INTEGRATED SEASONAL EPIGENOMICS AND TRANSCRIPTOMICS UNCOVER AND VALIDATE NOVEL THERMAL-RESPONSIVE MARKERS IN GILTHEAD SEA BREAM
Introduction
Global warming is rapidly reshaping marine ecosystems by increasing the frequency and intensity of thermal anomalies, thereby posing a major challenge to both wild and farmed fish in the Mediterranean basin, a recognised climate-change hotspot where warming rates exceed the global average (Atalah et al., 2024). Because temperature is a key driver of growth, metabolism, welfare, and disease resistance, Mediterranean aquaculture species such as gilthead sea bream (Sparus aurata) are particularly vulnerable to both seasonal variability and extreme thermal events. However, despite its importance, thermal tolerance remains a complex and multifactorial trait whose molecular basis is still poorly understood in most farmed fish (McCaw et al., 2020), especially with regard to the interplay between transcriptional and epigenetic regulation. In this context, integrative omics approaches have proven valuable for identifying robust welfare-related markers in gilthead sea bream (Belenguer et al., 2024), but their potential to elucidate the molecular mechanisms underlying seasonal thermal acclimation remains largely unexplored. To bridge this gap, we investigated a gilthead sea bream population that endured record marine heatwaves during the summer of 2022 while maintaining normal growth and production performance. Building on this, we integrated long-read epigenomic profiling with short-read transcriptomics across contrasting seasonal temperatures in metabolically active tissues. This approach enabled the identification of key molecular markers and regulatory mechanisms underlying thermal acclimation, which were subsequently validated in independent populations with the same genetic background under different warming and cooling scenarios.
Materials and methods
Gilthead sea bream from three production batches (2021, 2023, 2024) originating from the same Mediterranean hatchery (Avramar, Burriana, Spain) were reared from early life stages (~5 g) at the IATS experimental facilities. The first batch was assigned to the SEASON experiment, in which fish were maintained in 3000 L tanks from May 2022 to February 2023, with tissue sampling conducted in summer (July 2022; 28 °C; ~70 g) and winter (February 2023; 12.4 °C; ~340 g). Total DNA and RNA from white skeletal muscle (WSM) and liver were extracted and sequenced using Oxford Nanopore Technologies (PromethION 24) and Illumina NovaSeq 6000 platforms, respectively. DNA reads were basecalled with a modification-aware model, 5mC methylation (CG context) was identified using Modkit, and differentially methylated sites (DM5mC) were detected with MethylKit. RNA reads were mapped to the CSIC reference genome, and differentially expressed transcripts (DETs) were identified (FDR < 0.05). Integration of DETs with differentially methylated genes, combined with negative correlation analyses, regulatory-region filtering, and gene enrichment, enabled the identification of key molecular markers in both tissues. Additional filtering in WSM resulted in 23 candidate thermal-responsive genes (cTRGs), which were functionally validated as thermal responsive genes in separate trials, using fish from the 2023 and 2024 batches. In the WARMING-WINTER trial (February 2025), fish of ~360 g body weight were acclimated at 14 °C and then gradually exposed to 20 °C and 26 °C (~3 °C/day), with and intermediate sampling point. In the WARMING-SUMMER trial (June 2025), juvenile fish (~18 g) were exposed to either progressive warming (20–26 °C) or constant temperature (20 °C), followed by a common increase to 30 °C. In the COOLING-SUMMER trial (September 2025), fish (~184 g) acclimated at 28 °C were subjected to rapid cooling (~3 °C/day) to 22 °C and 16 °C, with sampling at each temperature step. Oxygen saturation remained above 75% in all trials.
Results and Discussion
Long-read ONT sequencing generated 825 Gb of data (avg. 29x genome coverage), detecting methylation at 78% of CG sites, with a prominent fraction found in gene bodies and promoter regions, confirming the suitability of the platform. Parallel RNA‑seq identified 33,807 active transcripts. Seasonal comparisons revealed 6,356 DETs and 195k DM5mC in liver and 7,651 DETs and 302k DM5mC in the WSM. Omics integration and robust filtering highlighted 148 differentially expressed and methylated genes (DEMGs), as a reason of 51 in Liver and 101 in WSM (Fig. 1). Indeed, muscular responses were higher at all layers and a diverse spectrum of functions, such as response to light, oxygen, and metabolic cues, were found as enriched. The 36 WSM cTRGs (23 from multi-omics and 13 extra genes) were further validated in the thermal-only trials. Of note, 31 out of 33 responded at least in one of the trials, becoming TRGs. PERMANOVA and PLSDA approaches showed a major temporal dynamic where the CTRL/initial sampling times were, regardless the trial, separated the most from the final points where temperature is maximum/minimum or has persisted. Once responsiveness and temporal dynamics were accredited for TRGs, our work further disclosed the major opposite expression change between WARMING and COOLING trials, mainly driven by up to 12 TRGs (cs, ghr1, ghr2, grp170, igf1r, pcna, slc27a4, sod2, spred2, stk11, usp4, usp7). These genes, involved in energetic, oxidative stress, cell proliferation and protein turnover cues, form an integrated molecular network that adjusts muscle physiology to thermal challenges. Altogether, this work opens novel perspectives to inform the effects of marine heatwaves in Mediterranean species and provides a mechanistic framework for understanding thermal resilience, supporting the development of genomic tools for climate‑resilient aquaculture.
Fig. 1. Graphical abstract of the main results in this study.
Funding: BreamHOLOBIONT (PID2023-146990OB-I00); TOMACUA (CIAICO/2024/281); CSIC-MOMENTUM (MMT24-IATS-01-01).
References: Atalah et al., 2024 Aquaculture, 588:740917; Belenguer et al., 2024 IJMS, 25:9836; McCaw et al., 2020 Integ. Comp. Biol. 60:1469-1480.