Molecular tracing confirms that infection with infectious pancreatic necrosis virus follows the smolt from hatchery to grow-out farm.

Infectious pancreatic necrosis (IPN) is an important restraint to production of salmonids in aquaculture globally. In order to implement efficacious mitigation strategies for control of this disease, it is important to understand infection routes under current production systems. IPN virus has been shown to be transmitted vertically in Rainbow trout, from broodstock to fingerlings in hatcheries, and there is circumstantial evidence suggesting that vertical transmission can also occur in Atlantic salmon, in addition to horizontal transmission between grow-out fish in farms. In this study, we show that the smolt carries infection with IPN from hatchery to the marine farm. We do this by comparing sequences from fish groups taken both in hatcheries and on corresponding marine grow-out farms. We use statistical analysis to prove that sequences obtained from the same fish group in both hatchery and marine farm are more similar than sequences obtained from random fish groups on hatcheries and marine farms.

Cardiac responses to elevated seawater temperature in Atlantic salmon.

BACKGROUND: Atlantic salmon aquaculture operations in the Northern hemisphere experience large seasonal fluctuations in seawater temperature. With summer temperatures often peaking around 18-20°C there is growing concern about the effects on fish health and performance. Since the heart has a major role in the physiological plasticity and acclimation to different thermal conditions in fish, we wanted to investigate how three and eight weeks exposure of adult Atlantic salmon to 19°C, previously shown to significantly reduce growth performance, affected expression of relevant genes and proteins in cardiac tissues under experimental conditions. RESULTS: Transcriptional responses in cardiac tissues after three and eight weeks exposure to 19°C (compared to thermal preference, 14°C) were analyzed with cDNA microarrays and validated by expression analysis of selected genes and proteins using real-time qPCR and immunofluorescence microscopy. Up-regulation of heat shock proteins and cell signaling genes may indicate involvement of the unfolded protein response in long-term acclimation to elevated temperature. Increased immunofluorescence staining of inducible nitric oxide synthase in spongy and compact myocardium as well as increased staining of vascular endothelial growth factor in epicardium could reflect induced vascularization and vasodilation, possibly related to increased oxygen demand. Increased staining of collagen I in the compact myocardium of 19°C fish may be indicative of a remodeling of connective tissue with long-term warm acclimation. Finally, higher abundance of transcripts for genes involved in innate cellular immunity and lower abundance of transcripts for humoral immune components implied altered immune competence in response to elevated temperature. CONCLUSIONS: Long-term exposure of Atlantic salmon to 19°C resulted in cardiac gene and protein expression changes indicating that the unfolded protein response, vascularization, remodeling of connective tissue and altered innate immune responses were part of the cardiac acclimation or response to elevated temperature.

N-acetylhistidine, a novel osmolyte in the lens of Atlantic salmon (Salmo salar L.).

Volume homeostasis is essential for the preservation of lens transparency and this is of particular significance to anadromous fish species where migration from freshwater to seawater presents severe osmotic challenges. In Atlantic salmon (Salmo salar L.), aqueous humor (AH) osmolality is greater in fish acclimated to seawater compared with young freshwater fish, and levels of lens N-acetylhistidine (NAH) are much higher in seawater fish. Here we investigate NAH as an osmolyte in the lenses of salmon receiving diets either with or without histidine supplementation. In the histidine-supplemented diet (HD) histidine content was 14.2 g/kg, and in the control diet (CD) histidine content was 8.9 g/kg. A transient increase in AH osmolality of 20 mmol/kg was observed in fish transferred from freshwater to seawater. In a lens culture model, temporary decreases in volume and transparency were observed when lenses were exposed to hyperosmotic conditions. A positive linear relationship between extracellular osmolality and lens NAH content was also observed, whereas there was no change in lens histidine content. Hypoosmotic exposure stimulated [(14)C]-histidine efflux by 9.2- and 2.6-fold in CD and HD lenses, respectively. NAH efflux, measured by HPLC, was stimulated by hypoosmotic exposure to a much greater extent in HD lenses. In vivo, lens NAH increased in response to elevated AH osmolality in HD but not CD fish. In conclusion, NAH has an important and novel role as a compatible osmolyte in salmon lens. Furthermore, it is the major osmolyte that balances increases in AH osmolality when fish move from freshwater to seawater. A deficiency in NAH would lead to a dysfunction of the normal osmoregulatory processes in the lens, and we propose that this would contribute to cataract formation in fish deficient in histidine.

Optimisation of gene expression analysis in Atlantic salmon lenses by refining sampling strategy and tissue storage.

Analysis of gene expression in the lens is one of the analytical tools employed to investigate cataract formation in Atlantic salmon (Salmo salar L.). High quality RNA preparations are an essential prerequisite for gene expression analysis. The first aim of the present study was to investigate the possible effects of two methods of tissue preservation on the quality of RNA extracted from Atlantic salmon lenses. RNA was extracted from lenses either stored in RNAlater or flash-frozen in liquid nitrogen. Both tissue preservation methods yielded RNA of similarly high quality. The second aim was to examine if stress related to fish handling and the choice of anaesthesia during the sampling procedure affected gene expression in the lens. Six sampling procedures were tested on groups of sea water adapted Atlantic salmon smolt. Fish were either killed instantaneously (control group) or sampled after 30 min anaesthetised with isoeugenol, after 30 min without anaesthesia, after 120 min anaesthetised with isoeugenol, after 120 min with 15 min anaesthesia with metacaine or after 120 min without anaesthesia. The expression levels of specific genes, of special interest in the study of molecular mechanisms of cataractogenesis, were analysed in lenses by real-time RT-PCR. Fish not anaesthetised had significantly increased levels of heat shock protein 70 (HSP70) mRNA after 30 min compared to the control group. Glutathione reductase (GR) and manganese superoxide dismutase (Mn-SOD) were expressed at significantly lower levels in groups of Atlantic salmon sampled after 120 min anaesthetised with isoeugenol or metacaine, and anaesthetised with isoeugenol, metacaine or without anaesthesia, respectively. The same expression patterns were found in corresponding gill tissues for these two antioxidant genes. In conclusion, preservation in liquid nitrogen instead of RNAlater is recommended due to practical conditions in RNA extraction. A quick sampling protocol with the use of anaesthetics and not exceeding 30 min should be preferred to avoid effects of the sampling procedure on lens gene expression in Atlantic salmon.

Genome-wide transcription analysis of histidine-related cataract in Atlantic salmon (Salmo salar L).

PURPOSE: Elevated levels of dietary histidine have previously been shown to prevent or mitigate cataract formation in farmed Atlantic salmon (Salmo salar L). The aim of this study was to shed light on the mechanisms by which histidine acts. Applying microarray analysis to the lens transcriptome, we screened for differentially expressed genes in search for a model explaining cataract development in Atlantic salmon and possible markers for early cataract diagnosis. METHODS: Adult Atlantic salmon (1.7 kg) were fed three standard commercial salmon diets only differing in the histidine content (9, 13, and 17 g histidine/kg diet) for four months. Individual cataract scores for both eyes were assessed by slit-lamp biomicroscopy. Lens N-acetyl histidine contents were measured by high performance liquid chromatography (HPLC). Total RNA extracted from whole lenses was analyzed using the GRASP 16K salmonid microarray. The microarray data were analyzed using J-Express Pro 2.7 and validated by quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: Fish developed cataracts with different severity in response to dietary histidine levels. Lens N-acetyl histidine contents reflected the dietary histidine levels and were negatively correlated to cataract scores. Significance analysis of microarrays (SAM) revealed 248 significantly up-regulated transcripts and 266 significantly down-regulated transcripts in fish that were fed a low level of histidine compared to fish fed a higher histidine level. Among the differentially expressed transcripts were metallothionein A and B as well as transcripts involved in lipid metabolism, carbohydrate metabolism, regulation of ion homeostasis, and protein degradation. Hierarchical clustering and correspondence analysis plot confirmed differences in gene expression between the feeding groups. The differentially expressed genes could be categorized as "early" and "late" responsive according to their expression pattern relative to progression in cataract formation. CONCLUSIONS: Dietary histidine regimes affected cataract formation and lens gene expression in adult Atlantic salmon. Regulated transcripts selected from the results of this genome-wide transcription analysis might be used as possible biological markers for cataract development in Atlantic salmon.

The impact of different water gas levels on cataract formation, muscle and lens free amino acids, and lens antioxidant enzymes and heat shock protein mRNA abundance in smolting Atlantic salmon, Salmo salar L.

The present experiment was conducted to examine if freshwater (FW) oxygen and carbon dioxide regimes cause physiological responses that lead to cataract formation in Atlantic salmon (Salmo salar L.) smolt. Duplicate groups of 50 g Atlantic salmon smolts were exposed to three freshwater oxygen saturation regimes (95, 112 or 125% saturation), with or without addition of carbon dioxide (measured 17-18 and 2-3 mg L(-1), respectively), for six weeks before transfer to seawater (SW). The FW exposure groups were followed up for another six weeks under a common SW regime. Fish were screened for cataract and sampled accordingly, at start, after 6 weeks in FW and after 6 weeks in SW. Increased growth related cataract incidences and severities were recorded in SW, mainly in the groups previously exposed to normoxic and hyperoxic conditions in FW, as compared to the respective groups added carbon dioxide. The concentration of histidine compounds (imidazoles) in muscle and lens tissue, used as quantitative risk markers of cataract, were lower than observed in earlier studies, however, neither were affected by the present water gas regimes in FW nor after follow up in SW. Independently of water oxygenation in FW, muscle free amino acid profiles in salmon groups concomitantly exposed to elevated carbon dioxide indicated use of selected free amino acids for energy purposes. Significantly lower abundance of heat shock protein 70 mRNA and trends towards stepwise reduction of antioxidant enzymes mRNA in the lens from fish exposed to increased water oxygenation were recorded, probably linked to increased growth and/or external stress during smoltification. This represents a first communication on using early molecular markers to express reduced protection of the fish lens against external stress to explain cataract development.

Short-term starvation at low temperature prior to harvest does not impact the health and acute stress response of adult Atlantic salmon.

A period of starvation is regarded as a sound practice in aquaculture prior to handling, transportation and harvest, to minimise impacts on welfare and ensure proper hygiene after harvest. However, documentation of welfare issues such as stress following starvation and handling in adult Atlantic salmon are lacking. This study aimed to examine gut emptying and potential stress during a two week starvation period, and whether this starvation period changed the tolerance for physical stress. The study confirmed slower emptying of the gut segments at low temperature. Plasma and bile cortisol, and selected clinical analyses were used to characterize potential stress, as well as the response to acute physical crowding stress during the starvation period. Neither the general stress level nor the ability to cope with handling stress was affected by a 14 day starvation period. Down-regulation of selected nutritional related gene markers in liver indicated classical starvation responses, with reduced metabolism and oxidative pressure, and sparing of nutrients. The response to acute handling stress was not affected by two weeks of starvation. There were minor effects of starvation on stress and health markers, as evaluated by plasma lysozyme activity and gene expression of selected inflammation marker proteins in heart and skin tissues.

Lens metabolomic profiling as a tool to understand cataractogenesis in Atlantic salmon and rainbow trout reared at optimum and high temperature.

Periods of high or fluctuating seawater temperatures result in several physiological challenges for farmed salmonids, including an increased prevalence and severity of cataracts. The aim of the present study was to compare cataractogenesis in Atlantic salmon (Salmo salar L.) and rainbow trout (Oncorhynchus mykiss) reared at two temperatures, and investigate whether temperature influences lens metabolism and cataract development. Atlantic salmon (101±2 g) and rainbow trout (125±3 g) were reared in seawater at either 13°C (optimum for growth) or 19°C during the 35 days experiment (n = 4 tanks for each treatment). At the end of the experiment, the prevalence of cataracts was nearly 100% for Atlantic salmon compared to ~50% for rainbow trout, irrespective of temperature. The severity of the cataracts, as evaluated by slit-lamp inspection of the lens, was almost three fold higher in Atlantic salmon compared to rainbow trout. The global metabolic profile revealed differences in lens composition and metabolism between the two species, which may explain the observed differences in cataract susceptibility between the species. The largest differences were seen in the metabolism of amino acids, especially the histidine metabolism, and this was confirmed by a separate quantitative analysis. The global metabolic profile showed temperature dependent differences in the lens carbohydrate metabolism, osmoregulation and redox homeostasis. The results from the present study give new insight in cataractogenesis in Atlantic salmon and rainbow trout reared at high temperature, in addition to identifying metabolic markers for cataract development.

A holistic approach to development of diets for Ballan wrasse (Labrus berggylta) - a new species in aquaculture.

Wild wrasses are used for delousing of farmed salmon but increasing demands have prompted the salmon industry to develop cultures of Ballan wrasse. One of the bottlenecks has been nutrition and feed intake in the juvenile phase, while broodstock nutrition is considered critical for production of viable offspring. The present study was aimed at developing functioning ongrowing and broodstock diets for Ballan wrasse. In juveniles the best lengthwise growth was identified at 65% dietary protein, 12% lipid and 16% carbohydrate. To investigate if the requirements for the other nutrients were covered by the diets developed for the species, the nutrient composition in juveniles (whole body) and broodstock (female gonad) were analyzed and compared to the composition in wild fish. We found that the levels of the lipid soluble Vitamins A, K and D were lower in cultured than in wild fish, however, the requirements for these nutrients in Ballan wrasse are not known. Other candidate nutrients for more in-depth investigation are the bone minerals, zinc, taurine and fatty acids.

GH-IGF system regulation of attenuated muscle growth and lipolysis in Atlantic salmon reared at elevated sea temperatures.

Growth regulation in adult Atlantic salmon (1.6 kg) was investigated during 45 days in seawater at 13, 15, 17, and 19 °C. We focused on feed intake, nutrient uptake, nutrient utilization, and endocrine regulation through growth hormone (GH), insulin-like growth factors (IGF), and IGF-binding proteins (IGFBP). During prolonged thermal exposure, salmon reduced feed intake and growth. Feed utilization was reduced at 19 °C after 45 days compared with fish at lower temperatures, and body lipid storage was depleted with increasing water temperature. Although plasma IGF-1 concentrations did not change, 32-Da and 43-kDa IGFBP increased in fish reared at ≤17 °C, and dropped in fish reared at 19 °C. Muscle igf1 mRNA levels were reduced at 15 and 45 days in fish reared at 15, 17, and 19 °C. Muscle igf2 mRNA levels did not change after 15 days in response to increasing temperature, but were reduced after 45 days. Although liver igf2 mRNA levels were reduced with increasing temperatures after 15 and 45 days, temperature had no effect on igf1 mRNA levels. The liver igfbp2b mRNA level, which corresponds to circulating 43-kDa IGFBP, exhibited similar responses after 45 days. IGFBP of 23 kDa was only detected in plasma in fish reared at 17 °C, and up-regulation of the corresponding igfbp1b gene indicated a time-dependent catabolic response, which was not observed in fish reared at 19 °C. However, higher muscle ghr mRNA levels were detected in fish at 17 and 19 °C than in fish at lower temperatures, indicating lipolytic regulation in muscle. These results show that the reduction of muscle growth in large salmon is mediated by decreased igf1 and igf2 mRNA levels in addition to GH-associated lipolytic action to cope with prolonged thermal exposure. Accordingly, 13 °C appears to be a more optimal temperature for the growth of adult Atlantic salmon at sea.

Effect of plant-based feed ingredients on osmoregulation in the Atlantic salmon lens.

Lenses of adult Atlantic salmon fed with a plant oil and plant protein-based diet (plant diet) were compared to lenses of fish fed a diet based on traditional marine ingredients (marine diet) with respect to biochemical composition and functionality ex vivo. After 12 months of feeding, plant diet-fed fish had smaller lenses with higher water contents and lower concentrations of histidine (His) and N-acetylhistidine (NAH) than fish fed with the marine diet. Cataract development in both dietary groups was minimal and no differences between the groups were observed. Lens fatty acid and lipid class composition differed minimally, although a significant increase in linoleic acid was observed. The lenses were examined for their ability to withstand osmotic disturbances ex vivo. Culture in hypoosmotic and hyperosmotic media led to increase and decrease of lens volume, respectively. Lenses from plant diet-fed fish were less resistant to swelling and shrinking, released less NAH into the culture medium, and accumulated His and NAH at higher rates than lenses from marine diet-fed fish. Culture in hypoosmotic medium resulted in higher cataract scores than in control and hyperosmotic medium. mRNA expression of selected genes, including glutathione peroxidase 4 and SPARC (secreted protein acidic and rich in cysteine), was affected by diet and osmotic treatment. It can be concluded that lenses of farmed Atlantic salmon are affected by the diet composition, both in biochemical composition and physiological functionality in relation to osmoregulation.

Modelling sources of variation and risk factors for spinal deformity in farmed Atlantic salmon using hierarchical- and cross-classified multilevel models.

Sources of variation and risk factors for spinal deformity were investigated in a 2002-2004 year-class database of farmed Atlantic salmon using multilevel modelling. The prevalence of spinal deformity, recorded on subsamples of Atlantic salmon at individual days of harvest, was used as the outcome variable in the study. The dataset consisted of a multilevel structure with days of harvest (n=1441) nested within sea water pens (n=544), which were nested within sea water sites (n=39), which again were cross-classified with fresh water plants (n=21). A four level combined hierarchical- and cross-classified linear mixed model was built in MLwiN using Markov chain Monte Carlo (MCMC) estimation of variance components and fixed effects. Results revealed that a large part of the variance could be explained as sampling and classification random errors, accounting for 32% of the variation in the random intercept model and 41% of the variation in the final mixed effect model. Of the remaining "biological variation" in the random intercept model, 33% was explained by fixed effects where both the use half-year and 1.5 year old photo-manipulated autumn smolts (compared to using one year old spring smolt), and the use of six component vaccines (compared to using four and five component vaccines), were significantly associated with spinal deformity. The results suggest that the physiological changes at time of smoltification make Atlantic salmon susceptible to stressors causing vertebral deformation and that this is most evident in photo-manipulated fish smoltifying when temperature and growth is at its peak. The study further shows the potential of using multilevel modelling in epidemiological studies based on data from industrial aquaculture.