Introduction
The application of continuous light (CL) acts as a potential regulator of delayed puberty in aquaculture Atlantic cod (Gadus morhua). However, the epigenetic mechanisms underlying this process have not yet been studied. DNA methylation is an epigenetic modification that enables organisms to respond to stress or environmental changes and regulates gene expression, thereby influencing the phenotype. It is known that gene expression can increase or decrease depending on the genomic location of DNA methylation, and it often causes gene silencing when present in promoter regions. In this context, this study analyzed the transcriptomic and epigenetic profile of Atlantic cod under CL conditions, with the aim of elucidating the molecular and hormonal factors that promote increased growth and delayed sexual maturation.
Materials & Methods
Sixteen-month old Atlantic cod were divided into two groups and exposed to either ambient light (AL) or CL for 6 months. To investigate the interplay between the epigenome and transcriptome, 36 liver samples (18 females and 18 males) were analyzed. Global DNA methylation was mapped at single-cytosine resolution via enzymatic methylation sequencing (EM-seq), with differentially methylated regions (DMRs) and sites (DMCs) identified using the DSS R package. RNA-seq libraries were prepared using the NEBNext Ultra II Directional kit and raw counts were used to identify differentially expressed genes (DEGs) across treatment groups using the R package Limma. To relate DNA methylation patterns to gene expression, genes exhibiting both differential methylation (DMG) and changes in DEG were selected and termed differentially methylated and differentially expressed genes (DEMG). The correlation between DMCs levels and DEG was evaluated using a integration analysis, with the following significance thresholds: DMCs were defined as sites with a minimum methylation difference of 15% and a False Discovery Rate (FDR) < 0.05, and a |Log2 fold change (FC) ≥ 1 and P adj < 0.05 (Benjamini–Hochberg multiple test correction method) for DEGs. The correlations and data distribution were visualized in the R using the ggplot2 package.
Results
Transcriptome analysis identified 1,527 DEGs, in females exposed to CL compared to their counterparts reared under AL. They were enriched in pathways such as "Biosynthesis of unsaturated fatty acids," "Fatty acid metabolism," and "PPAR signaling pathway". In males, 1,788 DEGs were identified, and the metabolic pathways were enriched for "DNA replication" and "ECM-receptor interaction", suggesting a sex-specific response to photoperiod. Methylation analysis identified a total of 155 DMCs, which were located in the following genomic regions: 88 in promoters, 25 in introns, and 28 in distal regions in females. In contrast, males exhibited a total of 201 DMCs, which were distributed across 76 introns, 68 promoters, and 57 distal regions. A comprehensive analysis of DNA methylation and transcriptome data from Atlantic cod liver identified genes that exhibited differential methylation and expression. In males, the intersection focused on cellular homeostasis and neural support genes, such as gpx3 (glutathione peroxidase 3) and smpl (schwann cell myelin protein-like). Conversely, females exhibited regulation of apoptotic and metabolic signaling pathways, highlighted by casp6 (caspase-6) and c1qtnf6b (C1q and TNF related 6b). These results demonstrate that CL triggers a complex epigenetic-transcriptomic interplay that may reconfigure liver metabolism, providing new insights into the sex-specific response to photoperiod, due to interactions between epigenetic modifications and gene expression.
Acknowledgment
This study received funding from the Research Council of Norway under the Researcher Project for Scientific Renewal (EPICOD, project No. 336112), the Spanish Ministry of Science and Innovation grant PID2023-146286OB-I00 "Holosex" and FPI contract PRE2022-101795 associated to grant CEX2019-000928-S-20-5 funded by AEI 10.13039/501100011033 (Spain).