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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - EPIFISH (INNOVATIVE EPIGENETIC MARKERS FOR FISH DOMESTICATION)

Teaser

Summary

Domestication of animals as a reliable food source was a critical point in human history and it is still shaping our society nowadays. Farming of domesticated fish species is one of the fastest growing food production sectors worldwide, since fisheries are unable to meet the increasing demand for fish protein to feed a growing global population. In order to ensure sustainability of the aquaculture sector it is critical to selectively improve the major commercial fish species. However, selective breeding has only been developed for relatively few fishes and the genetic markers used in these programmes cannot explain the observed variation in physical characteristics, such as body size. The EPIFISH project INNOVATIVE EPIGENETIC MARKERS FOR FISH DOMESTICATION adopts a multidisciplinary approach to address fish domestication and selection 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 captivity 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. Specific objectives are as follows:
1. To determine how selection for growth affects the epigenetic landscape (DNA methylation and hydroxymethylation) and the expression of non-coding RNAs (miRNAs and circRNAs) during domestication.
2. To perform a functional characterization of miRNA variants and epigenetic alleles associated with improved growth.
3. To validate the potential epigenetic markers of growth for future selective breeding programmes.

Work performed

The EPIFISH 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 (Figure 1). Fertilised eggs collected from the mouth of several females were transported to the research station at Nord University in BodÃ¸ (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. The same procedure was followed for two generations of selection and our selective breeding programme is now in its third generation. Samples of muscle and other tissues directly involved in regulating growth (e.g., liver and pituitary) were humanely collected from control and selected fish from each generation, as well as from wild fish.
Using next-generation sequencing to compare gene expression levels in muscle, we found over 2,700 protein-coding genes that were differentially expressed between wild tilapia females and their progeny reared in captivity. In general, domestication was associated with a decrease in transcript levels of genes involved in the immune response, which is likely explained by the reduced number of pathogenic bacteria, fungi, viruses and parasites 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. We are currently performing a large genome-wide association study to determine to what extent genetics can explain the changes in growth and muscle structure that occur during domestication.
While ascertaining the importance of non-coding RNAs in growth improvement, 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 expressed 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 are also investigating circRNAs, which are a relatively new type of non-coding RNAs that can act as miRNA sponges. Using our newly developed pipeline for in silico prediction of circRNAs, we have discovered that they are differentially expressed between wild females and their progeny reared in captivity.
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. While some were common to both males and females, there were several differentially methylated genes that were sex-specific, indicating that the epigenetic regulation of growth involves different networks in males and females. Another study comparing wild and domesticated fish revealed that differentially methylated genes were mainly associated with muscle growth, epigenetic mechanisms, immunity and diet (Figure 2). Interestingly, many genes that were related to growth were also involved in immune processes, suggesting that modulation of growth rate during the early stages of domestication could directly affect the immunological performance of fish. These results also indicate that diet likely modulates the muscle epigenome at early stages of domestication. We have also investigated a closely related 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 throughou

Final results

The EPIFISH project has made a major contribution to our current understanding of the epigenetic mechanisms associated with animal domestication and improved growth (Figure 3). In particular, the main results beyond the state of the art are the following:
1. The identification of differentially hydroxymethylated cytosines in muscle of Nile tilapia after a single generation in captivity provided the first experimental evidence that hydroxymethylation is likely involved in domestication.
2. The pioneer single-cytosine resolution studies of methylation in fast muscle of Nile tilapia in the context of growth and the early domestication revealed that the epigenetic regulation of growth in Nile tilapia involves different gene networks in males and females. Our results also highlighted the potential impact of diet on the epigenome at the very early stages of domestication.
3. The identification of novel candidate epimarkers (i.e., miRNAs and differentially methylated cytosines) associated with growth.
4. The discovery of circRNAs differentially expressed between wild fish and their offspring after just one generation in captivity adds a novel dimension to the complex molecular network underlying domestication.

In the near future, I will determine if there any changes in muscle structure during the domestication process. For the remainder of the project, I expect to perform a functional characterization of the key miRNAs and DNA methylation marks identified above. This 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. I will also validate the epimarkers in Nile tilapia and European sea bass, which will provide an innovative basis for a more effective genomic selection and increased sustainability of the aquaculture industry.