INNOVATIVE EPIGENETIC MARKERS FOR FISH DOMESTICATION

Domestication of animals as a reliable food source was a critical point in human history and it is still shaping our society nowadays. Fish farming is one of the fastest growing food production sectors worldwide, since fisheries are unable to meet the increasing demand for fish protein. In order to ensure sustainability of the aquaculture sector it is critical to selectively improve the major commercial fish species. The EPIFISH project INNOVATIVE EPIGENETIC MARKERS FOR FISH DOMESTICATION adopts a multidisciplinary approach to address fish domestication at the research frontier, based on the innovative hypothesis that epigenetic mechanisms (e.g., DNA modifications and non-coding RNAs) are involved in selection and adaptation of fish to farming conditions. The overarching aim of EPIFISH is to ascertain the importance of epigenetics in fish domestication using the Nile tilapia (Oreochromis niloticus) as model species, since tilapias are the second most important group of farmed fish. The project started with a field trip to Alexandria and Luxor in Egypt. The team spent 7 weeks with local fisherman capturing live Nile tilapia using traditional methods, such as traps. Fertilised eggs collected from the mouth of several females were transported to the research station at Nord University in Bodø (Norway), where they were reared in a state-of-the-art recirculating aquaculture system. At 5 months old, the fish were separated in two groups according to their weight: a control line comprising individuals of average weight and a selected line of fish that were at least 10% larger than average. We have completed the third generation of selection, which was the goal of our selective breeding program. All the required samples have been humanely collected and we are now determining how the muscle fibres changed. Using next-generation sequencing to compare gene expression levels in muscle, we found several thousand protein-coding genes that were differentially expressed between wild tilapia females and their progeny reared in captivity. In general, domestication was associated with reduced expression of genes involved in the immune response, which is likely explained by the lower number of pathogens in the research station compared to wild conditions. There was a concomitant up-regulation of genes related to metabolic processes and muscle development, which may account for the improved growth of domesticated fish and the observed changes in muscle cellularity. We have identified several miRNAs and isomiRs that are known to target genes involved in metabolic pathways and muscle development. Several of these miRNAs were expressed at different levels in muscle and blood serum between slow- and fast-growing Nile tilapia. They are promising candidates for epigenetic markers (epimarkers) of growth, since their expression is strongly correlated to fish weight. We have also expanded our research to circular RNAs (circRNAs), which are a novel type of non-coding RNAs. To this aim, we have developed a bioinformatic pipeline for circRNA host gene prediction and discovered several circRNAs that are related to growth. After optimising a protocol to cover a larger proportion of methylated cytosines in the genome, we have obtained a high-resolution, genome-wide methylation map in fast muscle of Nile tilapia. We have found many cytosines that were differentially methylated between small and large fish of the same age and that were associated with muscle growth. Another study comparing wild and domesticated fish revealed that differentially methylated genes were mainly associated with muscle growth, epigenetic regulation, immunity and diet. In parallel, we have obtained the first mitochondrial methylome map in a non-model fish species. We have also investigated a DNA modification termed hydroxymethylation, since there is increasing evidence that it is a stable epigenetic mark. Our genome-wide profiling revealed that hydroxymethylation is a ubiquitous DNA modification throughout the nuclear and mitochondrial genomes both in muscle and liver. Nile tilapia domestication was associated with a decrease in muscle hydroxymethylation, especially in genes related to growth and immunity. Several genes involved in metabolism and growth were differentially hydroxymethylated between pituitary, liver and muscle. Functional characterization of the key miRNAs, circRNAs and DNA methylation/ hydroxymethylation marks that we identified can be performed using the microinjection and in vitro fertilization protocols that we developed. These data will provide ground-breaking mechanistic insights into the role of epigenetics in fish domestication, which will surely open new horizons for research in transgenerational inheritance and nutritional epigenetics. Moreover, the novel epimarkers will provide an innovative basis for a more effective selection and increased sustainability of the aquaculture industry.

The outcomes from EPIFISH have provided novel mechanistic insights into the role of epigenetics in fish domestication. In particular, we showed for the first time that differentially hydroxymethylated cytosines between wild and captive fish are present in genes involved in immune-, growth- and neuronal-related pathways. We also published the first single-cytosine resolution study of methylation in fast muscle of Nile tilapia in the context of growth and the early domestication process in fish, reporting the identification of muscle-related genes that were differentially methylated between slow- and fast-growing fish. Our data demonstrated that the epigenetic regulation of growth in Nile tilapia most likely involves different gene networks in males and females. There were also several differences in expression levels of non-coding RNAs (miRNAs and circRNAs) that were linked to growth performance. These findings help us to understand how animals can adapt so rapidly to captivity. In addition to representing a leap forward from the state of the art in understanding the molecular basis of fish domestication and selective breeding, these results open new horizons for future frontier research in epigenetics, namely within the fields of evolutionary biology, transgenerational inheritance and nutritional epigenetics. The growth-related non-coding RNAs and epialleles identified in EPIFISH can have a major impact on aquaculture biotechnology, since they may enable the development and application of epigenomic selection as a new feature in future selective breeding programmes. These epimarkers have the potential to cover a substantial part of the unknown variability that is missed by current selection methods and will therefore provide a much more efficient selection for robust fish (e.g., disease resistant and high growth performance), which will translate into a tremendous gain in profitability and sustainability of the aquaculture sector. The results from EPIFISH directly contribute to the UN Sustainable Development Goals i) Zero hunger (SDG2), ii) responsible consumption and production (SDG12) and iii) Life below water (SDG14).

Domestication of plants and animals has been critical to expansion of the human population and emergence of modern society as we know it. Remarkably, the number of domesticated fish species of commercial relevance is very limited, even if aquaculture is the fastest growing food production sector in the world, accounting for almost 50% of the fish for human consumption worldwide. In order to ensure the sustainability of the aquaculture industry it is critical to domesticate and selectively improve the major commercial fish species. To date, the molecular basis of domestication is poorly understood and the genetic markers used in selective breeding of fish account only for a fraction of the observed phenotypic variation. EPIFISH is a scientifically innovative and timely project that will address fish domestication and selection from a new perspective using a multidisciplinary approach. The rapid pace of substantial phenotypic changes during adaptation to new environmental conditions in fish undergoing domestication raises the original hypothesis that epigenetic mechanisms are involved in this process. Thus, the overarching aim of EPIFISH is to ascertain the importance of epigenetics in fish domestication using the Nile tilapia (Oreochromis niloticus) as model species. The identification of epigenetic markers (DNA methylation marks and miRNA variants) will be a ground-breaking element of EPIFISH with major impact on aquaculture biotechnology, since they will enable the development and application of epigenomic selection as a new feature in future selective breeding programmes. Moreover, the project outcomes will provide novel mechanistic insights into the role of epigenetics in fish domestication, which will surely open new horizons for future frontier research in epigenetics, namely transgenerational inheritance and nutritional epigenetics.