Atlantic salmon aquaculture is an important food source globally, but its sustainability is challenged by environmental impacts and the nutritional demands of farmed fish, particularly when it comes to fatty acids. Salmon feeds still rely heavily on fish or soybean meal, which poses sustainability concerns due to overfishing and carbon footprint. Innovations in feed composition seek to address these challenges, e.g., by using more sustainable ingredients, but the impacts of alternative feeds on fish and environment can be hard to quantify. Here, we developed a model with detailed and flexible accounting for lipids – Simulated Salmon Lipid Metabolism (SimSaLipiM) – to predict the nutritional and environmental outcomes of feed formulations. Integrating SimSaLipiM with feed ingredient databases enabled detailed analysis of an in vivo feed trial in silico . The model predicted optimal feed efficiency in agreement with observations as well as a detailed energy budget and fish biomass lipid composition for each feed. We also used the model to formulate novel sustainable feeds and feed supplements by minimising CO2 footprint. Thus, SimSaLipiM makes it easy to identify recipes that optimize key feed properties such as efficiency and environmental impacts. This could be a valuable tool for feed manufacturers, guiding the formulation of feeds that are both sustainable and cost effective. By bridging the gap between feed formulation and the flexible growth and energy requirements of a fish, SimSaLipiM can contribute to advancing sustainable aquaculture. ### Competing Interest Statement The authors have declared no competing interest.

Astaxanthin (Ax) determines the flesh redness of a salmonid fish which is the most desirable quality indicator by consumers. Fish cannot synthesize Ax de novo, therefore, the only way to increase flesh redness is to increase dietary input or improve the absorption and retention rate of dietary Ax. As a hydrophobic carotenoid, the absorption of Ax can be modulated by other lipid molecules in the diet. The present study explored the effect of three lipids, cholesterol (CH), phytosterol (PS), and docosahexaenoic acid (DHA) on Ax absorption, transport, and retention in rainbow trout. Dietary CH significantly improved Ax absorption by elevating plasma Ax levels (p < 0.05); however, it had no effect on the whole body Ax or flesh color. Dietary PS appears to inhibit Ax absorption since fish had significantly (p < 0.05) reduced whole body Ax. Dietary DHA appeared to have no effect on Ax absorption or retention. By comparing intestinal transcriptomes, a low density lipoprotein receptor (ldlr) gene was significantly downregulated in fish fed the CH diet as compared to the control diet. Since LDLR protein plays a major role in plasma lipoprotein turnover, we hypothesized that the inhibition of ldlr gene by high dietary CH resulted in higher retention of plasma Ax. The elevation of plasma Ax was not reflected in higher flesh coloration, which suggested other limiting factors governing Ax retention in the muscle. On the other hand, the transcriptomic and proteomic analyses found no changes of genes or proteins involved in Ax absorption, transport, or excretion in fish fed PS or DHA diets as compared to the control diet. In conclusion, this study has suggested that CH promotes Ax absorption by regulating lipoprotein retention and provide evidence for improving Ax absorption via dietary modulation.

Genome-scale metabolic models are key biotechnology tools that can predict metabolic capabilities and growth for an organism. In particular, these models have become indispensable for metabolic analysis of microbial species and communities such as the gut microbiomes of humans and other animals. Accurate microbial models can be built automatically from genomes, but many microbes have only been observed through sequencing of marker genes such as 16S rRNA and thus remain inaccessible to genome-scale modeling. To extend the scope of genome-scale metabolic models to microbes that lack genomic information, we trained an artificial neural network to build microbial models from numeric representations of 16S rRNA gene sequences. Specifically, we built models and extracted 16S rRNA gene sequences from more than 15,000 reference and representative microbial genomes, computed multiple sequence alignments and large language model embeddings for the 16S rRNA gene sequences, and trained the neural network to predict metabolic reaction probabilities from sequences, alignments, or embeddings. Training was fast on a single graphics processing unit and trained networks predicted reaction probabilities accurately for unseen archaeal and bacterial sequences and species. This makes it possible to reconstruct microbial genome-scale metabolic networks from any 16S rRNA gene sequence and enables simulation of metabolism and growth for all observed microbial life.### Competing Interest StatementThe authors have declared no competing interest.

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.

The metabolic network of an organism can be analyzed as a constraint-based model. This analysis can be biased, optimizing an objective such as growth rate, or unbiased, aiming to describe the full feasible space of metabolic fluxes through pathway analysis or random flux sampling. In particular, pathway analysis can decompose the flux space into fundamental and formally defined metabolic pathways. Unbiased methods scale poorly with network size due to combinatorial explosion, but a promising approach to improve scalability is to focus on metabolic subnetworks, e.g., cells’ metabolite exchanges with each other and the environment, rather than the full metabolic networks. Here, we applied pathway enumeration and flux sampling to metabolite exchanges in microbial species and a microbial community, using models ranging from central carbon metabolism to genome-scale and focusing on pathway definitions that allow direct targeting of subnetworks such as metabolite exchanges (elementary conversion modes, elementary flux patterns, and minimal pathways). Enumerating growth-supporting metabolite exchanges, we found that metabolite exchanges from different pathway definitions were related through a hierarchy, and we show that this hierarchical relationship between pathways holds for metabolic networks and subnetworks more generally. Metabolite exchange frequencies, defined as the fraction of pathways in which each metabolite was exchanged, were similar across pathway definitions, with a few specific exchanges explaining large differences in pathway counts. This indicates that biological interpretation of predicted metabolite exchanges is robust to the choice of pathway definition, and it suggests strategies for more scalable pathway analysis. Our results also signal wider biological implications, facilitating detailed and interpretable analysis of metabolite exchanges and other subnetworks in fields such as metabolic engineering and synthetic biology.

Constraint-based models (CBMs) are used to study metabolic network structure and function in organisms ranging from microbes to multicellular eukaryotes. Published CBMs are usually generic rather than context-specific, meaning that they do not capture differences in reaction activities, which, in turn, determine metabolic capabilities, between cell types, tissues, environments, or other conditions. Only a subset of a CBM’s metabolic reactions and capabilities are likely to be active in any given context, and several methods have therefore been developed to extract context-specific models from generic CBMs through integration of omics data. We tested the ability of six model extraction methods (MEMs) to create functionally accurate context-specific models of Atlantic salmon using a generic CBM (SALARECON) and liver transcriptomics data from contexts differing in water salinity (life stage) and dietary lipids. Three MEMs (iMAT, INIT, and GIMME) outperformed the others in terms of functional accuracy, which we defined as the extracted models’ ability to perform context-specific metabolic tasks inferred directly from the data, and one MEM (GIMME) was faster than the others. Context-specific versions of SALARECON consistently outperformed the generic version, showing that context-specific modeling better captures salmon metabolism. Thus, we demonstrate that results from human studies also hold for a non-mammalian animal and major livestock species.

Motivation Constraint-based models (CBMs) are used to study the metabolic networks of organisms ranging from microbes to multicellular eukaryotes. Published CBMs are usually generic rather than context-specific, meaning that they do not capture metabolic differences between cell types, tissues, environments, or other conditions. However, only a subset of reactions in a model are likely to be active in any given context, and several methods have therefore been developed to extract context-specific models from generic CBMs through integration of omics data. Results We tested the ability of six model extraction methods (MEMs) to create functionally accurate context-specific models of Atlantic salmon using a generic CBM (SALARECON) and liver transcriptomics data from contexts differing in water salinity (life stage) and dietary lipids. Reaction contents and metabolic task feasibility predictions of context-specific CBMs were mainly determined by the MEM that was used, but life stage explained significant variance in both contents and predictions for some MEMs. Three MEMs clearly outperformed the others in terms of their ability to capture context-specific metabolic activities inferred directly from the data, and one of these (GIMME) was much faster than the others. Context-specific versions of SALARECON consistently outperformed the generic version, showing that context-specific modeling captures more realistic representations of Atlantic salmon metabolism. Contact jon.vik@nmbu.no

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.

Salmon is an important source of long-chain highly unsaturated fatty acids (LC-HUFAs) such as 22:6n-3 [docosahexaenoic acid (DHA)]. In the present study, we conducted two identical experiments on salmon in freshwater (FW) and seawater (SW) stages, with a diet switch from fish oil (high in LC-HUFA) to vegetable oil (low in LC-HUFA) and vice versa. Our aim was to investigate the diet and life stage-specific features of lipid uptake (gut), processing (liver), and deposition (muscle). The lipid composition changed much faster in the gut of SW fish relative to FW fish, suggesting that the former had a higher rate of lipid absorption and transport. SW fish also had higher expression of phospholipid synthesis and lipoprotein formation genes in the gut, whereas FW fish had higher expression of lipid synthesis genes in the liver. All phospholipids except PC-44:12 and PE-44:12 were less abundant in SW, suggesting that SW fish have a higher requirement for DHA.

Rapeseed meal and faba beans (RSM/FB) can serve as an alternative to imported soybean meal (SBM). In this study, forty Norwegian crossbred ([Landrace x Yorkshire] x Duroc) growing-finishing pigs (108.7 ± 4.2 kg final BW) were fed a diet with either SBM or RSM/FB as protein sources. RSM/FB increased feed conversion ratio (P = .04) in the finishing period, reduced lightness (P = .04) and yellowness (P = .004) of meat, changed amounts of individual fatty acids, but not of total SFA, MUFA and PUFA. Importantly, RSM/FB reduced the glucose level (P < .05) in meat. Lower pyroglutamic acid (P = .06) in RSM/FB indicate lower oxidative stress in pre-rigor muscle cell. Increased abundance of free amino acids, sweet tasting metabolites, reduced warmed-over flavor and flavor attributes indicated desirable properties of RSM/FB meat. To conclude, RSM/FB in pig diet supported growth performance and carcass quality comparable to SBM and had a positive effect on meat quality.

Domestication of animals imposes strong targeted selection for desired traits but can also result in unintended selection due to new domestic environments. Atlantic salmon (Salmo salmar) was domesticated in the 1970s and has subsequently been selected for faster growth in systematic breeding programmes. More recently, salmon aquaculture has replaced fish oils (FOs) with vegetable oils (VOs) in feed, radically changing the levels of essential long-chain polyunsaturated fatty acids (LC-PUFAs). Our aim here was to study the impact of domestication on metabolism and explore the hypothesis that the shift to VO diets has unintentionally selected for a domestication-specific lipid metabolism. We conducted a 96-day feeding trial of domesticated and wild salmon fed diets based on FOs, VOs or phospholipids, and compared transcriptomes and fatty acids in tissues involved in lipid absorption (pyloric caeca) and lipid turnover and synthesis (liver). Domesticated salmon had faster growth and higher gene expression in glucose and lipid metabolism compared to wild fish, possibly linked to differences in regulation of circadian rhythm pathways. Only the domesticated salmon increased expression of LC-PUFA synthesis genes when given VOs. This transcriptome response difference was mirrored at the physiological level, with domesticated salmon having higher LC-PUFA levels but lower 18:3n-3 and 18:2n-6 levels. In line with this, the VO diet decreased growth rate in wild but not domesticated salmon. Our study revealed a clear impact of domestication on transcriptomic regulation linked to metabolism and suggests that unintentional selection in the domestic environment has resulted in evolution of stronger compensatory mechanisms to a diet low in LC-PUFAs.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

FAIRDOM (Findable, Accessible, Interoperable, Reusable Data, Operating procedures and Models) is an initiative to establish sustained data, model and process management service to the European Systems Biology community ( https://fair-dom.org/ ). With the free SEEK ( https://seek4science.org/ ) data management software FAIRDOM offers a data management platform for interdisciplinary projects to support the storage and exchange of data and models from research partners based on the FAIR principles. SEEK can be installed, run and further developed as its own instance, and over 50 organisations have done that including many in ELIXIR Nodes. It is also the platform used for the web-accessible public Commons platform FAIRDOMHub ( https://fairdomhub.org/ ), offering public information and password protected user collaboration spaces. The Hub is a managed service hosted at HITS (ELIXIR-DE) and currently supports 185 projects.

With declining wild fish populations, farmed Atlantic salmon ( Salmo salar ) has gained popularity as a source for healthy long-chain highly unsaturated fatty acids (LC-HUFA) including 20:5n-3 and 22:6n-3. However, the introduction of plant-based oil in fish diets has reduced the content of these beneficial LC-HUFA. The capability of biosynthesis of LC-HUFAs depends on fatty acids supplied in diets and the genetic potential residing in the fish. Key proteins involved in LC-HUFA synthesis in salmon include fatty acid desaturases 2 (Fads2). In a recent study we used CRISPR/Cas9 to generate two F0 mutant strains of salmon, 1) Δ6abc/5 Mt with mutations in Δ5fads2, Δ6fads2-a, Δ6fads2-b and Δ6fads2-c genes, and 2) Δ6bc Mt with mutations in Δ6fads2-b and Δ6fads2-c genes. The CRISPR mutated salmon (crispants) had reduced levels of LC-HUFA and expression of targeted fads2 genes. In present study we apply whole transcriptome analysis on these fads2 crispants. Our purpose is to evaluate the genetic mosaicism in fads2 crispants and the effect these mutations had on other lipid metabolism pathways in fish. Both Δ6abc/5 Mt and Δ6bc Mt crispants demonstrated high percentage of indels within all intended target genes, though different indel types and percentage were observed between individuals. Skipping of a CRISPR-targeted exon was observed in Δ6fads2-a gene of Δ6abc/5 Mt salmon. The Δ6abc/5 Mt fish also 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 as well as genes involved in fatty acid de-novo synthesis, fatty acid β -oxidation and lipogenesis. Both elovl5 and elovl2 genes were not changed, suggesting that the genes were not targeted by Srebp1. The mutation of Δ6bc Mt surprisingly resulted in over 3000 DEGs which were enriched in factors encoding genes involved in mRNA regulation and stability.

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 β-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.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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) 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 three days in major metabolic pathways and stabilized through nine 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.

The onset of first feeding is a critical period for Atlantic salmon (Salmo salar). The fish goes through a dramatic transition, from using the yolk sac as an energy and nutrient resource to rely on exogenous food. However, the digestive system of salmon is not fully developed at start feeding. This is especially true for digestion and absorption of dietary lipids. To optimize lipid composition of the initial feed for better growth and development of salmon, it is important to have a systemic understanding of lipid metabolism. The present study combines transcriptomics and lipid composition data to obtain an overview of how lipid metabolism pathways change when salmon switch from endogenous to exogenous feed. Transition to exogenous feeding had higher influence on gene expression and fatty acids composition in pyloric caeca relative to liver, suggesting metabolism is more responsive in pyloric caeca when diet switches. The pathways of phospholipid and lipoprotein synthesis were both up-regulated in pyloric caeca, while phospholipid content unchanged. Because phospholipids are a major component of intestinal lipoproteins, it suggests that a higher level of dietary phospholipids is required for optimal lipid transport at first feeding stages. De-novo cholesterol synthesis pathway was up-regulated in pyloric caeca after exogenous feeding, while a down-regulation of bile acid synthesis pathway was found in liver. This suggests a higher requirement of cholesterol in salmon fry after switching to exogenous feeding. The present study has provided new insights on the systemic changes of lipid synthesis and transport pathways in salmon fry when switching from endogenous to exogenous feeding.

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) 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 three days in major metabolic pathways and stabilized through nine 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.