Epigenetic and transcriptional effects of optimizing 1-C nutrients to improve healthy growth in Atlantic salmon

Improving fish health through epigenetic modulation is a new approach for phenotypic plasticity. Nutrition-based epigenetic changes can lead to lifelong phenotypic changes in economically important traits for the aquaculture. Early stages of development and the smoltification process represent sensitive periods for epigenetic changes on the genetic material that guide a number of traits towards health. DNA methylation (meDNA) is an epigenetic mechanism that controls gene expression (GE) for tissue type and cell type, but it can also be used to regulate metabolism in relation to changes in the cellular environment such as altered micronutrient levels. In this project we have especially focused on the 1C nutrients (1C): methionine, folate, vitamin B6 and vitamin B12. The hypothesis was that 1C influences GE patterns for healthy salmon through epigenetic mechanisms, especially meDNA. The goal is to increase the potential for healthy growth in early life through the optimization of feed-induced epigenetic regulation. This was achieved by studying gonad / liver (WP1), eggs (WP2) and smoltification (WP3). In WP1, we studied the meDNA profile of salmon that have received three levels of 1C in the feed. The results show that 1C levels affect GE as well as epigenetic regulation in both liver and gonad tissues. Liver meDNA and GE are affected dose depending on 1C given in the feed (Saito et al., 2021a). In gonads, it was found that amount of 1C affects the meDNA of the DNA (Saito et al., 2021b). The overall picture shows that the two groups that received more 1C were more similar to each other in relation to the control feed that had the lowest level of 1C. This was different from liver which responded to the content of the feed in a more dose dependent meDNA response. We have shown here that the gonads of male fish respond to the feed and change the level of meDNA. This potentially influences growth and development for the next generation. The hypothesis required that we bioinformatically investigate epigenetic data from nucleotide sequencing and align to the genome-duplicated genetic material. The methods for this analysis have been reported. In WP2, the aim was to evaluate "seasonal variation" of nutrient status in offspring from AquaGen and correlate with GE and meDNA. We examined two groups of eggs that were spawned two months earlier or later than the normal spawning season in November. In addition, we had the opportunity to include an extra group of offspring. The last group was spawned five months before normal spawning in RAS systems. The studies show 1C nutritional differences between spawning season (Skjærven et al., 2020, Skjærven et al., submitted). At the European Aquaculture 2021, we presented that meDNA profiles and GE have changed significantly in the different batches of eggs (Skjaerven et al., 2021). For WP3, the goal was to optimize 1C in feed during smoltification to elucidate any changes in growth, 1C and epigenetic regulation. The feeding experiment was carried out by Skretting ARC. We had three levels of 1C that we fed six weeks before and three months after smoltification. The data showed that a little more 1C than what is recommended today for salmon gave better growth after smoltification. The fish that received the highest levels did not grow as well and are believed to use energy to get rid of the extra amounts of 1C. The 1C feed changed the levels of free amino acids (FAA) in both liver and muscle and more FAA was released for growth for the fish that got medium level in the feed. Furthermore, the analyzes for 1C and FAA in liver and muscle showed that the levels are affected by the feeds (Espe et al., 2020). Three months after smoltification, we saw major changes in how liver cells could cope with an immune response in cell culture study (Espe et al., 2019) as well as GE in muscle (Espe et al., 2020). Adam was granted a post-doc scholarship for further studies on muscle (NFR project: 295118). We have analyzed fish from all three feed groups for epigenetic analyzes which show that there are great effects of the feed on methylation of DNA. We have shown that micronutrient status affects the epigenome via nutrition-based programming for broodstock, early life stages (WP2), smoltification (WP3) and growth (WP1). For communication and dissemination, we arranged a kick-off meeting, we have had regular project meetings, and an epigenetic workshop in Tromsø. The publications have been presented on conferences, hi.no, forskning.no, intrafish.no and a popular science article in NFexperts. We published an chronicle on forskning.no with the title: "Count micronutrients instead of calories this Easter". The WP2 publication was also presented as a headline news item on hatcheryfm.com. This project here led to new collaborations both nationally and internationally. The findings have had positive effects for both the industry, new companies and for the research community.

We have had the opportunity to work interdisciplinary by combining nutrition and developmental biology with epigenetic analyzes for Atlantic salmon. The project outcomes provide wide range coverage in quality-guaranteed reports to the project participants and surroundings. We have shown that micronutrient status affects the epigenome via nutrition-based programming in different phases: broodstock (WP2), early life stages (WP2), smoltification (WP3) and growth (WP1). The knowledge is of interest for breeding and feed companies and led to new protocols that focus on handling procedures considering the epigenetic signatures of farmed fish. We have communicated and presented the results in both national and international conferences (WP4). The results gained have led to new projects and establishment of new companies which focus on the epigenetic profiles of aquaculture species. The projects have also led to new international collaborations with both other research groups and companies.

Increasing the health quality represent a potential for reducing the high mortality in Atlantic salmon production. Nutritional DNA methylation changes can lead to irreversible life-long phenotypic effects, and development represents a sensitive period for the establishment of improved health quality through new DNA methylation patterns. The 1-C nutrients, methionine, folate, vitamin B6 and B12, are responsible for the transfer of the methyl group to DNA and thereby directly influence the epigenetic regulation of the gene expression. The hypothesis of this project is that the composition of 1-C nutrients affects the gene expression patterns for healthy growth (increased growth/immune capacity, and decreased fat accumulation) through epigenetic mechanisms, particularly DNA methylation. The aim is to increase the potential for healthy growth in early life stages through optimizing the dietary-induced epigenetic regulation of gene expression. This will be achieved through improved 1-C nutrient composition in a) gonads, b) eggs and c) smoltification. This will be reached by linking metabolomics, transcriptomics and epigenomics with traditional growth and histological studies for in depth studies of nutritional composition of parental feed, yolk sac nutrients or smoltification feed that sets the future potential for healthy growth. This is in line with the needs for aquaculture which is knowledge on how micronutrient availability adjust the potential for healthy growth and reduce fat accumulation through epigenetic regulation of gene expression as recently shown by us, using zebrafish as a model. We have demonstrated dietary-induced manipulation of epigenetic regulation through generations for zebrafish, and now we apply to study epigenetic regulation for salmon.