EPIGENETIC EFFECTS OF ENVIRONMENTAL STRESS ON ATLANTIC SALMON AND COD

Introduction

Through epigenetic mechanisms gene expression patterns can permanently change without any modification of the underlying DNA sequence (Feil and Fraga, 2012). Teleost fish show a high degree of phenotypic plasticity in muscle development, growth rate and possibly stress coping abilities when subjected to different environmental conditions (e.g. Campos et al., 2013; Hutchings, 2011; Macqueen et al., 2008). Understanding the underlying epigenetic mechanisms is of great importance for the aquaculture industry but also for addressing the adverse effects of climate change on wild populations. We are currently investigating the epigenetic mechanisms in Atlantic salmon and cod subjected to handling or temperature stress during early life stages, and its effects on the phenotype in later stages.

Materials and Methods

A 2.5-year study with salmon is currently ongoing to assess the epigenetic effects of embryonic incubation temperature on muscle development and growth in 16 families selected for fast or slow growth rate. Eggs were incubated at 8°C or at 4°C until the eyed stage, thereafter all were reared at 8°C until start feeding followed by ambient temperatures (~2-13°C) and were transferred to sea cages in spring 2015. Samples wereobtained at consecutive time points.

Two short-term studies related to handling or temperature stress using salmon and cod were conducted. Salmon were either subjected to cold shocks and air-exposure once a week prior to hatching, post-hatching, pre- and post-hatching, or not (control). Cod embryos were reared at low (4°C) or high (10°C) temperature. At hatching, half of both groups were subjected to a temperature change, i.e. from low to high or high to low.

Using gene expression analysis (Illumina sequencing and qPCR) and methylation analysis (Reduced Representation Bisulfite Sequencing (RRBS) and pyrosequencing) we aim to profile the genetic patterns and gain insight in the mechanisms involved in muscle development and stress coping abilities. A bioinformatic pipeline for genome-wide methylation analysis has recently been established at Nofima.

Results and Discussion

Preliminary results from salmon subjected to different incubation temperatures show higher methylation levels in the promoter of the conserved muscle regulatory factor myogenin of the 4°C group compared to the 8°C group at start feeding stage (Fig. 1). Intriguingly, the methylation patterns in offspring from fish selected for slow growth rate were less affected by temperature. No or less pronounced differences in methylation levels between temperature or fast and slow growth groups were found at a later developmental stage, i.e. 5-10g juveniles. Methylation of the myogenin promoter in muscle tissue shows a decrease between start feeding larvae and 5-10g juveniles and is affected by temperature as well as growth phenotype. The relative expression of myogenin appears to confirm the methylation levels, although this needs to be further elucidated. A high positive correlation was found between myogenin and the DNA methyltransferase DNMT3a mRNA levels in both developmental stages, indicates the occurrence of de novo methylation. This correlation may indicate e.g. hypermethylation in the gene body which can result in activation of the gene (Jones, 2012), or other genes being down-regulated.

In handling stressed salmon, expression of stress-axis related genes was down regulated in the stress treated groups compared to that of the control. Additionally, methylation patterns of these groups show clear differences between stressed groups and the control. The epigenetic effect of the stressor appears to be maintained for at least two months and adaptation to the stressor is suggested. RRBS analysis of cod subjected to different temperature regimes revealed strong effects on the genome-wide methylation patterns.

Overall, the preliminary data show effects of environmental stress on gene methylation and expression patterns in salmon and cod.

References

Campos, C., Valente, L.M.P., Conceição, L.E.C, Engrola, S., Fernandes, J.M.O. 2013. Temperature affects methylation of the myogenin putative promotor, its expression and muscle cellularity in Senegalese sole larvae. Epigenetics, 8:389-397

Feil, R., Fraga, M.F. 2012. Epigenetics and the environment: emerging patterns and implications. Nature Reviews Genetics, 13:97-109

Jones, P.A. 2012. Functions of DNA methylation: islands, start sites, gene bodies and beyond. Nature Reviews Genetics, 13: 484-492

Hutchings, J.A. 2011. Old wines in new bottles: reaction norms in salmonid fishes. Heredity, 106:421-437

Macqueen, D.J., Robb, D.H.F., Olsen, T., Melstveit, L., Paxton, C.G.M., Johnston, I.A. 2008. Temperature until the 'eyed-stage' of embryogenesis programmes the growth trajectory and muscle phenotype of adult Atlantic salmon. Biology Letters, 4:294-298