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 Bodø (Norway), where they were reared in a state-of-the-art recirculating aquaculture system for three generations. 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.
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. There was an up-regulation of genes related to metabolic processes and muscle development, which may account for the improved growth of domesticated fish. A large genome-wide association study identified outliers that explain to what extent genetics contributes to growth changes during domestication.
We have identified several circRNAs, miRNAs and isomiRs that target genes involved in metabolic pathways and muscle development. Several of these non-coding RNAs are expressed at different levels in muscle and blood serum expressed between slow- and fast-growing Nile tilapia. Hence, they are promising candidates for epigenetic markers (epimarkers) of growth, since their expression is strongly correlated to fish weight. Using our newly developed pipeline for in silico prediction of circRNAs, we have discovered novel circRNAs associated with domestication.
After optimising a protocol to cover a larger proportion of methylated cytosines in the genome, we obtained a high-resolution, genome-wide methylation map in fast muscle of Nile tilapia. The mitochondrial methylome in fast muscle was determined by whole genome bisulfite sequencing. 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).
We 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 throughout the nuclear and mitochondrial genomes both in muscle and liver. Nile tilapia domestication was associated with a generalised decrease in muscle hydroxymethylation.
An artificial breeding protocol was developed to obtain fertilized eggs for the functional assays. After optimizing the microinjection and egg incubation methods, we microinjected sgRNA and Cas9mRNA into hormone-induced at the single-cell stage. Finally, high-resolution melting assays were developed to determine differences in levels of the candidate 5mC and 5hmC epimarkers in muscle from slow- and fast-growing fish, and primer sets for each miRNA target were optimized for specific quantification. Their potential as markers of growth in Nile tilapia has been assessed in muscle and blood serum with the aim of developing a reference panel of epimarkers.
The 21 publications from EPIFISH, including MSc and PhD theses, have been published as Gold Open Access or are registered in public databases, and next-generation sequencing data have been submitted to public repositories. The results were presented to the academic community in 47 communications, as well as in workshops, trade magazines, newspaper articles and open science events for the general public. We have worked closely with one of the major Nile tilapia producers (Genomar) and Nord Innovasjon to identify intellectual property assets during the project. A patent has been prepared for a multi-panel of epigenetic markers (non-coding RNAs and DNA methylation/ hydroxymethylation) of improved growth.
