Embracing prospects for reducing the numbers of animals used in aquaculture research.

The principles of three Rs-REPLACEMENT, REDUCTION, and REFINEMENT-govern the protection and use of animals, including fish, for research purposes in the European Union and Norway. In this paper, we discuss some straightforward steps to simplify the delivery of these principles at the idea stage and adapt some of these examples for conducting fish trials related to health and welfare. Although some of the approaches are well established in other animal science arenas, we believe there can be a timely recap of their key facets. We discuss a number of simple strategies to emphasize how a reduction in fish numbers can be achieved from initial project conception to implementation, highlighting not only their advantages but also their limitations. We also highlight the role that funding agencies can play in the implementation of the 3R principles in aquaculture research. These simple points can be used in frameworks to initiate a broader and dynamic intersectoral dialogue among stakeholders of aquaculture research on how to promote ethics and embrace opportunities for this within the tenets of the 3Rs.

Nasal responses to elevated temperature and Francisella noatunensis infection in Atlantic cod (Gadus morhua).

We report the histological and transcriptomic changes in the olfactory organ of Atlantic cod exposed to Francisella noatunensis. Experimental infection was performed at either 12 °C or 17 °C. Infected fish presented the classic gross pathologies of francisellosis. Nasal morpho-phenotypic parameters were not significantly affected by elevated temperature and infection, except for the number of mucus cells in the 12 °C group seven weeks after the challenge. A higher number of genes were altered through time in the group reared at 17 °C. At termination, the nasal transcriptome of infected fish in both groups was similar to the control. When both infected groups were compared, 754 DEGs were identified, many of which were involved in signalling, defence, transmembrane and enzymatic processes. In conclusion, the study reveals that elevated temperature could trigger responses in the olfactory organ of Atlantic cod and shape the nasal response to F. noatunensis infection.

Transcriptomic landscape of Atlantic salmon (Salmo salar L.) skin.

In this study, we present the first spatial transcriptomic atlas of Atlantic salmon skin using the Visium Spatial Gene Expression protocol. We utilized frozen skin tissue from 4 distinct sites, namely the operculum, pectoral and caudal fins, and scaly skin at the flank of the fish close to the lateral line, obtained from 2 Atlantic salmon (150 g). High-quality frozen tissue sections were obtained by embedding tissue in optimal cutting temperature media prior to freezing and sectioning. Further, we generated libraries and spatial transcriptomic maps, achieving a minimum of 80 million reads per sample with mapping efficiencies ranging from 79.3 to 89.4%. Our analysis revealed the detection of over 80,000 transcripts and nearly 30,000 genes in each sample. Among the tissue types observed in the skin, the epithelial tissues exhibited the highest number of transcripts (unique molecular identifier counts), followed by muscle tissue, loose and fibrous connective tissue, and bone. Notably, the widest nodes in the transcriptome network were shared among the epithelial clusters, while dermal tissues showed less consistency, which is likely attributable to the presence of multiple cell types at different body locations. Additionally, we identified collagen type 1 as the most prominent gene family in the skin, while keratins were found to be abundant in the epithelial tissue. Furthermore, we successfully identified gene markers specific to epithelial tissue, bone, and mesenchyme. To validate their expression patterns, we conducted a meta-analysis of the microarray database, which confirmed high expression levels of these markers in mucosal organs, skin, gills, and the olfactory rosette.

Welfare and performance of ballan wrasse (Labrus bergylta) reared at two different temperatures after a preparatory feeding trial with enhanced dietary eicosapentaenoic acid.

Concerns have long been raised about the welfare of ballan wrasse (Labrus bergylta) used for the biological control of sea lice in Atlantic salmon (Salmo salar) aquaculture. This study assessed the effect of increased dietary eicosapentaenoic acid (EPA) levels and initial condition factor (CF) on the subsequent performance and welfare of ballan wrasse farmed in high and low water temperatures. Fish were fed a diet with either commercial or high EPA levels for 3 months at 15°C. Subsequently, fish were tagged with a passive integrated transponder, measured for their CF and divided into two groups consisting of fish from both treatments and reared for 4.5 months at either 15 or 6°C fed a commercial diet. Each fish was categorized as high (≥2.7) or low CF (<2.7) fish based on the calculated average CF of the population. Dietary composition influenced the fatty acid (FA) profile of the stored lipids without affecting the growth or welfare of ballan wrasse. Fish reared at 15°C showed higher growth, more fat and energy reserves and less ash content. Fish reared at 6°C lost weight, using up their body lipids at the end of the temperature trial. Gene expression analyses showed upregulation of the positive growth marker (GHrα) and two genes involved in the synthesis and oxidation of FAs (elovl5, cpt1) and downregulation of the negative growth marker (mstn) in fish reared at 15°C compared to those reared at 6°C. Fish reared at 6°C showed upregulated levels of il-6 compared to those reared at 15°C, suggesting an enhanced immune reaction in response to low temperature. Fish with high CF showed better survival, growth and performance compared to those with low CF. External welfare scoring showed higher prevalence and severity in emaciation, scale loss and the sum index score (of all measured welfare parameters) in fish reared at 6°C compared to those reared at 15°C and better welfare in fish with high CF compared to those with low CF. Histological examination of the skin showed that fish reared at 6°C had decreased epidermal thickness, a lower overall number of mucous cells in the inner and outer epidermis and a different organization of mucous cells compared to fish reared at 15°C, indicating stress in fish reared at 6°C. Overall, low water temperatures had profound effects on the performance and external and internal welfare parameters of ballan wrasse and can be considered a stressor likely affecting the delousing efficacy. These findings support the seasonal use of different cleaner fish species. High CF, but not increased dietary EPA levels, appeared to help fish cope better with low water temperatures and should thus be assessed and considered before deploying them in salmon cages.

Long-Term Influence of PCB- and PBDE-Spiked Microplastic Spheres Fed through Rotifers to Atlantic Cod (Gadus morhua) Larvae.

Omnipresent microplastics (MPs) in marine ecosystems are ingested at all trophic levels and may be a vector for the transfer of persistent organic pollutants (POPs) through the food web. We fed rotifers polyethylene MPs (1-4 µm) spiked with seven congeners of polychlorinated biphenyls (PCBs) and two congeners of polybrominated diphenyl ethers (PBDEs). In turn, these rotifers were fed to cod larvae from 2-30 days post-hatching (dph), while the control groups were fed rotifers without MPs. After 30 dph, all the groups were fed the same feed without MPs. Whole-body larvae were sampled at 30 and 60 dph, and four months later the skin of 10 g juveniles was sampled. The PCBs and PBDEs concentrations were significantly higher in MP larvae compared to the control larvae at 30 dph, but the significance dissipated at 60 dph. Expression of stress-related genes in cod larvae at 30 and 60 dph showed inconclusive minor random effects. The skin of MP juveniles showed disrupted epithelial integrity, fewer club cells and downregulation of a suite of genes involved in immunity, metabolism and the development of skin. Our study showed that POPs were transferred through the food web and accumulated in the larvae, but that the level of pollutants decreased once the exposure was ceased, possibly related to growth dilution. Considering the transcriptomic and histological findings, POPs spiked to MPs and/or MPs themselves may have long-term effects in the skin barrier defense system, immune response and epithelium integrity, which may potentially reduce the robustness and overall fitness of the fish.

Investigation of the functions of n-3 very-long-chain PUFAs in skin using in vivo Atlantic salmon and in vitro human and fish skin models.

The purpose of this study was to investigate the effect of dietary n-3 very-long-chain PUFA (n-3 VLC-PUFA) on the maturation and development of skin tissue in juvenile Atlantic salmon (Salmo salar) in vivo, as well as their effects on skin keratocyte and human skin fibroblast cell migration in vitro. Atlantic salmon were fed different dietary levels of n-3 VLC-PUFA from an initial weight of 6 g to a final weight of 11 g. Changes in skin morphology were analysed at two time points during the experiment, and the effects on skin tissue fatty acid composition were determined. Additionally, in vitro experiments using human dermal fibroblasts and primary Atlantic salmon keratocytes were conducted to investigate the effect of VLC-PUFA on the migration capacity of the cells. The results demonstrated that increased dietary levels of n-3 VLC-PUFA led to an increased epidermis thickness and more rapid scale maturation in Atlantic salmon skin in vivo, leading to a more mature skin morphology, and possibly more robust skin, at an earlier life stage. Additionally, human skin fibroblasts and salmon skin keratocytes supplemented with n-3 VLC-PUFA in vitro showed more rapid migration, indicating potentially beneficial effects of VLC-PUFA in wound healing. In conclusion, VLC-PUFA may have beneficial effects on skin tissue development, function and integrity.

The skin mucosal barrier of lumpfish (Cyclopterus lumpus L.) is weakened by exposure to potential aquaculture production-related stressors.

Various cleaner fish species, such as the lumpfish (Cyclopterus lumpus L.), are used in the sea cage production of Atlantic salmon (Salmo salar L.) as a control measure against the ectoparasitic salmon louse (Lepeophtheirus salmonis). Nonetheless, during severe lice infestation, alternative treatments are required to control parasitic burden. The aim of this study was to gain insight into how lumpfish skin responds to different chemicals used to treat parasites. The authors collected skin from lumpfish from both research facilities (tank-reared fish) and commercial production (cage-reared fish) and used operational welfare indicators, in vitro models, histology and transcriptomics to study how the skin responded to two anti-parasitic oxidative chemicals, hydrogen peroxide (H2 O2 ) and peracetic acid. Lumpfish sampled from the farm were classified as clinically healthy or weak based on their morbidity status, and fish from each category were used to gain insight into how the therapeutics affect the skin barrier. Differences between healthy and weakened (moribund) fish, and between treated fish from each of the two groups, were observed. Histological examination showed an overall reduced skin quality in fish characterized as moribund, including different grades of exposed bony plates. In vitro oxidant-treated lumpfish skin had reduced the migration capacity of keratocytes, a weakened epidermal barrier, and altered gene transcription, changes that are known predisposing factors to secondary infections. Skin from non-treated, healthy fish sampled from commercial farms exhibited similar features and attributes to oxidant-exposed tank-reared fish from a research facility, suggesting that apparently healthy cage-held lumpfish exhibited stress responses in the epidermal barrier. The results of the study outline the risks and consequences lumpfish can face if accidentally subjected to potential anti-parasitic oxidant treatments aimed at Atlantic salmon. It also strengthens the evidence behind the requirement that lumpfish should be removed from the cages before being potentially exposed to this type of treatment and outlines the potential risks of differing husbandry practices upon lumpfish health, welfare and resilience.

PCB-126 spiked to polyethylene microplastic ingested by juvenile Atlantic cod (Gadus morhua) accumulates in liver and muscle tissues.

In the present study, polyethylene (PE) microplastics (150-300 µm) were added to Atlantic cod (Gadus morhua) feeds at 1 %, either in their present form (Virgin PE) or spiked with PCB-126 (Spiked PE). The feeds were given to juvenile cod for a 4-week period. The fish grew from 11 to 23 g with no significant difference between dietary treatments. Cod fed spiked PE showed a significantly higher concentration of PCB-126 in liver and muscle samples compared to control and fish ingesting virgin PE. In accordance with the accumulation of PCB-126 in the liver, the expression of hepatic cyp1a was higher in cod fed spiked PE. Notably, we observed that spiked PE, as well as virgin PE, have an effect on skin. Overall changes indicated a reduced skin barrier in fish fed a diet containing PE. Indicating that PE itself through interaction with gut tissue may influence skin health in fish.

Transcriptome Responses of Atlantic Salmon (Salmo salar L.) to Viral and Bacterial Pathogens, Inflammation, and Stress.

Transcriptomics provides valuable data for functional annotations of genes, the discovery of biomarkers, and quantitative assessment of responses to challenges. Meta-analysis of Nofima's Atlantic salmon microarray database was performed for the selection of genes that have shown strong and reproducible expression changes. Using data from 127 experiments including 6440 microarrays, four transcription modules (TM) were identified with a total of 902 annotated genes: 161 virus responsive genes - VRG (activated with five viruses and poly I:C), genes that responded to three pathogenic bacteria (523 up and 33 down-regulated genes), inflammation not caused by infections - wounds, melanized foci in skeletal muscle and exposure to PAMP (180 up and 72 down-regulated genes), and stress by exercise, crowding and cortisol implants (33 genes). To assist the selection of gene markers, genes in each TM were ranked according to the scale of expression changes. In terms of functional annotations, association with diseases and stress was unknown or not reflected in public databases for a large part of genes, including several genes with the highest ranks. A set of multifunctional genes was discovered. Cholesterol 25-hydroxylase was present in all TM and 22 genes, including most differentially expressed matrix metalloproteinases 9 and 13 were assigned to three TMs. The meta-analysis has improved understanding of the defense strategies in Atlantic salmon. VRG have demonstrated equal or similar responses to RNA (SAV, IPNV, PRV, and ISAV), and DNA (gill pox) viruses, injection of bacterial DNA (plasmid) and exposure of cells to PAMP (CpG and gardiquimod) and relatively low sensitivity to inflammation and bacteria. Genes of the highest rank show preferential expression in erythrocytes. This group includes multigene families (gig and several trim families) and many paralogs. Of pathogen recognition receptors, only RNA helicases have shown strong expression changes. Most VRG (82%) are effectors with a preponderance of ubiquitin-related genes, GTPases, and genes of nucleotide metabolism. Many VRG have unknown roles. The identification of TMs makes possible quantification of responses and assessment of their interactions. Based on this, we are able to separate pathogen-specific responses from general inflammation and stress.

Wound healing in post-smolt Atlantic salmon (Salmo salar L.).

Skin biopsies (5 mm) taken from behind the dorsal fin on Atlantic salmon post-smolts were followed over a 2 month period. The healing process was dominated by hemostasis, acute inflammation, and epidermal repair the first 14 days post wounding (dpw), as shown through imaging, histological evaluation, and transcriptomics. Most of the immune genes showed decreased expression after two weeks, approaching the levels of intact skin, as also reflected in sections where reduced inflammation in the wound bed was observed. Transcriptional events suggest recruitment of lymphocytes to the wound site during the acute phase, with activation of humoral responses from 14 dpw and onward. From the histology, a more adherent mucus was observed that correlated with altered transcription of glycosyltransferases. This may indicate different properties and functions of the mucus during the wound healing process. Wound contraction started between 14 and 36 dpw. The occurrence of these events was concurrent with granulation tissue formation, melanocyte migration and up-regulation of genes involved in extracellular matrix formation. The presented description of the wound healing processes in Atlantic salmon gives insight into comparative ulcerative biology in mammals and fish and provides both novel and updated knowledge that can be applied for improved best operational practices for fish welfare in aquaculture.

Preparation and Culturing of Atlantic Salmon Muscle Cells for In Vitro Studies.

This chapter outlines methods for isolating myosatellites from Atlantic salmon (Salmo salar), how to keep them in culture and differentiate them into mature myocytes. The protocol further describes how to trans-differentiate the myocytes into osteoblasts (bone cells).

High fish density delays wound healing in Atlantic salmon (Salmo salar).

In this study, we look closer at how high fish densities influence wound repair mechanisms in post-smolt Atlantic salmon. The fish were wounded with a 5 mm skin punch biopsy needle and stocked at two different densities, a high fish density (100 kg/m3) treatment and a low fish density treatment (20 kg/m3) serving as the control. The healing wounds were followed for 57 days with samples taken 1, 3, 7, 14, 36, 43 and 57 days post wounding. The transcriptomic analysis suggests that high fish density enhance inflammation and represses cell proliferation, tissue secretion and collagen synthesis in the healing wounds. The histological analysis further showed delayed epidermal and dermal repair in the high fish density treatment compared to control. The overall wound contraction was also altered by the treatment. In conclusion, high fish density enhances immune responses and delay tissue repair, which ultimately results in delayed wound healing.

Atlantic salmon skin barrier functions gradually enhance after seawater transfer.

Atlantic salmon farming operates with high production intensities where skin integrity is recognized as a central factor and indicator for animal health and welfare. In the described trial, the skin development and its immune status in healthy Atlantic salmon reared in two different systems, a traditional open net-pen system and a semi-closed containment system, were investigated. Freshwater smolts were compared to post-smolts after 1 and 4 months in seawater. Growth performance, when adjusted for temperature, was equal between the systems. Skin analyses, including epidermis and dermis, showed that thickness and mucus cell numbers increased in pace with the growth and time post seawater transfer (PST). Gene expression changes suggested similar processes with development of connective tissue, formation of extracellular matrix and augmented cutaneous secretion, changes in mucus protein composition and overall increased immune activity related to gradually enforced protection against pathogens. Results suggest a gradual morphological development in skin with a delayed recovery of immune functions PST. It is possible that Atlantic salmon could experience increased susceptibility to infectious agents and risk of diseases during the first post-smolt period.

Genome-wide analysis of Atlantic salmon (Salmo salar) mucin genes and their role as biomarkers.

The aim of this study was to identify potential mucin genes in the Atlantic salmon genome and evaluate tissue-specific distribution and transcriptional regulation in response to aquaculture-relevant stress conditions in post-smolts. Seven secreted gel-forming mucin genes were identified based on several layers of evidence; annotation, transcription, phylogeny and domain structure. Two genes were annotated as muc2 and five genes as muc5. The muc2 genes were predominantly transcribed in the intestinal region while the different genes in the muc5 family were mainly transcribed in either skin, gill or pyloric caeca. In order to investigate transcriptional regulation of mucins during stress conditions, two controlled experiments were conducted. In the first experiment, handling stress induced mucin transcription in the gill, while transcription decreased in the skin and intestine. In the second experiment, long term intensive rearing conditions (fish biomass ~125 kg/m3) interrupted by additional confinement led to increased transcription of mucin genes in the skin at one, seven and fourteen days post-confinement.

Precursor cells from Atlantic salmon (Salmo salar) visceral fat holds the plasticity to differentiate into the osteogenic lineage.

In order to study the potential plasticity of Atlantic salmon (Salmo salar) precursor cells (aSPCs) from the adipogenic mesenchyme cell lineage to differentiate to the osteogenic lineage, aSPCs were isolated and cultivated under either osteogenic or adipogenic promoting conditions. The results strengthen the hypothesis that aSPCs most likely are predestined to the adipogenic lineage, but they also hold the flexibility to turn into other lineages given the right stimuli. This assumption is supported by the fact that the transcription factor pparγ , important for regulation of adiopogenesis, was silent in aSPCs grown in osteogenic media, while runx2, important for osteogenic differentiation, was not expressed in aSPCs cultivated in adipogenic media. After 2 weeks in osteogenic promoting conditions the cells started to deposit extracellular matrix and after 4 weeks, the cells started mineralizing secreted matrix. Microarray analyses revealed large-scale transcriptome responses to osteogenic medium after 2 days, changes remained stable at day 15 and decreased by magnitude at day 30. Induction was observed in many genes involved in osteogenic differentiation, growth factors, regulators of development, transporters and production of extracellular matrix. Transcriptome profile in differentiating adipocytes was markedly different from differentiating osteoblasts with far fewer genes changing activity. The number of regulated genes slowly increased at the mature stage, when adipocytes increased in size and accumulated lipids. This is the first report on in vitro differentiation of aSPCs from Atlantic salmon to mineralizing osteogenic cells. This cell model system provides a new valuable tool for studying osteoblastogenesis in fish.

Sulphated glycosaminoglycans and proteoglycans in the developing vertebral column of juvenile Atlantic salmon (Salmo salar).

In the present study, the distribution of sulphated glycosaminoglycans (GAGs) in the developing vertebral column of Atlantic salmon (Salmo salar) at 700, 900, 1100 and 1400 d° was examined by light microscopy. The mineralization pattern was outlined by Alizarin red S and soft structures by Alcian blue. The temporal and spatial distribution patterns of different types of GAGs: chondroitin-4-sulphate/dermatan sulphate, chondroitin-6-sulphate, chondroitin-0-sulphate and keratan sulphate were addressed by immunohistochemistry using monoclonal antibodies against the different GAGs. The specific pattern obtained with the different antibodies suggests a unique role of the different GAG types in pattern formation and mineralization. In addition, the distribution of the different GAG types in normal and malformed vertebral columns from 15 g salmon was compared. A changed expression pattern of GAGs was found in the malformed vertebrae, indicating the involvement of these molecules during the pathogenesis. The molecular size of proteoglycans (PGs) in the vertebrae carrying GAGs was analysed with western blotting, and mRNA transcription of the PGs aggrecan, decorin, biglycan, fibromodulin and lumican by real-time qPCR. Our study reveals the importance of GAGs in development of vertebral column also in Atlantic salmon and indicates that a more comprehensive approach is necessary to completely understand the processes involved.

Small leucine-rich proteoglycans in the vertebrae of Atlantic salmon Salmo salar.

We analysed the distribution and expression of the small leucine-rich proteoglycans (SLRPs) decorin, biglycan and lumican in vertebral columns of Atlantic salmon Salmo salar L. with and without radiographically detectable deformities. Vertebral deformities are a reoccurring problem in salmon and other intensively farmed species, and an understanding of the components involved in the pathologic development of the vertebrae is important in order to find adequate solutions to this problem. Using immunohistology and light microscopy, we found that in non-deformed vertebrae biglycan, lumican and decorin were all expressed in osteoblasts at the vertebral growth zones and at the ossification front of the chondrocytic arches. Hence, the SLRPs are expressed in regions where intramembranous and endochondral ossification take place. In addition, mRNA expression of biglycan, decorin and lumican was demonstrated in a primary osteoblast culture established from Atlantic salmon, supporting the in vivo findings. Transcription of the SLRPs increased during differentiation of the osteoblasts in vitro and where lumican mRNA expression increased later in the differentiation compared with decorin and biglycan. Intriguingly, in vertebral fusions, biglycan, decorin and lumican protein expression was extended to trans-differentiating cells at the border between arch centra and osteoblast growth zones. In addition, mRNA expression of biglycan, decorin and lumican differed between non-deformed and fused vertebrae, as shown by quantitative PCR (qPCR). Western blotting revealed an additional band of biglycan in fused vertebrae which had a higher molecular weight than in non-deformed vertebrae. Fourier-transform infrared (FTIR) spectroscopy revealed more spectral focality in the endplates of vertebral fusions and significantly more non-reducible collagen crosslinks compared with non-deformed vertebrae, thus identifying differences in bone structure.

Exercise induced mechano-sensing and substance P mediated bone modeling in Atlantic salmon.

Mechanical stress plays a vital role in maintaining bone architecture. The process by which osteogenic cells convert the mechanical signal into a biochemical response governing bone modeling is not clear, however. In this study, we investigated how Atlantic salmon (Salmo salar) vertebra responds to exercise-induced mechanical loading. Bone formation in the vertebrae was favored through increased expression of genes involved in osteoid production. Fourier transform infrared spectroscopy (FT-IR) showed that bone matrix secreted both before and during sustained swimming had different properties after increased load compared to control, suggesting that both new and old bones are affected. Concomitantly, both osteoblasts and osteocytes in exercised salmon showed increased expression of the receptor nk-1 and its ligand substance P (SP), both known to be involved in osteogenesis. Moreover, in situ hybridization disclosed SP mRNA in osteoblasts and osteocytes, supporting an autocrine function. The functional role of SP was investigated in vitro using osteoblasts depleted for SP. The cells showed severely reduced transcription of genes involved in mineralization, demonstrating a regulatory role for SP in salmon osteoblasts. Investigation of α-tubulin stained osteocytes revealed cilia-like structures. Together with SP, cilia may link mechanical responses to osteogenic processes in the absence of a canaliculi network. Our results imply that salmon vertebral bone responds to mechanical load through a highly interconnected and complex signal and detection system, with SP as a key factor for initializing mechanically-induced bone formation in bone lacking the canaliculi system.

Molecular and physiological responses to long-term sublethal ammonia exposure in Atlantic salmon (Salmo salar).

The objective of this study was to determine the underlying physiological and molecular responses to long-term sublethal ammonia exposure in Atlantic salmon (Salmo salar) parr. Previous studies have predominately focused on mechanisms during acute, short-term exposure. For that purpose Atlantic salmon parr were exposed to four ammonia concentrations between 4 and 1800 µmol l(-1) total ammonia nitrogen (TAN), and subjected to two feeding regimes for 15 weeks. Elevated environmental ammonia and full feeding strength caused an initial increase in plasma ammonia levels ([T(amm)]) after 22 days of exposure, which thereafter declined and remained similar to the control animals towards the end of the study. On the other hand, a progressive decrease in plasma urea levels was evident throughout the entire exposure period and depended on the concentration of environmental ammonia, with the largest decrease in urea levels observed at the highest ammonia concentrations (1700 and 1800 µmol l(-1) TAN). We hypothesized that the successful adaptation to long-term elevated ammonia levels would involve an increased capacity for carrier-facilitated branchial excretion. This hypothesis was strengthened by the first evidence of an up-regulation of branchial transcription of the genes encoding the Rhesus (Rh) glycoproteins, Rhcg1 and Rhcg2, urea transporter (UT) and aquaporin 3a (Aqp3a), during long-term exposure. Of the Rhesus glycoprotein (Rh) mRNAs, Rhcg1 was up-regulated at all tested ammonia levels, while Rhcg2 showed a concentration-sensitive increase. Increased transcription levels of V-type H(+)-ATPase (H(+)-ATPase) were observed at the highest ammonia concentrations (1700 and 1800 µmol l(-1) TAN) and coincided with an up-regulation of Rhcg2 at these concentrations. Transcription of UT and Aqp3a was increased after 15 weeks of exposure to low ammonia levels (470 and 480 µmol l(-1) TAN). A significant increase in brain glutamine (Gln) concentration was observed for full fed Atlantic salmon after 22 days and in fish with restricted feeding after 105 days of exposure to 1800 and 1700 µmol l(-1) TAN, respectively, without any concomitant decrease in brain glutamate (Glu) concentrations. These results suggest that Gln synthesis is an ammonia detoxifying strategy employed in the brain of Atlantic salmon parr during long-term sublethal ammonia exposure. Full feed strength had an additive effect on plasma [T(amm)], while the restricted feeding regime postponed the majority of the observed physiological and molecular responses. In conclusion, Atlantic salmon parr adapts to the long-term sublethal ammonia concentrations with increased branchial transcription levels of ammonia and urea transporting proteins and ammonia detoxification in the brain.