Introduction
In the current global population growth and climate change, there is an urgent need to supply the global fish demand while addressing key challenges in aquaculture , particularly those related to the immune response in farmed fish, which affects their vulnerability to infections. The European sea bass (Dicentrarchus labrax) is one of the most widely cultured species. According to APROMAR (2023 ), in 2021, this species ranked as the fifth most farmed in Europe and has shown a steady increase in production, rising from 82,825 tonnes in 2020 to 96,647 tonnes in 2021 across Europe.
Sexual dimorphism has been shown to influence vertebrate immunity through various factors, whose effects are mediated by epigenetic mechanisms, such as DNA methylation, non-coding RNAs, and histone modifications (Sasaki and Matsui, 2008). However, this relationship in fish remains underestimated and poorly studied (Caballero-Huertas et al., 2024). Our study uses cutting-edge techniques to investigate differences in genome-wide
DNA methylation patterns in the context of a sex-related immune response.
Methodology
Samples f rom the gonads of European sea bass were collected 48 hours after bacterial infection with Vibrio anguillarum . Subsequently, genome-wide DNA methylation analysis was performed using the NEBNext® Enzymatic Methyl-seq Kit (EM-seq™) protocol with triplicate samples of each condition and gonadal tissue. Methylation patterns were assessed at each CpG site. Differentially methylated regions (DMRs) and differentially methylated loci (DMLs) were then identified for each comparison: between treatment conditions within the same sex (INF_F vs CTRL_F and INF_M vs CTRL_M), and between sexes under the same condition (CTRL_F vs CTRL_M and INF_F vs INF_M). After that, the different gene regions that overlapped with the DMRs and DMLs were found. Finally, these genes were annotated using the European sea bass genome as a reference, followed by functional enrichment analyses to identify associated Gene Ontology (GO) terms.
Results
Vibrio anguillarum induced changes in the gonadal DNA methylation pattern across all comparisons. The highest number of differentially methylated CpG sites was observed when comparing sexes under the same condition, specifically in CTRL_F vs CTRL_M and INF_F vs INF_M, with 88,029 and 105,053 hypermethylated CpGs , and 2,829,573 and 2,752,371 hypomethylated CpGs, respectively. Our results suggested that there are more differences in DNA methylation between sexes under basal and infected conditions than after immune activation within the same gonadal tissue. Notably, the highest number of differentially hypermethylated CpGs was found after immune activation when comparing females to males, further highlighting the importance of sexual dimorphism in immune responses. When comparing the infected group against the control group within the same sex, in testes, we observed a higher number of differentially hypomethylated CpGs (53,489) than hypermethylated ones (50,099). In contrast, in ovaries, a higher number of differentially hypermethylated CpGs (94,329) compared to hypomethylated ones (68,876) was observed.
Discussion
These differences in the number of differentially methylated CpG sites suggest that sexual dimorphism plays a significant role in the methylation pattern of European sea bass, with females being hypomethylated compared to males under basal conditions. Furthermore, changes in the methylation pattern after the immune activation show a different tendency in each sex. Overall, in males, immune activation appears to induce a higher number of hypomethylated CpG sites compared to controls. In contrast, females show a tendency towards hyper methylation, which may be associated with gene repression. These findings highlight the importance of sex influencing the methylation status.
References
APROMAR (2023). https://apromar.es/wp-content/uploads/2023/09/La_acuicultura_Espana_2023_APROMAR.pdf.
Caballero-Huertas et al. (2024). DOI: 10.1080/23308249.2024.2390965.
Gelderen , T.A. van, et al. (2025) . DOI: 10.1007/s10142-025-01537-w.
Sasaki, H. and Matsui, Y. (2008) . DOI: 10.1038/NRG2295.
Acknowledgements
SL-C is supported by a predoctoral contract (PREP202293-002132) funded by MICIU/AEI/10.13039/501100011033 and the ESF+ ("European Social Fund Plus").
Institutional support to CNAG was provided by the Spanish Ministry of Science and Innovation through the Instituto de Salud Carlos III, and by the Generalitat de Catalunya through the Departament de Salut and the Departament de Recerca i Universitats.
This study was supported by the Spanish Ministry of Science and Innovation grant 2PID2020-113781RB-I00 “MicroMet” and PID2023-146286OB-I00 “Holosex” to LR.