Immersion challenge model for Flavobacterium psychrophilum infection of Atlantic salmon (Salmo salar L.) fry.

Flavobacterium psychrophilum is the causative agent of bacterial cold-water disease (CWBD) and rainbow trout fry syndrome (RTFS), which affect salmonids. To better understand this pathogen and its interaction with the host during infection, including to support the development of resistant breeds and new vaccines and treatments, there is a pressing need for reliable and reproducible immersion challenge models that more closely mimic natural routes of infection. The aim of this present study was to evaluate a challenge model developed previously for rainbow trout for use in Atlantic salmon. First, preliminary challenges were conducted in Atlantic salmon (n = 120) and rainbow trout (n = 80) fry using two F. psychrophilum isolates collected from each fish species, respectively; fish had been pretreated with 200 mg/L hydrogen peroxide for 1 h. Thereafter, the main challenge was performed for just one F. psychrophilum isolate for each species (at 2 × 107 CFU/mL) but using larger cohorts (Atlantic salmon: n = 1187; rainbow trout: n = 2701). Survival in the main challenge was 81.2% in Atlantic salmon (21 days post-challenge) and 45.3% in rainbow trout (31 days post-challenge). Mortalities progressed similarly during the preliminary and main challenges for both species, demonstrating the reproducibility of this model. This is the first immersion challenge model of F. psychrophilum to be developed successfully for Atlantic salmon.

Targeted mutagenesis of ∆5 and ∆6 fatty acyl desaturases induce dysregulation of lipid metabolism in Atlantic salmon (Salmo salar).

BACKGROUND: With declining wild fish populations, farmed salmon has gained popularity as a source for healthy long-chain highly unsaturated fatty acids (LC-HUFA). However, the introduction of plant oil in farmed salmon feeds has reduced the content of these beneficial LC-HUFA. The synthetic capability for LC-HUFAs depends upon the dietary precursor fatty acids and the genetic potential, thus there is a need for in-depth understanding of LC-HUFA synthetic genes and their interactions with other genes involved in lipid metabolism. Several key genes of LC-HUFA synthesis in salmon belong to the fatty acid desaturases 2 (fads2) family. The present study applied whole transcriptome analysis on two CRISPR-mutated salmon strains (crispants), 1) Δ6abc/5Mt with mutations in Δ5fads2, Δ6fads2-a, Δ6fads2-b and Δ6fads2-c genes, and 2) Δ6bcMt with mutations in Δ6fads2-b and Δ6fads2-c genes. Our purpose is to evaluate the genetic effect fads2 mutations have on other lipid metabolism pathways in fish, as well as to investigate mosaicism in a commercial species with a very long embryonal period. RESULTS: Both Δ6abc/5Mt and Δ6bcMt crispants demonstrated high percentage of indels within all intended target genes, though different indel types and percentage were observed between individuals. The Δ6abc/5Mt fish displayed several disruptive indels which resulted in over 100 differentially expressed genes (DEGs) enriched in lipid metabolism pathways in liver. This includes up-regulation of srebp1 genes which are known key transcription regulators of lipid metabolism as well as a number of down-stream genes involved in fatty acid de-novo synthesis, fatty acid ß-oxidation and lipogenesis. Both elovl5 and elovl2 genes were not changed, suggesting that the genes were not targeted by Srebp1. The mutation of Δ6bcMt surprisingly resulted in over 3000 DEGs which were enriched in factors encoding genes involved in mRNA regulation and stability. CONCLUSIONS: CRISPR-Cas9 can efficiently mutate multiple fads2 genes simultaneously in salmon. The results of the present study have provided new information on the transcriptional regulations of lipid metabolism genes after reduction of LC-HUFA synthesis pathways in salmon.

Lipid Deposition and Mobilisation in Atlantic Salmon Adipocytes.

The present study aimed to elucidate how Atlantic salmon adipocytes pre-enriched with palmitic (16:0, PA), oleic (18:1n-9, OA), or eicosapentaenoic (20:5n-3, EPA) acid respond to a fasting condition mimicked by nutrient deprivation and glucagon. All experimental groups were supplemented with radiolabeled PA to trace secreted lipids and distribution of radioactivity in different lipid classes. There was a higher content of intracellular lipid droplets in adipocytes pre-enriched with OA than in adipocytes pre-enriched with PA or EPA. In the EPA group, the radiolabeled PA was mainly esterified in phospholipids and triacylglycerols, whereas in the OA and PA groups, the radioactivity was mainly recovered in phospholipids and cholesterol-ester. By subjecting the experimental groups to nutrient-deprived media supplemented with glucagon, lipolysis occurred in all groups, although to a lower extent in the OA group. The lipids were mainly secreted as esterified lipids in triacylglycerols and phospholipids, indicating mobilization in lipoproteins. A significant proportion was secreted as free fatty acids and glycerol. Leptin secretion was reduced in all experimental groups in response to fasting, while the mitochondria area responded to changes in the energy supply and demand by increasing after 3 h of fasting. Overall, different lipid classes in adipocytes influenced their mobilization during fasting.

DHA Modulates Immune Response and Mitochondrial Function of Atlantic Salmon Adipocytes after LPS Treatment.

Adipocytes play a central role in overall energy homeostasis and are important contributors to the immune system. Fatty acids (FAs) act as signaling molecules capable to modulate adipocyte metabolism and functions. To identify the effects of two commonly used FAs in Atlantic salmon diets, primary adipocytes were cultured in the presence of oleic (OA) or docosahexaenoic (DHA) acid. DHA decreased adipocyte lipid droplet number and area compared to OA. The increase in lipid load in OA treated adipocytes was paralleled by an increase in iNOS activity and mitochondrial SOD2-GFP activity, which was probably directed to counteract increase in oxidative stress. Under lipopolysaccharide (LPS)-induced inflammation, DHA had a greater anti-inflammatory effect than OA, as evidenced by the higher SOD2 activity and the transcriptional regulation of antioxidant enzymes and pro- and anti-inflammatory markers. In addition, DHA maintained a healthy mitochondrial structure under induced inflammation while OA led to elongated mitochondria with a thin thread like structures in adipocytes exposed to LPS. Overall, DHA possess anti-inflammatory properties and protects Atlantic salmon against oxidative stress and limits lipid deposition. Furthermore, DHA plays a key role in protecting mitochondria shape and function.

Transcriptional development of phospholipid and lipoprotein metabolism in different intestinal regions of Atlantic salmon (Salmo salar) fry.

BACKGROUND: It has been suggested that the high phospholipid (PL) requirement in Atlantic salmon (Salmo salar) fry is due to insufficient intestinal de-novo synthesis causing low lipoprotein (LP) production and reduced transport capacity of dietary lipids. However, in-depth ontogenetic analysis of intestinal PL and LP synthesis with the development of salmon has yet to be performed. Therefore, in this paper we used RNA-Seq technology to investigate the expression of genes involved in PL synthesis and LP formation throughout early developmental stages and associate insufficient expression of synthesis pathways in salmon fry with its higher dietary PL requirement. There was a special focus on the understanding homologous genes, especially those from salmonid-specific fourth vertebrate whole-genome duplication (Ss4R), and their contribution to salmonid specific features of regulation of PL metabolic pathways. Salmon fry were sampled at 0.16 g (1 day before first-feeding), 2.5 and 10 g stages of development and transcriptomic analysis was applied separately on stomach, pyloric caeca and hindgut of the fish. RESULTS: In general, we found up-regulated pathways involved in synthesis of phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn), and LP in pyloric caeca of salmon between 0.16 and 10 g. Thirteen differentially expressed genes (q < 0.05) in these pathways were highly up-regulated in 2.5 g salmon compared to 0.16 g, while only five more differentially expressed (q < 0.05) genes were found when the fish grew up to 10 g. Different homologous genes were found dominating in stomach, pyloric caeca and hindgut. However, the expression of dominating genes in pathways of PL and LP synthesis were much higher in pyloric caeca than stomach and hindgut. Salmon-specific homologous genes (Ss4R) had similar expression during development, while other homologs had more diverged expression. CONCLUSIONS: The up-regulation of the de-novo PtdCho and PtdEtn pathways confirm that salmon have decreasing requirement for dietary PL as the fish develops. The similar expressions between Ss4R homologous genes suggest that the functional divergence of these genes was incomplete compared to homologs derived from other genome duplication. The results of the present study have provided new information on the molecular mechanisms of phospholipid synthesis and lipoprotein formation in fish.

Life-stage-associated remodelling of lipid metabolism regulation in Atlantic salmon.

Atlantic salmon migrates from rivers to sea to feed, grow and develop gonads before returning to spawn in freshwater. The transition to marine habitats is associated with dramatic changes in the environment, including water salinity, exposure to pathogens and shift in dietary lipid availability. Many changes in physiology and metabolism occur across this life-stage transition, but little is known about the molecular nature of these changes. Here, we use a long-term feeding experiment to study transcriptional regulation of lipid metabolism in Atlantic salmon gut and liver in both fresh- and saltwater. We find that lipid metabolism becomes significantly less plastic to differences in dietary lipid composition when salmon transitions to saltwater and experiences increased dietary lipid availability. Expression of genes in liver relating to lipogenesis and lipid transport decreases overall and becomes less responsive to diet, while genes for lipid uptake in gut become more highly expressed. Finally, analyses of evolutionary consequences of the salmonid-specific whole-genome duplication on lipid metabolism reveal several pathways with significantly different (p < .05) duplicate retention or duplicate regulatory conservation. We also find a limited number of cases where the whole-genome duplication has resulted in an increased gene dosage. In conclusion, we find variable and pathway-specific effects of the salmonid genome duplication on lipid metabolism genes. A clear life-stage-associated shift in lipid metabolism regulation is evident, and we hypothesize this to be, at least partly, driven by nondietary factors such as the preparatory remodelling of gene regulation and physiology prior to sea migration.

De novo lipogenesis in Atlantic salmon adipocytes.

BACKGROUND: Carnivorous teleost fish utilize glucose poorly, and the reason for this is not known. It is possible that the capacity of adipocytes to synthesize lipids from carbohydrate precursors through a process known as "de novo lipogenesis" (DNL) is one of the factors that contributes to glucose intolerance in Atlantic salmon. METHODS: Primary adipocytes from Atlantic salmon differentiated in vitro were incubated with radiolabelled glucose in order to explore the capacity of salmon adipocytes to synthesize and deposit lipids from glucose through DNL. The lipid-storage capacity of adipocytes incubated with glucose was compared with that of cells incubated with the fatty acid palmitic acid. Quantitative PCR and immunohistochemistry were used to assess changes of genes and proteins involved in glucose and lipid transport and metabolism. RESULTS: Less than 0.1% of the radiolabelled glucose was metabolized to the fatty acids 16:0 and the stearoyl-CoA desaturase products 16:1 and 18:1 by DNL, whereas approximately 40% was converted to glycerol to form the triacylglycerol backbone of lipids. Transcriptional analysis indicated that adipocytes ensure the availability of necessary cofactors and other substrates for lipid synthesis and storage from glycolysis, the pentose phosphate pathway and glyceroneogenesis. CONCLUSIONS: We have shown for the first time that the DNL pathway is active in fish adipocytes. The capacity of the pathway to convert glucose into cellular lipids for storage is relatively low. GENERAL SIGNIFICANCE: The limited capacity of adipocytes to utilize glucose as a substrate for lipid deposition may contribute to glucose intolerance in salmonids.

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.

Divergent and sex-dimorphic expression of the paralogs of the Sox9-Amh-Cyp19a1 regulatory cascade in developing and adult atlantic cod (Gadus morhua L.).

The factors of the Sox9-Amh-Cyp19a1 cascade play a crucial role in the complex process of sex differentiation in mammals. The involvement of Sox9 and Cyp19a1 paralogs and the single Amh ortholog in sex differentiation and development of the gonads and the brain in Atlantic cod was examined by analyzing bimodal and sex-dimorphic gene expression patterns, respectively, during early stages and in maturing males and females. Expression of sox9a and sox9b were initiated at blastulation, and both paralogs were expressed in chondrogenic tissue in the hatched larvae. The male-specific expression of sox9a in the adult gonads supports a conserved role in testis function, while sox9b was expressed in the maturing testes and ovaries at similar levels. Amh was expressed at low, but variable, levels from late gastrulation prior to the onset of cyp19a1a and cyp19a1b expression. Male-biased amh expression was found in the maturing gonads, but the increased ovarian levels during maturation suggest a role also in females. The larval expression of cyp19a1a and cyp19a1b increased at the expected time of sex differentiation, but showed large individual variation. The ovarian expression of cyp19a1a and amh increased concomitant with increased plasma estradiol levels during vitellogenesis. The testis-specific cyp19a1b expression supports the importance of estrogen in the spermatogenesis, while abundant expression in the male and female brain is probably related to the continuous neurogenesis in fish. These divergent and sex-dimorphic expression patterns of the cod sox9 and cyp19a1 paralogs demonstrate the complexity of the genetic network regulating sexual development in fish.

Efficient transfection of Atlantic salmon primary hepatocyte cells for functional assays and gene editing.

The expansion of genomic resources for Atlantic salmon over the past half decade has enabled efficient interrogation of genetic traits by large-scale correlation of genotype to phenotype. Moving from correlation to causation will require genotype-phenotype relationships to be tested experimentally in a cost-efficient and cell context-relevant manner. To enable such future experiments, we have developed a method for the isolation and genetic manipulation of primary hepatocytes from Atlantic salmon for use in heterologous expression, reporter assay, and gene editing experiments. We chose the liver as the tissue of interest because it is the metabolic hub and many current Atlantic salmon research projects focus on understanding metabolic processes to improve traits such as the growth rate, total fat content, and omega-3 content. We find that isolated primary hepatocytes are optimally transfected with both plasmid and ribonucleoprotein using a Neon electroporator at 1,400 V, 10 ms, and 2 pulses. Transfection efficiency with plasmid and cutting efficiency with ribonucleoprotein were optimally 46% and 60%, respectively. We also demonstrate a 26 times increase in luciferase expression under the promoter of the key liver metabolic gene, elovl5b, compared to an empty vector, in line with expected liver-specific expression. Taken together, this work provides a valuable resource enabling transfection and gene editing experiments in a context-relevant and cost-effective system.

In vivo and in vitro insulin and fasting control of the transmembrane fatty acid transport proteins in Atlantic salmon (Salmo salar).

We have examined the nutritional and insulin regulation of the mRNA expression of transmembrane fatty acid (FA) transporters [FA transport protein-1 (FATP1) and CD36] together with the lipoprotein lipase (LPL), the cytosolic FA carrier FA binding protein (FABP3), and mitochondrial FA-CoA and -carnitine palmitoyl transferase carriers (CPT)1 and -2 in Atlantic salmon tissues and myocyte cell culture. Two weeks of fasting diminished FATP1, CD36, and LPL in adipose tissue, suggesting a reduction in FA uptake, while FABP3 increased in liver, probably enhancing the transport of FA to the mitochondria. Insulin injection decreased FATP1 and CD36 in white and red muscles, while both transporters were upregulated in the adipose tissue in agreement with the role of insulin-inhibiting muscle FA oxidation and stimulating adipose fat stores. Serum deprivation of 48 h in Atlantic salmon myotubes increased FATP1, FABP3, and CPT-2, while CPT-1 was diminished. In myotubes, insulin induced FATP1 expression but decreased CD36, FABP3, and LPL, suggesting that FATP1 could be more involved in the insulin-stimulated FA uptake. Insulin increased the FA uptake in myotubes mediated, at least in part, through the relocation of FATP1 protein to the plasma membrane. Overall, Atlantic salmon FA transporters are regulated by fasting and insulin on in vivo and in vitro models.

Intracellular localisation and innate immune responses following Francisella noatunensis infection of Atlantic cod (Gadus morhua) macrophages.

The facultative intracellular bacterium Francisella noatunensis causes francisellosis in Atlantic cod (Gadus morhua), but little is known about its survival strategies or how these bacteria evade the host immune response. In this study we show intracellular localisation of F. noatunensis in cod macrophages using indirect immunofluorescence techniques and green fluorescent labelled bacteria. Transmission electron microscopy revealed that F. noatunensis was enclosed by a phagosomal membrane during the initial phase of infection. Bacteria were at a later stage of the infection found in large electron-lucent zones, apparently surrounded by a partially intact or disintegrated membrane. Immune electron microscopy demonstrated the release of bacterial derived vesicles from intracellular F. noatunensis, an event suspected of promoting phagosomal membrane degradation and allowing escape of the bacteria to cytoplasm. Studies of macrophages infected with F. noatunensis demonstrated a weak activation of the inflammatory response genes as measured by increased expression of the Interleukin (IL)-1ß and IL-8. In comparison, a stronger induction of gene expression was found for the anti-inflammatory IL-10 indicating that the bacterium exhibits a role in down-regulating the inflammatory response. Expression of the p40 subunit of IL-12/IL-17 genes was highly induced during infection suggesting that F. noatunensis promotes T cell polarisation. The host macrophage responses studied here showed low ability to distinguish between live and inactivated bacteria, although other types of responses could be of importance for such discriminations. The immunoreactivity of F. noatunensis lipopolysaccharide (LPS) was very modest, in contrast to the strong capacity of Escherichia coli LPS to induce inflammatory responsive genes. These results suggest that F. noatunensis virulence mechanisms cover many strategies for intracellular survival in cod macrophages.

Remodeling of the notochord during development of vertebral fusions in Atlantic salmon (Salmo salar).

Histological characterization of spinal fusions in Atlantic salmon (Salmo salar) has demonstrated shape alterations of vertebral body endplates, a reduced intervertebral space, and replacement of intervertebral cells by ectopic bone. However, the significance of the notochord during the fusion process has not been addressed. We have therefore investigated structural and cellular events in the notochord during the development of vertebral fusions. In order to induce vertebral fusions, Atlantic salmon were exposed to elevated temperatures from fertilization until they attained a size of 15g. Based on results from radiography, intermediate and terminal stages of the fusion process were investigated by immunohistochemistry and real-time quantitative polymerase chain reaction. Examination of structural extracellular matrix proteins such as Perlecan, Aggrecan, Elastin, and Laminin revealed reduced activity and reorganization at early stages in the pathology. Staining for elastic fibers visualized a thinner elastic membrane surrounding the notochord of developing fusions, and immunohistochemistry for Perlecan showed that the notochordal sheath was stretched during fusion. These findings in the outer notochord correlated with the loss of Aggrecan- and Substance-P-positive signals and the further loss of vacuoles from the chordocytes in the central notochord. At more progressed stages of fusion, chordocytes condensed, and the expression of Aggrecan and Substance P reappeared. The hyperdense regions seem to be of importance for the formation of notochordal tissue into bone. Thus, the remodeling of notochord integrity by reduced elasticity, structural alterations, and cellular changes is probably involved in the development of vertebral fusions.

Exposure to lipopolysaccharide induces immune genes in cultured preadipocytes of Atlantic salmon.

In addition to its central role of energy storage and release, white adipose tissue (WAT) performs complex endocrine and immune activities. WAT produces physiologically active secretory proteins, including cytokines and complement factors. Furthermore, treatment of mammalian adipocytes with cytokines and inflammatory stimulators induces immune genes, suppresses regulators of adipocyte differentiation and activates lipolysis. Previously we reported up-regulation of immune genes in the course of in vitro development of Atlantic salmon white adipocytes. If WAT is immunoactive tissue in fish, excessive deposition of fat resulting from lipid-rich diets may imply risk for health of farmed fish. In this paper we investigated how lipopolysaccharide (LPS) affects immune activity in the adipose tissue-derived stromo-vascular fraction (aSVF) of Atlantic salmon. Experiments were performed with confluent cultures of proliferating preadipocytes. Exposure to LPS induced expression of immune genes, including TNFalpha and TNF-dependent genes, chemokines and receptors, NFkappaB related genes, matrix metalloproteinases and genes involved in eicosanoid metabolism. LPS decreased expression of adipocyte markers and genes involved in lipid metabolism, however, in parallel, it accelerated a number of transcriptional events that take place during the adipogenic differentiation of aSVF.

Morphological and molecular characterization of developing vertebral fusions using a teleost model.

BACKGROUND: Spinal disorders are a major cause of disability for humans and an important health problem for intensively farmed animals. Experiments have shown that vertebral deformities present a complex but comparable etiology across species. However, the underlying molecular mechanisms involved in bone deformities are still far from understood. To further explicate the mechanisms involved, we have examined the fundamental aspects of bone metabolism and pathogenesis of vertebral fusions in Atlantic salmon (Salmo salar). RESULTS: Experimentally, juvenile salmon were subjected to hyperthermic conditions where more than 28% developed fused vertebral bodies. To characterize the fusion process we analyzed an intermediate and a terminal stage of the pathology by using x-ray, histology, immunohistochemistry, real-time quantitative PCR and in situ hybridization. At early stage in the fusion process, disorganized and proliferating osteoblasts were prominent at the growth zones of the vertebral body endplates. PCNA positive cells further extended along the rims of fusing vertebral bodies. During the developing pathology, the marked border between the osteoblast growth zones and the chondrocytic areas connected to the arches became less distinct, as proliferating cells and chondrocytes blended through an intermediate zone. This cell proliferation appeared to be closely linked to fusion of opposing arch centra. During the fusion process a metaplastic shift appeared in the arch centra where cells in the intermediate zone between osteoblasts and chondrocytes co-expressed mixed signals of chondrogenic and osteogenic markers. A similar shift also occurred in the notochord where proliferating chordoblasts changed transcription profile from chondrogenic to also include osteogenic marker genes. In progressed fusions, arch centra and intervertebral space mineralized. CONCLUSION: Loss of cell integrity through cell proliferation and metaplastic shifts seem to be key events in the fusion process. The fusion process involves molecular regulation and cellular changes similar to those found in mammalian deformities, indicating that salmon is suitable for studying general bone development and to be a comparative model for spinal deformities.

Molecular pathology of vertebral deformities in hyperthermic Atlantic salmon (Salmo salar).

BACKGROUND: Hyperthermia has been shown in a number of organisms to induce developmental defects as a result of changes in cell proliferation, differentiation and gene expression. In spite of this, salmon aquaculture commonly uses high water temperature to speed up developmental rate in intensive production systems, resulting in an increased frequency of skeletal deformities. In order to study the molecular pathology of vertebral deformities, Atlantic salmon was subjected to hyperthermic conditions from fertilization until after the juvenile stage. RESULTS: Fish exposed to the high temperature regime showed a markedly higher growth rate and a significant higher percentage of deformities in the spinal column than fish reared at low temperatures. By analyzing phenotypically normal spinal columns from the two temperature regimes, we found that the increased risk of developing vertebral deformities was linked to an altered gene transcription. In particular, down-regulation of extracellular matrix (ECM) genes such as col1a1, osteocalcin, osteonectin and decorin, indicated that maturation and mineralization of osteoblasts were restrained. Moreover, histological staining and in situ hybridization visualized areas with distorted chondrocytes and an increased population of hypertrophic cells. These findings were further confirmed by an up-regulation of mef2c and col10a, genes involved in chondrocyte hypertrophy. CONCLUSION: The presented data strongly indicates that temperature induced fast growth is severely affecting gene transcription in osteoblasts and chondrocytes; hence change in the vertebral tissue structure and composition. A disrupted bone and cartilage production was detected, which most likely is involved in the higher rate of deformities developed in the high intensive group. Our results are of basic interest for bone metabolism and contribute to the understanding of the mechanisms involved in development of temperature induced vertebral pathology. The findings may further conduce to future molecular tools for assessing fish welfare in practical farming.

Localization of mRNAs and proteins in methyl methacrylate-embedded tissues.

Precise localization of proteins and mRNA in histological sections is necessary for evaluating spatial gene expression patterns. Here we report sensitive detection of the gene products in fish tissues by immunohistochemistry (IHC) and in situ hybridization (ISH) assays on sections of whole specimens and vertebra embedded in methyl methacrylate (MMA) resin. This plastic resin favors easy preparation of various specimen types and enables preparation of large sections with well-preserved cell morphology. IHC analysis of the muscle regulatory factor MyoD in transverse sections of juvenile cod revealed MyoD-positive cells in the dorsolateral parts of the adaxial muscle. ISH revealed less spatially restricted signals of the bone morphogenic protein bmp4 in muscle and brain. To assess the applicability of ISH on sections of bony tissue, col1a1 and col2a1 expression was investigated in non-decalcified vertebra sections of Atlantic salmon. The former was identified in both chondrocytes and osteoblasts, whereas the latter was mostly evident in chondrocytes. We conclude that MMA resin offers easy preparation of large and problematic tissues and the possibility of carrying out both IHC and ISH analyses using standard protocols.

Gene expression analyses of essential catch factors in the smooth and striated adductor muscles of larval, juvenile and adult great scallop (Pecten maximus).

The scallop adductor muscle consists of striated fibres responsible for the fast closure of the shells, and smooth fibres able to maintain tension in a prolonged state of contraction called catch. Formation of the force-bearing catch linkages has been demonstrated to be initiated by dephosphorylation of the key catch-regulating factor twitchin by a calcineurin-like phosphatase, while the involvement of other thick filament proteins is uncertain. Here we report on the development of catchability of the adductor smooth muscle in the great scallop (Pecten maximus) by analysing the spatio-temporal gene expression patterns of the myosin regulatory light chain (MLCr), twitchin, myorod and calcineurin using whole mount in situ hybridization and real-time quantitative PCR. The MLCr signal was identified in the retractor and adductor muscles of the pediveliger larvae, and the juvenile and adult scallop displayed abundant mRNA levels of MLCr in the smooth and striated adductor muscles. Twitchin was mainly expressed in the smooth adductor muscle during metamorphosis, whereas the adult striated adductor muscle contained seven-folds higher twitchin mRNA levels compared to the smooth portion. Calcineurin expression predominated in the gonads and in the smooth adductor, and five-folds higher mRNA levels were measured in the smooth than in the striated fibres at the adult stage. In contrast to the other genes examined, the expression of myorod was confined to the smooth adductor muscle suggesting that myorod plays a permissive role in the molluscan catch muscles, which are first required at the vulnerable settlement stage as a component of the predator defence system.

Liver slice culture as a model for lipid metabolism in fish.

Hepatic lipid metabolism is traditionally investigated in vitro using hepatocyte monocultures lacking the complex three-dimensional structure and interacting cell types essential liver function. Precision cut liver slice (PCLS) culture represents an alternative in vitro system, which benefits from retention of tissue architecture. Here, we present the first comprehensive evaluation of the PCLS method in fish (Atlantic salmon, Salmo salar L.) and validate it in the context of lipid metabolism using feeding trials, extensive transcriptomic data, and fatty acid measurements. We observe an initial period of post-slicing global transcriptome adjustment, which plateaued after 3 days in major metabolic pathways and stabilized through 9 days. PCLS fed alpha-linolenic acid (ALA) and insulin responded in a liver-like manner, increasing lipid biosynthesis gene expression. We identify interactions between insulin and ALA, where two PUFA biosynthesis genes that were induced by insulin or ALA alone, were highly down-regulated when insulin and ALA were combined. We also find that transcriptomic profiles of liver slices are exceedingly more similar to whole liver than hepatocyte monocultures, both for lipid metabolism and liver marker genes. PCLS culture opens new avenues for high throughput experimentation on the effect of "novel feed composition" and represent a promising new strategy for studying genotype-specific molecular features of metabolism.

Genomic and functional gene studies suggest a key role of beta-carotene oxygenase 1 like (bco1l) gene in salmon flesh color.

Red coloration of muscle tissue (flesh) is a unique trait in several salmonid genera, including Atlantic salmon. The color results from dietary carotenoids deposited in the flesh, whereas the color intensity is affected both by diet and genetic components. Herein we report on a genome-wide association study (GWAS) to identify genetic variation underlying this trait. Two SNPs on ssa26 showed strong associations to the flesh color in salmon. Two genes known to be involved in carotenoid metabolism were located in this QTL- region: beta-carotene oxygenase 1 (bco1) and beta-carotene oxygenase 1 like (bco1l). To determine whether flesh color variation is caused by one, or both, of these genes, functional studies were carried out including mRNA and protein expression in fish with red and pale flesh color. The catalytic abilities of these two genes were also tested with different carotenoids. Our results suggest bco1l to be the most likely gene to explain the flesh color variation observed in this population.