Impact of photoperiod and sexual maturation on growth performance of Atlantic cod - a genomic perspective

The Growcod project was very successful and we have obtained a number of novel and exciting results, as summarised below. WP1 - TOOLBOX Several high-throughput next generation sequencing techniques (Illumina, 454, Ion Torrent and SOLiD) have been used to determine the gonadal and muscle transcriptomes of protein-coding and miRNA genes in Atlantic cod under different conditions. In addition, we have cloned and sequenced key muscle- and reproduction-related genes, including follistatin, myosin heavy chain, insulin growth factors, DNA methyltransferases (Dnmt), mixed linage leukaemia histone methyltransferases (Mll) and oestrogen receptors. These data constitute a powerful molecular toolbox, which will be most useful for further gene expression studies involving various aspects of muscle growth, circadian rhythmicity and reproduction in Atlantic cod and other fish species. We have also developed a myosatellite cell culture and muscle explants as in vitro models of muscle growth plasticity and circadian rhythmicity in Atlantic cod. WP2 - PHOTOPERIOD AND GROWTH/ WP4 - GENE CHARACTERIZATION In order to understand how light stimulates somatic growth, we have kept one group of juvenile cod under a simulated natural photoperiod for Bodø, whereas their counterparts were reared under continuous illumination. It was found that continuous light stimulated growth concomitantly with a decrease in mll and an increase in dnmt transcript levels. Moreover, we examined global mRNA and miRNA expression changes using next-generation sequencing. Our data revealed potential novel markers of growth and suggested that DNA and histone methylation may be involved in the epigenetic regulation of muscle growth by photoperiod in Atlantic cod. We have characterized the main molecular components of the circadian system in cod and in vitro models were used to study the effect of photoperiod manipulation on rhythmicity of these 21 clock-related genes. A transcriptome-wide approach unveiled that several mRNA and miRNA genes are expressed in a cyclic manner throughout the day/night cycle. One muscle gene in particular (unc45b) displayed conserved daily rhythmicity with its zebrafish counterpart but the morpholino knock-down did not produce an obvious phenotype in zebrafish. Our results point to the possible presence of a non-autonomous but functional clock system in cod muscle, which contributes significantly to our understanding of muscle growth regulation by photoperiod in this species. WP3 - MATURATION AND GROWTH/ WP4 - GENE CHARACTERIZATION We have also examined the global expression of mRNAs and miRNAs in fast muscle of Atlantic cod throughout a reproductive cycle in order to identify key genes that may account for the observed decrease in growth during sexual maturation. In particular, numerous myosin heavy chain genes were found to be differentially expressed, which may play a key role in muscle physiology and growth. Among the most abundant miRNAs throughout the reproductive cycle, mir-1 is especially interesting because it was up-regulated during the spawning season in female muscle and it may affect growth. We have found that three oestrogen receptors (esr) are differentially expressed between male and female cod and that they play an important role in differentiation of the gonads throughout their annual reproductive cycle. We have also compared miRNA profiles in ovaries and testis of immature versus mature fish. Several miRNAs were found to be sex-specific and may be related to sexual maturation in cod. We also discovered novel putative miRNAs whose precursors have a typical hairpin structure. If validated, these miRNAs may prove useful as sex-specific molecular markers, which could enable us to sex the fish before they mature and to characterize their maturation status.

Production of cod in aquaculture has seen a notable increase since 2000 and it is poised to expand, as wild cod stocks are at record low levels. Norway is the main cod producer worldwide but profitability of the industry is restricted by the reduction in growth associated with premature sexual maturation. This adds to production time, resulting in significant economic losses. In order to address this serious bottleneck we need to understand the molecular basis of muscle growth in cod but at the moment our knowledge in this area is very limited. We propose to identify the networks of genes that regulate growth in cod and to investigate how they are influenced by light and sexual maturation. The first step is to obtain the tools required for this study. In addition to a microarray chip, we will clone the cod key genes involved in muscle growth, development and biological rhythms. This molecular toolbox will then be used to examine the expression of over 5000 genes in response to light manipulation, to help us understand how it can have a direct influence on growth. We will also investigate expression changes during a whole maturation cycle, in order to identify genes involved in the growth impairment associated with maturation. Data from these large-scale g ene expression studies will be integrated with muscle growth patterns at a cellular level. The most promising candidate genes identified above will be further characterized. In particular, we will look at their expression in response to sex steroids, whic h are known to be present at high levels during maturation. Finally, we will examine the potential use of different light sources to control maturation in aquaculture conditions and their effect on expression of selected genes and muscle growth. This proj ect has potential applications in cod farming, since it may identify growth markers for selective breeding programmes and new targets to minimise the negative impact of maturation on growth performance.