Claudin-15 and -25b expression in the intestinal tract of Atlantic salmon in response to seawater acclimation,smoltification and hormone treatment

In seawater fishes, osmotic homeostasis requires uptake of ions and water in the intestine and these processes are governed by the combined trans- and paracellular pathways. The current study examined mRNA expression of two tight junction proteins (claudin-15 and -25b) predominantly expressed in the intestine of Atlantic salmon. We examined the response in pyloric caecae, middle and posterior intestine to seawater challenge, during smoltification and after injection with osmoregulatory hormones. Seawater (SW) transfer elevated levels of claudin-15 and -25b while no change was induced throughout the smolt stage. In freshwater, cortisol and growth hormone inhibited claudin-15 expression in the two anterior segments. Claudin-25b was elevated in all intestinal segments by growth hormone, while cortisol had an inhibitory effect. In seawater, prolactin and cortisol inhibited claudin expression. The data suggest that claudin expression is involved in the reorganisation of intestinal epithelium and possibly change paracellular permeability during SW acclimation. The lack of preparatory change during smoltification suggests that this process is not completed during smolt development. The effects of the tested hormones cannot explain the sum of changes induced by salinity, which, like the smoltification data, suggests the importance of additional factors and possibly contact with the imbibed SW per se.     

Ionoregulatory changes in the gill epithelia of coho salmon during seawater acclimation

Short-term exposure of coho salmon smolts (Oncorhynchus kisutch) to a gradual increase in salinity over 2 d (0 per thousand -32 per thousand ) resulted in a decrease in proton pump abundance, detected as changes in immunoreactivity with a polyclonal antibody against subunit A of bovine brain vacuolar H(+)-ATPase. N-ethylmaleimide (NEM)-sensitive H(+)-ATPase activities in gill homogenates remained unchanged over 8 d to coincide with a 3.5-fold increase in Na(+)/K(+)-ATPase activities. A transient increase in plasma [Na(+)] and [Cl(-)] levels over the 8-d period was preceded by a 10-fold increase in plasma cortisol levels, which peaked after 12 h. Long-term (1 mo) acclimation to seawater resulted in the loss of apical immunoreactivity for vH(+)-ATPase and band 3-like anion exchanger in the mitochondria-rich cells identified by high levels of Na(+)/K(+)-ATPase immunoreactivity. The polyclonal antibody Ab597 recognized a Na(+)/H(+) exchanger (NHE-2)-like protein in what appears to be an accessory cell (AC) type. Populations of these ACs were found associated with Na(+)/K(+)-ATPase rich chloride cells in both freshwater- and seawater-acclimated animals.     

Gene expression patterns in Atlantic salmon Salmo salar: gene expression during osmoregulation in intestine tissue

Ireland has the world's largest stocks of wild Atlantic salmon. A better understanding of gene expression will benefit conservation of wild stock as well as salmon aquaculture. We describe the PRTLI project designed to advance the fundamental understanding of the genome of Atlantic Salmon Salmo salar . The major objective is to create the first comprehensive database of gene expression and functional information using cDNA libraries and Microarray technology. One key area of interest to salmon biology is osmoregulation, which is critical to the ability of salmon to adapt in seawater. Tissues implicated in this process are the gills, intestine and skin. To initiate studies, SSH (suppression subtractive hybridization) libraries were constructed from intestine RNA extracted from smolts sampled in January and May. A number of potentially interesting clones have been identified, among those a heat shock protein, hsp90 in the reverse library. Others SSH libraries from various tissues (pituitary, hypothalamus, brain, gill, intestine, head kidney and spleen) have also been constructed and will be used to construct a 5000 clone microarray slide. This slide will then be used to elucidate gene expression profiles in various tissues. Further sample collection has been carried out to answer questions regarding biologicaldifferences between one‐ year and two‐year old parr and wild and hatchery smolt.     

Functional characterization and localization of a gill-specific claudin isoform in Atlantic salmon

Claudins are the major determinants of paracellular epithelial permeability in multicellular organisms. In Atlantic salmon (Salmo salar L.), we previously found that mRNA expression of the abundant gill-specific claudin 30 decreases during seawater (SW) acclimation, suggesting that this claudin is associated with remodeling of the epithelium during salinity change. This study investigated localization, protein expression, and function of claudin 30. Confocal microscopy showed that claudin 30 protein was located at cell-cell interfaces in the gill filament in SW- and fresh water (FW)-acclimated salmon, with the same distribution, overall, as the tight junction protein ZO-1. Claudin 30 was located at the apical tight junction interface and in cell membranes deeper in the epithelia. Colocalization with the α-subunit of the Na(+)-K(+)-ATPase was negligible, suggesting limited association with mitochondria-rich cells. Immunoblotting of gill samples showed lower claudin 30 protein expression in SW than FW fish. Retroviral transduction of claudin 30 into Madin-Darby canine kidney cells resulted in a decreased conductance of 19%. The decreased conductance correlated with a decreased permeability of the cell monolayer to monovalent cations, whereas permeability to chloride was unaffected. Confocal microscopy revealed that claudin 30 was expressed in the lateral membrane, as well as in tight junctions of Madin-Darby canine kidney cells, thereby paralleling the findings in the native gill. This study suggests that claudin 30 functions as a cation barrier between pavement cells in the gill and also has a general role in cell-cell adhesion in deeper layers of the epithelium.     

Seasonal growth of wild Atlantic salmon juveniles and implications on age at smoltification

The seasonal growth trajectories of wild Atlantic salmon Salmo salar juveniles by age group within the Margaree River, Canada, are described. Circuli counts from scales were used to infer growth rates at different ages and these were used to predict the proportions of age 2‐ and 3‐year old smolts from different portions of the watershed. In the wild Atlantic salmon juveniles from the Margaree River, there was no bimodality in fork length frequencies and no 1 year old smolts were produced. Water temperature differences during the growing season were insufficient to explain the differences in growth rates and size at age among the sites sampled. There was a positive association between the growth rate in the first year and the subsequent age at smoltification. In the Margaree River, differences in tributary specific growth rates and size at age were expected to produce important differences in the relative ages at smoltification.     

Body temperature and seawater adaptation in farmed Atlantic salmon and rainbow trout during prolonged chilling

The core temperature of the rainbow trout Oncorhynchus mykiss (3·5 kg) dropped to 1·0° C during the first 6 h of chilling at 0·5° C, remained stable until 24 h, and dropped significantly to 0·7° C after 39 h. Blood plasma osmolality increased and muscle moisture content decreased gradually with increasing chilling time. After 39 h of chilling, the rainbow trout experienced 40 mosmol l^-1 higher blood plasma osmolality and 2·8% less muscle moisture content compared with initial values. In the Atlantic salmon Salmo salar (5·3 kg), core temperature dropped to 1·3° C and blood plasma osmolality increased significantly during the first 6 h of chilling at 0·5° C, but remained relatively stable throughout the rest of the experimental period. After 39 h of chilling, the salmon experienced 20 mosmol l^-1 higher blood plasma osmolality and 0·5% less muscle moisture content compared with initial values. In rainbow trout muscle moisture content was inversely related to blood plasma osmolality indicating reduced seawater adaptation with increasing hours of chilling. No such relationship was observed in the Atlantic salmon. Hence, changes in plasma osmolality and muscle moisture in the Atlantic salmon do not indicate osmoregulatory failure since the new levels, once established, were maintained throughout the chilling time.     

Ultrastructural features of chloride cells in the gill epithelium of the Atlantic salmon, Salmo salar, and their modifications during smoltification

To elucidate the ultrastructural modifications of the gill epithelium during smoltification, gills of the Atlantic salmon (Salmo salar) were examined by electron microscopy at three stages of this process, which were defined as follows: "parrs" were freshwater fish that had not yet started their transformation; "freshwater smolts" were freshwater fish that were ready to enter seawater; and "seawater smolts" were smolts that had been transferred from fresh water and maintained for 4 days in seawater (35%). In the gill epithelium of parrs, there were two types of chloride cells. The large chloride cells contained deeply stained mitochondria and numerous apical, irregular, dense, membrane-bound bodies that formed 77% of the chloride cell population and were distinguished easily from small chloride cells that have distinctly paler mitochondria and no dense bodies in their apical cytoplasm. In freshwater smolts, the large chloride cells formed 95% of the chloride-cell population. In contrast to the small chloride cells that were not modified, they almost doubled in size. Their tubular system developed extensively to form a tight network with regular meshes significantly smaller than those observed in parr chloride cells. Forty percent of the large chloride cells were associated with a new type of cell, the accessory cell, to which they were bound by shallow apical junctions. Half of these accessory cells were not seen to be in contact with the external medium. In seawater smolts, 80% of the large chloride cells were associated with accessory cells. Most accessory cells reached the external medium and sent numerous cytoplasmic interdigitations within the apical portion of the adjacent chloride cells. As a result, a section through the apical portion of the chloride cells and their associated accessory cells revealed a mosaic of interlocked cell processes bound together by an extended, shallow apical junction. It was concluded that the Atlantic salmon develops in fresh water most of the ultrastructural modifications of the gill epithelium which in most euryhaline fish are triggered by exposure to seawater. The effective transfer into seawater would act only as a final stimulus to achieve some adequacy between the freshwater smolt and its new environment.     

Changes in mucus cell numbers in the epidermis of the Atlantic salmon at the onset of smoltification

A sharp decrease to <50% in the numbers of mucus cells mm⁻² of epidermis was observed in the skin of Atlantic salmon Salmo salar at the beginning of the smoltification period in 1997 and 1999. Average numbers decreased in 1997 from 662 mm⁻² on 6 March to 275 mm⁻² on 20 March and in 1999 from 828 mm⁻² on 3 March to 388 mm² on 24 March. A second smaller decrease was observed towards the end of smoltification in both years.     

Differential transferrin gene expression in Atlantic salmon (Salmo salar L.) freshwater parr and seawater smolts

The Atlantic salmon has a complex life-cycle in which it encounters a salinity barrier initially upon migration to the sea as a young smolt and later as an adult salmon returning to its natal river. Concurrent with seawater migration is a process termed smoltification which is a series of metabolic changes which transform the freshwater parr into smolts adapted for life in the marine environment. To gain an understanding of events occurring at the molecular level in the salmon liver during this developmental process, a cDNA library prepared from post-smolt salmon liver mRNA was screened with total liver cDNA probes synthesised from parr and smolts. Clones which hybridised more strongly to the smolt probe than the parr probe were chosen as candidates, for an analysis of liver gene expression implicated in seawater adaptation. Many of these cDNA clones encoded the iron binding protein transferrin. Transferrin mRNA levels were determined to be significantly higher in seawater smolt salmon than in freshwater smolts implying that transferrin may play a role in seawater adaptation.     

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.     

Life-history patterns in a southern population of Atlantic salmon

Juvenile Atlantic salmon Salmo salar in the river Esva, Asturias, north-west Spain, developed a bimodal growth pattern during their first growing season. Segregation between the two modes was apparent by late autumn. All fish in the upper modal group (UMG) grew throughout the year and migrated downstream in the following spring (by April) at the age of 14 months. Some lower modal group (LMG) fish (56% of the main stem, 31% and 50% of the two tributaries) silvered like smolts and apparently migrated downstream the same spring, although c. 1·5 months after UMG fish. Larger LMG fish appeared more likely than smaller ones to migrate. LMG fish did not grow in winter, but they grew fast between March and May. Maturing male parr were detected first at 5 months old in July, and they occurred initially among the faster growers, >6·8 cm long. In spite of this, length of maturing males did not change from late summer and throughout the winter, whereas that of non-maturing UMG and LMG fishes continued to diverge from September onwards. These findings suggest that favourable conditions for growth (very early start of a long growing season) at the southern limit of the species' range may influence the life-history pattern of this population.     

An epidemiological study of cataracts in seawater farmed Atlantic salmon Salmo salar

Cataracts in farmed Atlantic salmon have been known for many years, but the aetiology and importance of the disease have not been clarified. A cross-sectional field study of 51 cages of Atlantic salmon at 49 randomly selected sea sites was performed during the summer of 1998. The target population was spring and autumn entry groups of the 1997 generation salmon. Approximately 15 fish from each cage, altogether 777 fish, were autopsied by the same person. Each eye of the fish was scored for cataracts on a scale from 0 to 4 using an otoscope lamp with magnification. The weight and length of each fish were measured. The prevalence of cataracts was 83 % and 79% in spring entry groups and autumn entry groups, respectively. The overall mean cataract index (mean score of both eyes) was 1.23, being significantly higher in the spring entry groups (1.36) than the autumn entry groups (0.85). The final results in the spring entry groups showed that the fish groups with higher weight at sea transfer also had a higher cataract index at inspection. The risk of development of cataracts varied significantly among the offspring from the 5 strains represented in the study. Fish from sites located in 2 counties in the southern part of Norway had a significantly higher cataract index than fish farmed in the northernmost county in the study. For the autumn entry groups none of the explanatory variables was significant. In the spring entry groups a significant negative relationship was observed between the cataract score and the weight of the fish at the time of inspection (Pearson's r = -0.17), while the corresponding correlation for the autumn released groups was r = -0.10. Among the spring entry groups the average weight of the fish with the highest cataract score was estimated to about a third of the weight of the fish with no visible cataracts.     

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.     

mRNA expression of antioxidant enzymes (SOD, CAT and GSH-Px) and lipid peroxidative stress in liver of Atlantic salmon (Salmo salar) exposed to hyperoxic water during smoltification

The mRNA levels of three antioxidant genes, Cu/Zn superoxide dismutase (SOD), catalase (CAT) and phospholipid hydroperoxide glutathione peroxidase (GSH-Px), were quantified with real-time qRT-PCR in liver of Atlantic salmon Salmo salar exposed to 80% (normoxia), 105% and 130% O2 saturation for 54 days. The salmon were then translocated and exposed to 90% and 130% O2 saturation for additional 72 days during smoltification. TBARS and vitamin E levels in liver and the levels of oxidized glutathione (GSSG), total glutathione (GSH) and the resulting oxidative stress index (OSI) in blood were quantified as traditional oxidative stress markers. No significant mean normalized expression (MNE) differences of SOD, CAT or GSH-Px were found in liver after hyperoxia exposure at the two sampling times. Significantly decreased OSI was found in smolt exposed to 130% O2 saturation after 126 days (n = 18, P < 0.0001), indicating hyperoxia-induced oxidative stress. No effects were seen on growth, or on the levels of thiobarbituric reactive substances (TBARS) and vitamin E in liver after the exposure experiment. Overall, the mRNA expression of SOD, CAT and GSH-Px in liver related poorly with the hyperoxic exposure regimes, and more knowledge are needed before the expressed levels of these antioxidant genes can be applied as biomarkers of hyperoxia in Atlantic salmon.     

Replication banding patterns in Atlantic salmon (Salmo salar).

Replication banding patterns have been obtained from in vivo treatment of Salmo salar using a modification of the 5-BrdU technique and in kidney cultures using the FPG staining method. Most of the chromosome pairs were identified in the karyotype based on the banding pattern, chromosome size, and centromere position. C-banding and replication banding patterns were compared.     

Changes in body composition associated with smoltification and premature transfer to seawater in Coho salmon (Oncorhynchus kisutch) and King salmon (O. tschawytscha)

Serum protein, glucose and fat levels were determined for Coho salmon parrs, freshwater smolts, seawater smolts, seawater stunts and freshwater desmolts. Liver and muscle glycogen, fat, protein and water concentrations were also calculated. Serum protein, glucose and fat levels were significantly lower in the freshwater smolts than in the parrs. Furthermore, both liver and muscle total fat levels were markedly decreased in the smolts, suggesting that smoltification is associated with increased catabolism. Smolts that failed to reach seawater by late summer reverted to a parr-like appearance and also regained the biochemical characteristics of parrs (high body fat and glycogen). Premature transfer of Coho presmolts into seawater caused impaired growth (stunting). Stunted Coho had higher muscle protein levels than normal smolts and parrs. Tissue water levels were not significantly different between stunts and normal seawater smolts, suggesting that the stunting phenomenon may not be caused primarily by osmoregulatory failure but may be due to shifts in metabolic patterns. In contrast to the pronounced changes seen in Coho salmon, transfer of King salmon to sea water did not result in increased catabolism of body reserves or in the production of stunts.     

Physiological seawater adaptation in juvenile Atlantic salmon (Salmo salar) autumn migrants

Summary

• 1 About 25 % of juvenile Atlantic salmon (Salmo salar) migrating downstream in the River Frome in southern England do so in the autumn rather than in the spring. Here, we examine the physiological status of these fish with regard to those features that adapt them to sea water during the parr–smolt transformation (i.e. gill Na⁺K⁺ ATPase activity; the number, size and type of chloride cells on the gill lamellae; salinity tolerance and relative plasma thyroid levels).
• 2 Autumn migrants, and those fish which subsequently reside in the tidal reaches during the winter, are not sufficiently physiologically adapted to permit permanent or early, entry into the marine environment.
• 3 It is not known what proportion of autumn migrating fish survive and return to spawn as adults. If significant numbers do return, however, the production from tidal reach habitats must be taken into account in the development of salmon stock management strategies, especially monitoring and assessment programmes, and in the evaluation of factors affecting stocks.