls. It seems likely that the enhanced intestinal surface area is a compensatory feature that is manifested in fast growing salmonids.     

Changes in intestinal fluid transport associated with smoltification and seawater adaptation in coho salmon, Oncorhynchus kisutch (Walbaum)

The intestinal role in osmoregulatory preadaptation of freshwater-adapted (FW) yearling coho salmon during smoltification was examined. Measurements of intestinal net fluid absorption (J_V) using an in vitro sac preparation showed that J_V of FW parr was significantly less than that of either FW or seawater-adapted (SW) smolts. From two to four weeks following the springtime thyroxin surge, J_V increases in FW coho to a level comparable with that observed for SW smolts. J_V remained elevated in FW smolts throughout most of the summer and then decreased in the autumn. Two months after the thyroxin peak, intestinal wet weight per unit serosal surface area increases. Coho stunts resulting from premature transfer into sea water had significantly higher J_V than that seen in SW smolts. The elevated J_V of stunts is only partially explicable on the basis of differences in body weight between stunts and smolts.
Our observations indicate that an increase in intestinal fluid absorption is a preadaptive change associated with smoltification occurring in concert with alterations in renal and branchial osmoregulatory mechanisms. The timing of the increase in J_V suggests a phase relationship to the thyroxin surge. This temporal relationship may help to explain the efficacy of using changes in plasma thyroxin levels to predict hatchery-fish release dates that may optimize eventual seawater survival.