Introduction
Atlantic salmon ( Salmo salar ) is an anadromous fish species, born in freshwater , spend most of the life in seawater and thereafter return to freshwater to spawn. For salmon aquaculture, female broodstock are kept in seawater for initial sexual maturation and transferred to freshwater few months prior to spawning. To keep the continuation during production cycle for aquaculture, it is important to have access to new generations of salmon throughout the year.
By adjusting the time for transferring sexually mature broodstock female from sea cages to land-based freshwater cages, the breeding companies have developed protocols to expand the spawning season. Water temperature, feeding and light regimes are abiotic environmental factors that can either accelerate or prolong the time until spawning. However, adjustments in production protocols might introduce both beneficial traits but also functional changes in tissues due to poor nutrition during organ development for the new generation according to the Barker hypothesis (Reviewed by Heindel and Vandenberg, 2015) . This phenomenon is called intergenerational programming whereby the nutritional status of the parents influences the next generation ( Reviewed by Heard and Martienssen, 2014).
The 1C metabolism, which includes choline, vitamin B6, vitamin B12, and folate, as well as the amino acid methionine, has previously shown to influence broodstock fecundity, but also liver lipid phenotype, metabolism and epigenetic gene regulation in mature zebrafish progeny (Skjærven et al., 2018) . For s almon, a shift from normal spawning period by five months in RAS systems disturbs the nutritional status and gene expression in both the female broodstock and their offspring, which results in less allocated nutrients into RAS spawned eggs followed by a deprived growth by the time for first feeding (Skjaerven et al., 2020). Here, we continue by investigating the nutritional status in broodstock and offspring when transferring sexually mature female from sea cages to land-based freshwater cages but thereafter adjusting the abiotic factors to obtain two months earlier and later spawning than normal spawning in November. The aim of this project was to investigate if the offspring seasonal groups had differences in their epigenetic DNA methylation profile, gene expression regulation and nutritional status and also link these results with offspring growth.
Material and methods
Atlantic salmon broodstock normal spawning in November was compared with two months earlier or later spawning . We measured the nutritional status in broodstock liver and muscle, and the offspring nutritional profile during development. DNA and mRNA were extracted from offspring liver tissue to investigate the epigenetic DNA methylation profile (method: Reduced R epresentation B isulfite S equences (RRBS)) and gene expression regulation (method: RNA sequencing).
Results
The present study revealed that the seasonal changes significantly alter the nutritional level of the nutrients in both broodstock and offspring. Early spawning broodstock incorporated less nutrients into the eggs but the measures revealed a sufficient broodstock nutrient status. Late season spawners had similar nutritional status in offspring as normal spawn ers. Broodstock liver and muscle from late season broodstock indicate hunger. Enrichment analyses from mRNA sequencing revealed that genes controlling cell cycle and proliferation were significantly altered between spawning groups . Comparing early and normal spawning season revealed around 3500 differentially methylated cytosines. Especially interesting was the gene ctl2b , encoding the choline transporter, which had 9 significant hypermethylated CpG sites in promoter , 5 of which had 20% more methylation in the early spawning group.
Discussion and conclusion
We believe our results provide an important understanding of the interplay between the abiotic environmental factors and nutritional status which together control the intermediary metabolism regulating growth and robustness from broodstock to next generation. Our in-depth study not just indicates a need for adjusting the broodstock feed to comply with the nutritional needs when changing abiotic factors, but also shows us how fragile and sensitive marine organisms are to a changing climate.
Acknowledgements
We are thankful to the technical staff at IMR, Nord University and AquaGen AS for assistance . This research was financed by IMRs Nutritional programming project and The Research Council of Norway (NutrEpi 267787).
References
Heindel and Vandenberg, 2015. Developmental Origins of Health and Disease: A Paradigm for Understanding Disease Etiology and Prevention , Curr Opin Pediatr. Doi: 10.1097/MOP.0000000000000191
Heard and Martienssen, 2014. Transgenerational epigenetic inheritance: myths and mechanisms. Cell 157, 95-109. Doi: 10.1016/j.cell.2014.02.045
Skjærven, Jakt., Fernandes, Dahl, Adam, Klughammer, Bock, Espe, 2018. Parental micronutrient deficiency distorts liver DNA methylation and expression of lipid genes associated with a fatty-liver-like phenotype in offspring. Scientific Reports 8. Doi: 10.1038/s41598-018-21211-5
Skjærven., Oveland, Mommens, Samori, Saito, Adam, Espe, 2020. Out-of-season spawning affects the nutritional status and gene expression in both Atlantic salmon female broodstock and their offspring. Comp Biochem Phys A 247. Doi: 10.1016/j.cbpa.2020.110717