Damaging effect of the fish pathogen <Emphasis Type="Italic">Aeromonas salmonicida</Emphasis> ssp. <Emphasis Type="Italic">salmonicida</Emphasis> on intestinal enterocytes of Atlantic salmon (<Emphasis Type="Italic">Salmo salar</Emphasis> L.)

In fish, bacterial pathogens can enter the host by one or more of three different routes: (a) skin, (b) gills and (c) gastrointestinal tract. Bacteria can cross the gastrointestinal lining in three different ways. In undamaged tissue, bacteria can translocate by transcellular or paracellular routes. Alternatively, bacteria can damage the intestinal lining with extracellular enzymes or toxins before entering. Using an in vitro (Ussing chamber) model, this paper describes intestinal cell damage in Atlantic salmon (Salmo salar L.) caused by the fish pathogen Aeromonas salmonicida ssp. salmonicida, the causative agent of furunculosis. The in vitro method clearly demonstrated substantial detachment of enterocytes from anterior region of the intestine (foregut) upon exposure to the pathogen. In the hindgut (posterior part of the intestine), little detachment was observed but cellular damage involved microvilli, desmosomes and tight junctions. Based on these findings, we suggest that A. salmonicida may obtain entry to the fish by seriously damaging the intestinal lining. Translocation of bacteria through the foregut (rather than the hindgut) is a more likely infection route for A. salmonicida infections in Atlantic salmon.Financial support from the Commission of the European Communities, quality of Life and Management of Living Resources programme, project Q5RT-2000-31656 Gastrointestinal Functions and Food Intake Regulation in Salmonids: Impact of Dietary vegetable Lipids (GUTINTEGRITY) and from Magnus Bergvalls Stiftelse for KS, is acknowledged.This work does not represent the opinion of the European Community, which is thus not responsible for any use of the data presented.     

Tolbutamide hydroxylation by hepatic microsomes from Atlantic salmon (<Emphasis Type="Italic">Salmo salar</Emphasis> L.)

Metabolic transformations of two substrates for human cytochrome P450 (CYP450) 2C9, tolbutamide and diclofenac, were investigated in hepatic microsomes from Atlantic salmon (Salmo salar L.). Tolbutamide hydroxylation followed Michaelis–Menten kinetics. Mean apparent Michaelis–Menten constant (K_m) and maximum reaction velocity (V_max) values for 4-hydroxytolbutamide (TBOH) formation were 0.09 ± 0.031 mM and 49.5 ± 6.03 pmol/min/mg, respectively. Addition of sulfaphenazole, an inhibitor for mammalian CYP2C9, in a range from 1 to 200 μM decreased formation of TBOH in a concentration-dependent manner, but not to 50%. Neither fluconazole, an inhibitor of human CYP2C9, nor ketoconazole, inhibitor of CYP1A and CYP3A in fish, affected TBOH formation. In contrast ellipticine, an inhibitor of CYP1A in fish inhibited TBOH formation with the IC₅₀ value of 12.1 μM. The rate of TBOH formation was competitively inhibited by 100 μM of sesamin in the incubations, but the degree of inhibition did not increase with increased sesamin concentration. Ethoxyresorufin hydroxylase (EROD) activity was inhibited by tolbutamide in a non-competitive manner (inhibition constant K_i = 218 μM). Our data suggest that tolbutamide is metabolized by salmon microsomes with formation of TBOH. CYP1A might be involved in this reaction as suggested by decreased TBOH formation in the presence of ellipticine and decreased EROD activity in the presence of tolbutamide. Incubation of diclofenac with the microsomes yielded no metabolite formation, suggesting that salmon does not possess diclofenac-metabolizing activity.     

Feeding of wild and hatchery reared Atlantic salmon (<Emphasis Type="Italic">Salmo salar</Emphasis> L.) smolts during downstream migration

In general, hatchery salmonid smolts experience higher mortality during migration than wild smolts, which is suggested to be due to domestication effects and that hatchery fish lack experience of the natural environment. However, possible differences in feeding during smolt migration between hatchery and wild smolts have rarely been addressed. We compared the number of feeding smolts and stomach fullness among wild Atlantic salmon smolts, hatchery-reared smolts released as 1-year-old parr, and hatchery-reared smolts released as 2-year-old smolts during their descent to sea in River Tornionjoki. In addition, estimations of prey selection among the smolt groups were conducted. A high proportion of wild smolts and smolts stocked as parr actively fed during the smolt migration. A lower proportion of smolts stocked as smolts was feeding and their stomach fullness were much reduced in comparison with the two other groups. The study also indicated that the feeding of migrating smolts is selective rather than opportunistic. In conclusion, this study suggests that stocked 2-year-old smolts may enter sea with an inferior foraging behaviour and it is a possibility that this may contribute to the observed low post-smolt survival in the Baltic Sea.     

Reduced anti-predator responses in multi-generational hybrids of farmed and wild Atlantic salmon (<Emphasis Type="Italic">Salmo salar</Emphasis> L.)

Cultured organisms undergo genetically-based behavioural changes that may reduce their ability to survive in the wild. This has raised concerns that interbreeding between escaped cultured and wild organisms will generate hybrids exhibiting maladaptive behaviours which may ultimately reduce the fitness of the wild counterpart. We compared anti-predator responses in Atlantic salmon (Salmo salar) from two wild North American populations, the major farmed strain used in regional aquaculture, and their wild-farmed hybrids (F₁, F₂, and wild backcross). Anti-predator responses of fry (age 0+ parr) were measured under common environmental conditions, using a model of a natural predator (belted kingfisher, Ceryle alcyon). Farmed fry exhibited significantly reduced anti-predator responses relative to fry from both wild populations. The anti-predator responses of wild-farmed hybrid fry were intermediate to those of the parental populations (pure farmed or wild). The magnitude by which wild-farmed hybrids differed in anti-predator responses from pure wild fish also depended on the wild population. These results suggest that: (1) the observed behavioural differences have a genetic basis; (2) wild-farmed hybrids have, on average, reduced anti-predator responses relative to wild fish; and that (3) the effects of wild-farmed interbreeding on anti-predator responses will differ between wild populations. Our study is consistent with the general hypothesis that continual farmed-wild interbreeding may have detrimental effects on the fitness of wild organisms.     

Sequencing the genome of the Atlantic salmon (<Emphasis Type="Italic">Salmo salar</Emphasis>)

The International Collaboration to Sequence the Atlantic Salmon Genome (ICSASG) will produce a genome sequence that identifies and physically maps all genes in the Atlantic salmon genome and acts as a reference sequence for other salmonids.     

An extended panel of single nucleotide polymorphisms in Atlantic salmon (<Emphasis Type="Italic">Salmo salar</Emphasis> L.) mitochondrial DNA

A total of 6,324 base pairs, distributed on eight different regions of the mitochondrial DNA (mtDNA) were re-sequenced for 32 specimens of Norwegian farmed and wild Atlantic salmon. A total of 16 new and three previously reported single nucleotide polymorphisms (SNPs) were detected. These SNPs were validated using 240 farm and 119 wild Norwegian salmon. Alignments of sequences obtained in this study and previously published sequences revealed another 15 previously unreported SNPs. The panel of mitochondrial SNPs detected in the present study will, in combination with previously identified SNPs, prove useful in designing efficient assays for analyzes of mtDNA variation.     

Genetic markers in population studies of Atlantic salmon <Emphasis Type="Italic">Salmo salar</Emphasis> L.: Analysis of DNA sequences

The review, which consist of two parts, summarizes literature data on all genetic markers used in population studies of Atlantic salmon. The second part of the review concerns analysis of DNA sequences; fragments of known genes, anonymous genome sequences, mini-and microsatellites, mitochondrial DNA. The main results of studies of the Atlantic salmon gene poll using DNA markers are discussed. Most of the markers examined in certain conditions may be under selection. The resolution power of various methods of DNA analysis and the fields of their use are considered in reference to Atlantic salmon.     

Etho-biochemical mechanisms of early differentiation in juveniles of the Atlantic salmon <Emphasis Type="Italic">Salmo salar</Emphasis>

The social status of hatchery-reared juveniles of Atlantic salmon Salmo salar at the age of 11–19 months in conditions of lack of shelters was investigated experimentally. It was demonstrated that biochemical differentiation in such juveniles begins at the age of 13 months—one month before the first differences in the social status of fish and four months prior to the beginning of smoltification. The dominants occupy bottom areas with shelters and extrude the subordinates to the water column. The subordinates undergo smoltification or die from aggression of the dominants. Concentrations of dopamine, noradrenalin, and of their metabolites in the brain of fish in the beginning of smoltification were higher in the dominants and, by the end of smoltification, were higher in smolts. The social behavior of fish is considered to be one of the ethological mechanisms of differentiation of salmon juveniles into smolts and parr.     

Deep-diving of Atlantic salmon (<Emphasis Type="Italic">Salmo salar</Emphasis>) during their marine feeding migrations

Data from seven data storage tags recovered from Atlantic salmon marked as smolts were analyzed for depth movements and patterns of deep diving during the marine migration. The salmon mostly stayed at the surface and showed diurnal activity especially from autumn until spring. During the first months at sea the salmon stayed at shallower depths (<100 m). The salmon took short deep dives (>100 m), that were rare or absent during the first summer at sea but increased in frequency and duration especially in late winter. The maximum depth of the dives varied from 419 to 1187 m. Most of dives were short, (<5 h) but could last up to 33 h. The duration of dives increased in late winter until spring and the overall depth and maximum depth per dive increased exponentially over time. The initiation of the dives was more common in evenings and at night, suggesting nocturnal diving. We hypothesized that deep diving is related to feeding of salmon as mesopelagic fish can be important food for salmon during winter.     

Histological changes in intestine of Atlantic salmon (<Emphasis Type="Italic">Salmo salar</Emphasis> L.) following in vitro exposure to pathogenic and probiotic bacterial strains

Furunculosis and vibriosis are diseases that cause severe economic losses in the fish-farming industry. The foregut of the Atlantic salmon (Salmo salar L.) was exposed in vitro to two fish pathogens, Aeromonas salmonicida (causative agent of furunculosis) and Vibrio anguillarum (causative agent of vibriosis), and to one probiotic strain, Carnobacterium divergens, at 6 × 10⁴ or 6 × 10⁶ viable bacteria per milliliter. Histological changes following bacterial exposure were assessed by light and electron microscopy. Control samples (foregut exposed to Ringer’s solution only) and samples exposed only to C. divergens had a similar appearance to intact intestinal mucosal epithelium, with no signs of damage. However, exposure of the foregut to the pathogenic bacteria resulted in damaged epithelial cells, cell debris in the lumen, and disorganization of the microvilli. Co-incubation of the foregut with a pathogen and C. divergens did not reverse the damaging effects caused by the pathogen, although these were alleviated when probiotic bacteria were used. Based on these results, we suggest that the probiotic bacterium, C. divergens, is able to prevent, to some extent, pathogen-induced damage in the Atlantic salmon foregut. I. Salinas thanks Fundacion Seneca for a PhD studentship.     

Development of ethologo-physiological differentiation between parrs and smolts of the Atlantic salmon <Emphasis Type="Italic">Salmo salar</Emphasis>

Dynamics of the level of monoamines and their metabolites in the brain of juveniles of the Atlantic salmon Salmo salar is investigated in the initial period of differentiation of the progeny into parrs and smolts. The first differences in the level of metabolism of noradrenaline (NA) and 5-HT arise in the optical tectum of the brain before the beginning of smolting. Appearance of the first signs of smolting in juveniles is accompanied by the increase of differences in the level of activity of HA-and 5-HT-ergic systems in the forebrain and hypothalamus. The first differences in the activity of the DAergic system between parrs and smolts were observed in the period of visible differences in fish coloration. In the same period, considerable differences in the level of adrenaline and noradrenaline in the fish blood were recorded. In experiments on a prolonged exposure of fish to a current of different intensity, it was shown that behavior and physiological response of the fish organism are different: in parrs, the stressogenous response is developed, they stop feeding, weight loss takes place, and they cannot withstand the water flow; smolts adapt to the current using heterogeneity of water movements.     

Molecular faunistics of accidental infections of <Emphasis Type="Italic">Gyrodactylus</Emphasis> Nordmann, 1832 (Monogenea) parasitic on salmon <Emphasis Type="Italic">Salmo salar</Emphasis> L. and brown trout <Emphasis Type="Italic">Salmo trutta</Emphasis> L. in NW Russia

Salmon Salmo salar L. and brown trout S. trutta L. juveniles were examined for the presence of accidental monogenean ectoparasitic species of Gyrodactylus Nordmann, 1832 in the Baltic and White Sea basins of Russian Karelia in order to estimate the frequency of host-switching attempts on an ecological timescale. To collect phylogeographical information and for exact species identification, the parasites were characterised by nuclear internal transcribed spacer sequences of rDNA (ITS) and, for some species, also by their mitochondrial DNA (CO1 gene) sequences. Four accidental Gyrodactylus species were observed on salmon and brown trout. A few specimens of G. aphyae Malmberg, 1957, the normal host of which is the Eurasian minnow Phoxinus phoxinus (L.), were observed on lake salmon from the Rivers Kurzhma (Lake Kuito, White Sea basin) and Vidlitsa (Lake Ladoga, Baltic basin). G. lucii Kulakovskaya, 1952, a parasite of the northern pike Esox lucius L., was observed on salmon in the Kurzhma. In the River Vidlitsa, two specimens of G. papernai Ergens & Bychowsky, 1967, normally on stone loach Barbatula barbatula (L.), were found on salmon. On anadromous White Sea salmon in the River Pulonga in Chupa Bay, a few salmon parr carried small colonies of G. arcuatus Bychowsky, 1933, which were shown to have originated from the local three-spined stickleback Gasterosteus aculeatus L. consumed as prey. No specimens of Gyrodactylus salaris Malmberg, 1957 were observed, although the Pulonga is the nearest salmon spawning river to the River Keret', which is heavily infected with introduced G. salaris. In the River Satulinoja, Lake Ladoga, three specimens of G. lotae Gusev, 1953, from burbot Lota lota (L.), were collected from a single brown trout S. trutta. All nonspecific gyrodactylid infections on salmonids were judged to be temporary, because only a few specimens were observed on each of the small number of infected fishes. The prevalence of endemic G. salaris was also low, only 1% (Nfish = 296) in Lake Onega and 0.7% (Nfish = 255) in Lake Ladoga, while brown trout specific Gyrodactylus species were not observed on any of the 429 trout examined from the Ladoga basin. The host-specific and unspecific burden of Gyrodactylus spp. on these 'glacial relict' populations of salmon and brown trout was very low, suggesting a generalised resistance against the co-evolved freshwater parasite community, or some kind of 'vaccination' effect. These hypotheses deserve further testing.     

Dynamics of lipid content during early development of freshwater salmon <Emphasis Type="Italic">Salmo salar</Emphasis> L.

Dynamics of lipid and phospholipid content was studied during early development of freshwater salmon Salmo salar L. from blastodisc formation (3 h) to hatching (108 days) as well as in eggs before fertilization. High and stable content of total lipids including structural phospholipids as well as relatively high content of triglycerides and its slight increase at the time of hatching have been demonstrated, which can indicate their utilization as the main energy source after hatching under conditions of deficient food and low fry activity for some time. Accumulation of a certain level of lipids in eggs before spawning is required for embryonic development and high survival after hatching. The significance of increasing and decreasing levels of structural lipids modulating membrane enzyme activities in metabolic changes before hatching is discussed.     

Protein degradation in the skeletal muscles of parrs and smolts of the Atlantic salmon <Emphasis Type="Italic">Salmo salar</Emphasis> L.

This paper reports a study of the activity of calcium-dependent proteases (calpains) and proteasome in the skeletal muscles of parrs and smolts of the Atlantic salmon Salmo salar L. from the Indera River (the Kola Peninsula). It was demonstrated that the growth rate and protein mass accumulation during smoltification in salmonid fishes are inversely related to the activity of proteases in their muscles. It was noted that decreased protein degradation in the skeletal muscles of salmon juveniles serves as a mechanism ensuring the intensive growth and physiological maturity necessary for migration from the river to the sea.     

Molecular Analysis of Microbiota Along the Digestive Tract of Juvenile Atlantic Salmon (<Emphasis Type="Italic">Salmo salar</Emphasis> L.)

Dominant bacterial microbiota of the gut of juvenile farmed Atlantic salmon was investigated using a combination of molecular approaches. Bacterial community composition from the stomach, the pyloric caeca, and the intestine was assessed by extracting DNA directly from each gut compartment. Temporal temperature gradient gel electrophoresis (TTGE) analysis of 16S ribosomal DNA (rDNA) amplicons showed very similar bacterial compositions throughout the digestive tract. Band sequencing revealed a narrow diversity of species with a dominance of Pseudomonas in the three compartments. However, cloning revealed more diversity among the Pseudomonas sequences. To confirm these results, we analyzed the bacterial community by amplifying the variable 16S–23S rDNA intergenic spacer region (ITS). Similar ITS profiles were observed among gastrointestinal compartments of salmon, confirming the TTGE results. Moreover, the dominant ITS band at 650 bp, identified as Pseudomonas, was observed in the ITS profile from fish collected in two seasons (July 2003 and 2004). In contrast, aerobic culture analysis revealed Shewanella spp. as the most prevalent isolate. This discrepancy was resolved by evaluating 16S rDNA and ITS polymerase chain reaction amplification efficiency from both Shewanella and Pseudomonas isolates. Very similar efficiencies were observed in the two bacteria. Hence, this discrepancy may be explained by preferential cultivation of Shewanella spp. under the experimental conditions. Also, we included analyses of pelleted feed and the water influent to explore environmental influences on the bacterial composition of the gut microbiota. Overall, these results indicate a homogeneous composition of the bacterial community composition along the gastrointestinal tract of reared juvenile salmon. This community is mainly composed of Pseudomonas spp., which could be derived from water influent and may be selectively associated with salmon in this hatchery.     

Influence of shelters and food resources on smoltification of juveniles of the Atlantic salmon <Emphasis Type="Italic">Salmo salar</Emphasis>

Influence of feeding conditions and of the presence of a various number of shelters in basins on development of differentiation of juveniles of Salmo salar at the age 1+ and formation of smolts were studied under experimental conditions. The experiments had been carried out on groups of ten fish. The influence of investigated factors turned out to be significant. Under normal feeding four times a day, the quantity of formed smolts depends on the number of shelters: the more shelters that are available, the fewer smolts appear in groups. The presence of one shelter is a strong stimulant enhancing aggressive activity towards conspecific individuals, resulting in destruction of some fish in the group. Under feeding once a day, i.e., under food shortage, more fish perished, and the number of smolts decreased as the fish did not accumulate a sufficient energy potential for the morphophysiological transformation. One of the potential mechanisms of development of differentiation of fish juveniles into parrs and future smolts is discussed, based on domination of some fish possessing the individual territory and exclusion of subdominant specimens into open water as a result of social interaction.