The genome regulatory landscape of Atlantic salmon liver through smoltification.

The anadromous Atlantic salmon undergo a preparatory physiological transformation before seawater entry, referred to as smoltification. Key molecular developmental processes involved in this life stage transition, such as remodeling of gill functions, are known to be synchronized and modulated by environmental cues like photoperiod. However, little is known about the photoperiod influence and genome regulatory processes driving other canonical aspects of smoltification such as the large-scale changes in lipid metabolism and energy homeostasis in the developing smolt liver. Here we generate transcriptome, DNA methylation, and chromatin accessibility data from salmon livers across smoltification under different photoperiod regimes. We find a systematic reduction of expression levels of genes with a metabolic function, such as lipid metabolism, and increased expression of energy related genes such as oxidative phosphorylation, during smolt development in freshwater. However, in contrast to similar studies of the gill, smolt liver gene expression prior to seawater transfer was not impacted by photoperiodic history. Integrated analyses of gene expression, chromatin accessibility, and transcription factor (TF) binding signatures highlight chromatin remodeling and TF dynamics underlying smolt gene regulatory changes. Differential peak accessibility patterns largely matched differential gene expression patterns during smoltification and we infer that ZNF682, KLFs, and NFY TFs are important in driving a liver metabolic shift from synthesis to break down of organic compounds in freshwater. Overall, chromatin accessibility and TFBS occupancy were highly correlated to changes in gene expression. On the other hand, we identified numerous differential methylation patterns across the genome, but associated genes were not functionally enriched or correlated to observed gene expression changes across smolt development. Taken together, this work highlights the relative importance of chromatin remodeling during smoltification and demonstrates that metabolic remodeling occurs as a preadaptation to life at sea that is not to a large extent driven by photoperiod history.

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.

SALARECON connects the Atlantic salmon genome to growth and feed efficiency.

Atlantic salmon (Salmo salar) is the most valuable farmed fish globally and there is much interest in optimizing its genetics and rearing conditions for growth and feed efficiency. Marine feed ingredients must be replaced to meet global demand, with challenges for fish health and sustainability. Metabolic models can address this by connecting genomes to metabolism, which converts nutrients in the feed to energy and biomass, but such models are currently not available for major aquaculture species such as salmon. We present SALARECON, a model focusing on energy, amino acid, and nucleotide metabolism that links the Atlantic salmon genome to metabolic fluxes and growth. It performs well in standardized tests and captures expected metabolic (in)capabilities. We show that it can explain observed hypoxic growth in terms of metabolic fluxes and apply it to aquaculture by simulating growth with commercial feed ingredients. Predicted limiting amino acids and feed efficiencies agree with data, and the model suggests that marine feed efficiency can be achieved by supplementing a few amino acids to plant- and insect-based feeds. SALARECON is a high-quality model that makes it possible to simulate Atlantic salmon metabolism and growth. It can be used to explain Atlantic salmon physiology and address key challenges in aquaculture such as development of sustainable feeds.

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.

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.

Gamma radiation induces life stage-dependent reprotoxicity in Caenorhabditis elegans via impairment of spermatogenesis.

The current study investigated life stage, tissue and cell dependent sensitivity to ionizing radiation of the nematode Caenorhabditis elegans. Results showed that irradiation of post mitotic L4 stage larvae induced no significant effects with respect to mortality, morbidity or reproduction at either acute dose ≤6 Gy (1500 mGy·h-1) or chronic exposure ≤15 Gy (≤100 mGy·h-1). In contrast, chronic exposure from the embryo to the L4-young adult stage caused a dose and dose-rate dependent reprotoxicity with 43% reduction in total brood size at 6.7 Gy (108 mGy·h-1). Systematic irradiation of the different developmental stages showed that the most sensitive life stage was L1 to young L4. Exposure during these stages was associated with dose-rate dependent genotoxic effects, resulting in a 1.8 to 2 fold increase in germ cell apoptosis in larvae subjected to 40 or 100 mGy·h-1, respectively. This was accompanied by a dose-rate dependent reduction in the number of spermatids, which was positively correlated to the reprotoxic effect (0.99, PCC). RNAseq analysis of nematodes irradiated from L1 to L4 stage revealed a significant enrichment of differentially expressed genes related to both male and hermaphrodite reproductive processes. Gene network analysis revealed effects related to down-regulation of genes required for spindle formation and sperm meiosis/maturation, including smz-1, smz-2 and htas-1. Furthermore, the expression of a subset of 28 set-17 regulated Major Sperm Proteins (MSP) required for spermatid production was correlated (R2 0.80) to the reduction in reproduction and the number of spermatids. Collectively these observations corroborate the impairment of spermatogenesis as the major cause of gamma radiation induced life-stage dependent reprotoxic effect. Furthermore, the progeny of irradiated nematodes showed significant embryonal DNA damage that was associated with persistent effect on somatic growth. Unexpectedly, these nematodes maintained much of their reproductive capacity in spite of the reduced growth.

CRISPR/Cas9-mediated ablation of elovl2 in Atlantic salmon (Salmo salar L.) inhibits elongation of polyunsaturated fatty acids and induces Srebp-1 and target genes.

Atlantic salmon can synthesize polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (20:5n-3), arachidonic acid (20:4n-6) and docosahexaenoic acid (22:6n-3) via activities of very long chain fatty acyl elongases (Elovls) and fatty acyl desaturases (Fads), albeit to a limited degree. Understanding molecular mechanisms of PUFA biosynthesis and regulation is a pre-requisite for sustainable use of vegetable oils in aquafeeds as current sources of fish oils are unable to meet increasing demands for omega-3 PUFAs. By generating CRISPR-mediated elovl2 partial knockout (KO), we have shown that elovl2 is crucial for multi-tissue synthesis of 22:6n-3 in vivo and that endogenously synthesized PUFAs are important for transcriptional regulation of lipogenic genes in Atlantic salmon. The elovl2-KOs showed reduced levels of 22:6n-3 and accumulation of 20:5n-3 and docosapentaenoic acid (22:5n-3) in the liver, brain and white muscle, suggesting inhibition of elongation. Additionally, elovl2-KO salmon showed accumulation of 20:4n-6 in brain and white muscle. The impaired synthesis of 22:6n-3 induced hepatic expression of sterol regulatory element binding protein-1 (srebp-1), fatty acid synthase-b, Δ6fad-a, Δ5fad and elovl5. Our study demonstrates key roles of elovl2 at two penultimate steps of PUFA synthesis in vivo and suggests Srebp-1 as a main regulator of endogenous PUFA synthesis in Atlantic salmon.

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.

Lineage-specific rediploidization is a mechanism to explain time-lags between genome duplication and evolutionary diversification.

BACKGROUND: The functional divergence of duplicate genes (ohnologues) retained from whole genome duplication (WGD) is thought to promote evolutionary diversification. However, species radiation and phenotypic diversification are often temporally separated from WGD. Salmonid fish, whose ancestor underwent WGD by autotetraploidization ~95 million years ago, fit such a 'time-lag' model of post-WGD radiation, which occurred alongside a major delay in the rediploidization process. Here we propose a model, 'lineage-specific ohnologue resolution' (LORe), to address the consequences of delayed rediploidization. Under LORe, speciation precedes rediploidization, allowing independent ohnologue divergence in sister lineages sharing an ancestral WGD event. RESULTS: Using cross-species sequence capture, phylogenomics and genome-wide analyses of ohnologue expression divergence, we demonstrate the major impact of LORe on salmonid evolution. One-quarter of each salmonid genome, harbouring at least 4550 ohnologues, has evolved under LORe, with rediploidization and functional divergence occurring on multiple independent occasions >50 million years post-WGD. We demonstrate the existence and regulatory divergence of many LORe ohnologues with functions in lineage-specific physiological adaptations that potentially facilitated salmonid species radiation. We show that LORe ohnologues are enriched for different functions than 'older' ohnologues that began diverging in the salmonid ancestor. CONCLUSIONS: LORe has unappreciated significance as a nested component of post-WGD divergence that impacts the functional properties of genes, whilst providing ohnologues available solely for lineage-specific adaptation. Under LORe, which is predicted following many WGD events, the functional outcomes of WGD need not appear 'explosively', but can arise gradually over tens of millions of years, promoting lineage-specific diversification regimes under prevailing ecological pressures.

SalmoBase: an integrated molecular data resource for Salmonid species.

BACKGROUND: Salmonids are ray-finned fishes which constitute 11 genera and at least 70 species including Atlantic salmon, whitefishes, graylings, rainbow trout, and char. The common ancestor of all Salmonidae experienced a whole genome duplication (WGD) ~80 million years ago, resulting in an autotetraploid genome. Genomic rediplodization is still going on in salmonid species, providing an unique system for studying evolutionary consequences of whole genome duplication. In recent years, high quality genome sequences of Atlantic salmon and Rainbow trout has been established, due to their scientific and commercial values. In this paper we introduce SalmoBase ( http://www.salmobase.org/ ), a tool for making molecular resources for salmonids public available in a framework of visualizations and analytic tools. RESULTS: SalmoBase has been developed as a part of the ELIXIR.NO project. Currently, SalmoBase contains molecular resources for Atlantic salmon and Rainbow trout. Data can be accessed through BLAST, Genome Browser (GBrowse), Genetic Variation Browser (GVBrowse) and Gene Expression Browser (GEBrowse). CONCLUSIONS: To the best of our knowledge, SalmoBase is the first database which integrates salmonids data and allow users to study salmonids in an integrated framework. The database and its tools (e.g., comparative genomics tools, synteny browsers) will be expanded as additional public resources describing other Salmonidae genomes become available.

Dietary Butyrate Helps to Restore the Intestinal Status of a Marine Teleost (Sparus aurata) Fed Extreme Diets Low in Fish Meal and Fish Oil.

There is a constant need to find feed additives that improve health and nutrition of farmed fish and lessen the intestinal inflammation induced by plant-based ingredients. The objective of this study was to evaluate the effects of adding an organic acid salt to alleviate some of the detrimental effects of extreme plant-ingredient substitution of fish meal (FM) and fish oil (FO) in gilthead sea bream diet. Three experiments were conducted. In a first trial (T1), the best dose (0.4%) of sodium butyrate (BP-70 ®NOREL) was chosen after a short (9-weeks) feeding period. In a second longer trial (T2) (8 months), four diets were used: a control diet containing 25% FM (T2-D1) and three experimental diets containing 5% FM (T2-D2, T2-D3, T2-D4). FO was the only added oil in D1, while a blend of plant oils replaced 58% and 84% of FO in T2-D2, and T2-D3 and T2-D4, respectively. The latter was supplemented with 0.4% BP-70. In a third trial (T3), two groups of fish were fed for 12 and 38 months with D1, D3 and D4 diets of T2. The effects of dietary changes were studied using histochemical, immunohistochemical, molecular and electrophysiological tools. The extreme diet (T2-D3) modified significantly the transcriptomic profile, especially at the anterior intestine, up-regulating the expression of inflammatory markers, in coincidence with a higher presence of granulocytes and lymphocytes in the submucosa, and changing genes involved in antioxidant defences, epithelial permeability and mucus production. Trans-epithelial electrical resistance (Rt) was also decreased (T3-D3). Most of these modifications were returned to control values with the addition of BP-70. None of the experimental diets modified the staining pattern of PCNA, FABP2 or ALPI. These results further confirm the potential of this additive to improve or reverse the detrimental effects of extreme fish diet formulations.

The Atlantic salmon genome provides insights into rediploidization.

The whole-genome duplication 80 million years ago of the common ancestor of salmonids (salmonid-specific fourth vertebrate whole-genome duplication, Ss4R) provides unique opportunities to learn about the evolutionary fate of a duplicated vertebrate genome in 70 extant lineages. Here we present a high-quality genome assembly for Atlantic salmon (Salmo salar), and show that large genomic reorganizations, coinciding with bursts of transposon-mediated repeat expansions, were crucial for the post-Ss4R rediploidization process. Comparisons of duplicate gene expression patterns across a wide range of tissues with orthologous genes from a pre-Ss4R outgroup unexpectedly demonstrate far more instances of neofunctionalization than subfunctionalization. Surprisingly, we find that genes that were retained as duplicates after the teleost-specific whole-genome duplication 320 million years ago were not more likely to be retained after the Ss4R, and that the duplicate retention was not influenced to a great extent by the nature of the predicted protein interactions of the gene products. Finally, we demonstrate that the Atlantic salmon assembly can serve as a reference sequence for the study of other salmonids for a range of purposes.

Candida utilis and Chlorella vulgaris counteract intestinal inflammation in Atlantic salmon (Salmo salar L.).

Intestinal inflammation, caused by impaired intestinal homeostasis, is a serious condition in both animals and humans. The use of conventional extracted soybean meal (SBM) in diets for Atlantic salmon and several other fish species is known to induce enteropathy in the distal intestine, a condition often referred to as SBM induced enteropathy (SBMIE). In the present study, we investigated the potential of different microbial ingredients to alleviate SBMIE in Atlantic salmon, as a model of feed-induced inflammation. The dietary treatments consisted of a negative control based on fish meal (FM), a positive control based on 20% SBM, and four experimental diets combining 20% SBM with either one of the three yeasts Candida utilis (CU), Kluyveromyces marxianus (KM), Saccharomyces cerevisiae (SC) or the microalgae Chlorella vulgaris (CV). Histopathological examination of the distal intestine showed that all fish fed the SC or SBM diets developed characteristic signs of SBMIE, while those fed the FM, CV or CU diets showed a healthy intestine. Fish fed the KM diet showed intermediate signs of SBMIE. Corroborating results were obtained when measuring the relative length of PCNA positive cells in the crypts of the distal intestine. Gene set enrichment analysis revealed decreased expression of amino acid, fat and drug metabolism pathways as well as increased expression of the pathways for NOD-like receptor signalling and chemokine signalling in both the SC and SBM groups while CV and CU were similar to FM and KM was intermediate. Gene expression of antimicrobial peptides was reduced in the groups showing SBMIE. The characterisation of microbial communities using PCR-DGGE showed a relative increased abundance of Firmicutes bacteria in fish fed the SC or SBM diets. Overall, our results show that both CU and CV were highly effective to counteract SBMIE, while KM had less effect and SC had no functional effects.

Genome wide response to dietary tetradecylthioacetic acid supplementation in the heart of Atlantic Salmon (Salmo salar L).

BACKGROUND: Under-dimensioned hearts causing functional problems are associated with higher mortality rates in intensive Atlantic salmon aquaculture. Previous studies have indicated that tetradecylthioacetic acid (TTA) induces cardiac growth and also stimulates transcription of peroxisome proliferator activated receptors (PPAR) αand ßin the Atlantic salmon heart. Since cardiac and transcriptional responses to feed are of high interest in aquaculture, the objective of this study was to characterize the transcriptional mechanisms induced by TTA in the heart of Atlantic salmon. RESULTS: Atlantic salmon were kept at sea for 17 weeks. During the first 8 weeks the fish received a TTA supplemented diet. Using microarrays, profound transcriptional effects were observed in the heart at the end of the experiment, 9 weeks after the feeding of TTA stopped. Approximately 90% of the significant genes were expressed higher in the TTA group. Hypergeometric testing revealed the over-representation of 35 gene ontology terms in the TTA fed group. The GO terms were generally categorized into cardiac performance, lipid catabolism, glycolysis and TCA cycle. CONCLUSIONS: Our results indicate that TTA has profound effects on cardiac performance based on results from microarray and qRT-PCR analysis. The gene expression profile favors a scenario of "physiological"lright hypertrophy recognized by increased oxidative fatty acid metabolism, glycolysis and TCA cycle activity as well as cardiac growth and contractility in the heart ventricle. Increased cardiac efficiency may offer significant benefits in the demanding Aquaculture situations.

Anti-inflammatory effects of tetradecylthioacetic acid (TTA) in macrophage-like cells from Atlantic salmon (Salmo salar L.).

BACKGROUND: Commercial Atlantic salmon is fed diets with high fat levels to promote fast and cost-effective growth. To avoid negative impact of obesity, food additives that stimulate fat metabolism and immune function are of high interest. TTA, tetradecylthioacetic acid, is a synthetic fatty acid that stimulates mitochondrial ß-oxidation most likely by activation of peroxysome proliferator-activated receptors (PPARs). PPARs are important transcription factors regulating multiple functions including fat metabolism and immune responses. Atlantic salmon experiments have shown that TTA supplemented diets significantly reduce mortality during natural outbreaks of viral diseases, suggesting a modulatory role of the immune system. RESULTS: To gain new insights into TTA effects on the Atlantic salmon immune system, a factorial, high-throughput microarray experiment was conducted using a 44K oligo nucleotide salmon microarray SIQ2.0 and the Atlantic salmon macrophage-like cell line ASK. The experiment was used to determine the transcriptional effects of TTA, the effects of TTA in poly(I:C) elicited cells and the effects of pretreating the cells with TTA. The expression patterns revealed that a large proportion of genes regulated by TTA were related to lipid metabolism and increased mitochondrial ß-oxidation. In addition we found that for a subset of genes TTA antagonized the transcriptional effects of poly(I:C). This, together with the results from qRT-PCR showing an increased transcription of anti-inflammatory IL10 by TTA, indicates anti-inflammatory effects. CONCLUSIONS: We demonstrate that TTA has significant effects on macrophage-like salmon cells that are challenged by the artificial dsRNA poly(I:C). The immune stimulatory effect of TTA in macrophages involves increased lipid metabolism and suppressed inflammatory status. Thus, suggesting that TTA directs the macrophage-like cells towards alternative, anti-inflammatory, activation. This has positive implications for TTA as a feed additive.