Production of the global salmonid aquaculture industry now exceeds 2.4 megatons per annum. Major European producers expect to expand their outputs between 30-50% over the next five years. Ambitions for expansion on this scale create major concerns around fish welfare, ecological impact and the sustainability of salmon feed components. Nutrition lies at the heart of the issue. In the wild, Atlantic salmon are specialized carnivores. In aquaculture, in a move away from the unsustainable use of wild fish protein and oil, proteins of plant origin now constitute the majority (>60%) of their diet. Associated digestive abnormalities are common. In addition, plant-based diets may affect the rate at which nutrients are absorbed and the associated growth rate of salmon, which determines how quickly they grow in marine cages. Rapid marine growth is desirable since it permits more extensive fallowing of coastal aquaculture sites, which reduces the impact of farmed fish on the marine environment (pathogen transfer, nutrient pollution). Finally, while wild fish protein can be replaced by plant protein in salmon diets, oils cannot. Key omega-3 fatty acids must be sourced from the marine environment; a significant burden on wild fisheries. Ensuring the efficient assimilation of fatty acid components from salmonid diets is of paramount importance to safeguard the sustainable exploitation of marine resources. Salmon energetic phenotypes are composites of several interlinked traits: metabolic rate, body fat content, growth, energy harvest from food, energy economy in times of starvation. These traits underpin concerns around salmon nutrition. Significant energetic variation exists in both wild and farmed salmon with multiple possible drivers - both genetic and environmental. Importantly a wealth of new data indicates a role for intestinal microbiota - the bacteria that live in the guts of all vertebrates - in determining host energy metabolism. Understanding how gut bacteria influence Atlantic salmon energetics is thus fundamental to understanding their role in nutrition. This is the principal aim of this project. To achieve this we will establish the influence of gut bacteria on the energetics of salmon living in salt and freshwater, in both wild and aquaculture settings. First, in a unique experimental river system established in Burrishoole, Mayo (Marine Institute/University College Cork, ROI) we will track introduced juvenile salmon through their freshwater lifecycle, measuring both metabolic and gut microbiome variation. This 'wild' cohort will include wild and released farmed fish to establish whether differences between gut microbiota contribute to the poor performance of farmed juveniles in the wild. Secondly, we will undertake parallel freshwater experiments in simulated aquaculture conditions in the laboratory at the University of Glasgow (UoG),UK. Finally, in association with Marine Harvest, we will carry out corresponding experiments on saltwater phase pre-adult salmon in Norway. Alongside experiments with live fish, we propose to harness bioengineering expertise at the School of Engineering, UoG and biotechnological expertise via industry partner Alltech to build an artificial salmon gut system. Via the transfer and maintenance of gut bacteria from metabolically different fish from farmed and wild settings into our gut model we aim to establish how bacterial fermentation underpins differences in energy harvest from feed. Once these 'artificial' bacterial communities are established, a final exploratory phase of the project will involve their transplantation into laboratory reared juvenile salmon to evaluate their potential impact on host metabolism. Understanding how salmon gut bacteria change energetic phenotypes will open new avenues to improve fish health, nutrition and productivity. A model Atlantic salmon gut in a world class UK bio-engineering laboratory puts in place an invaluable tool for salmon aquaculture in the UK

Farmed salmon is a major source of high quality protein and fatty acids essential for human health. Salmon aquaculture is worth approximately £1Bn to the UK economy, and supports many rural and coastal communities. However, disease outbreaks have a major negative effect on salmon production and animal welfare. Infectious salmon anaemia (ISA) is one such disease, and is sometimes dubbed 'salmon flu' because it is caused by a virus (ISAV) that is similar in to influenza. At present, ISA is a notifiable disease in the UK, meaning farmers are obliged to cull their stock in the event of an outbreak. Vaccination and biosecurity cannot fully prevent outbreaks, and developing disease resistant strains of salmon is high priority. Selective breeding can result in moderate improvements in disease resistance of salmon stocks and may take many generations. However, a revolutionary approach known as genome editing has potential to rapidly increase the rate at which disease resistant salmon can be produced. Genome editing involves the use of "gene scissors" to precisely cut the genome at a specific location, leading to small-scale targeted changes in the DNA sequence. In this proposal, genome editing technology will be used to investigate genes underlying resistance to ISAV, and potentially to produce a disease-resistant salmon. The first stage of the project is to identify target genes that will be edited. This will be achieved by measuring the ISAV resistance in a selective breeding program. Genetic markers dispersed throughout the salmon genome will then be used to map individual genes that contribute to variation in resistance in the population. Salmon from resistant and susceptible families will also be sequenced and to identify candidate genes and mutations causing this genetic effect on resistance to ISAV. In parallel to the 'forward genetic' approach described above, a 'reverse genetic' approach to identifying ISAV resistance candidates will be employed using cell culture models. A genome editing method known as CRISPR-Cas9 will be applied to destroy the function of key candidate ISAV resistance genes in the cell lines. Two methods of choosing candidate genes will be used. The first is based on prior knowledge of the biology of the interaction between the virus and the host cell, partly harnessing extensive research which has been performed on ISAV's close relative influenza. The second is to use the genes affecting natural resistance identified in the forward genetic screen described above. These edited cell lines will be infected with ISAV, and the impact of the edited gene on ISAV resistance and cellular response to infection will be assessed. This will build on an ongoing project to develop genome editing for salmon cell lines. Finally, genome editing will be used in Atlantic salmon embryos to test the highest priority ISAV resistance genes, especially where knockout of the gene has an impact on resistance in cell culture. Targeted editing of the genes will be performed by microinjecting newly fertilised embryos, which will be reared until the freshwater fry stage. These edited embryos, and unedited controls from the same family, will be challenged with ISAV. The nature and frequency of the edited genes in the resistant and susceptible salmon will be measured. This proposal has potential to create Atlantic salmon with resistance to a problematic viral disease (ISA) using a novel breeding technology. As such, it could have major animal welfare and economic impacts via prevention of outbreaks and subsequent culling of stocks. The approaches will be directly relevant to other viral disease in fish aquaculture. While the regulatory landscape for application of edited animals in food production is uncertain, a successful outcome of this proposal will provide a high profile example of the power of this technology to understand biology and to improve food security and animal health.

Farmed salmon is a major source of high quality protein and fatty acids essential for human health. Salmon aquaculture is worth approximately £1Bn to the UK economy, and supports many rural and coastal communities. However, sea lice are a major perennial problem for salmon aquaculture worldwide. These parasites attach to the skin of salmon and feed on tissue, mucus and blood. Infected fish show impaired growth and increased occurrence of secondary infections. They cause significant negative impacts on salmonid health and welfare, while lice prevention and treatment costs are a large economic burden for salmon farming, over £800M per annum. Encouragingly there is substantial genetic variation in resistance to sea lice both within and across salmonid species. While the commonly farmed Atlantic salmon are generally susceptible to infection, other salmonid species such as coho salmon are fully resistant. Improving the innate genetic resistance of the farmed salmon to sea lice is an environmentally friendly, but underexploited approach to lice control. Incremental improvements have been achieved via selective breeding of Atlantic salmon, but their long generation interval slows progress. Genome editing raises the possibility of rapidly increasing the resistance of salmon via precise targeted changes to their genomes; the key is knowing which specific genes to target. This project focusses on understanding the genetic mechanisms underlying resistance to sea lice, and identifying gene targets for genome editing to develop lice-resistant Atlantic salmon. To identify target lice resistance genes for editing, several different approaches will be taken, each exploiting the latest genomic technologies. Firstly, whole genome sequences will be obtained from a large population of farmed salmon on which sea lice counts following challenge have been collected. These will be used to map individual genes that contribute to variation in resistance in the commercial Atlantic salmon population. Secondly, it is known that the mechanisms underlying resistance to sea lice are due to a successful localised immune response close to the attachment site of the louse. Therefore, a detailed gene expression comparison of the immune response of Atlantic and coho salmon in the first four days following a lice challenge will be undertaken, using single cell sequencing approaches to highlight different responses in distinct cell populations at louse attachment sites. This will be complemented by profiling of the gene expression of the lice, and identification of potential immunomodulatory proteins and their targets in the host. Thirdly, genome editing approaches will be used to assess the impact of perturbing candidate resistance genes on response to sea lice both in cell culture and in the fish themselves. The former will be used to assess the cellular response to proteins secreted from the sea lice, and the consequences of knocking out each of the target genes on that response. This will lead to a final set of target genes for editing in salmon embryos, after which the edited fish will be challenged with sea lice. The resistance of the edited fish compared to full sibling control fish will then be assessed. The scientific programme of the project will be complemented by co-development of a strategy for the breeding and dissemination of edited lice resistant salmon, together with industrial partner Benchmark PLC. Furthermore, public and stakeholder engagement events are planned to communicate the research plans and outcomes, with a particular focus on the benefits and risks of genome editing in aquaculture. A successful outcome of lice resistant salmon would have major animal welfare and economic impacts via prevention of outbreaks, and removal of the need for chemical treatments. It would also provide a high profile example of the power of genome editing technology to understand biology and to improve food security and animal health.