17 pages, 6 figures, 1 table, supplementary material https://doi.org/10.1186/s12864-025-11551-8.-- Data availability: Raw sequencing data generated during the current study can be found in NCBI SRA repository with the accession number: PRJNA1008584. The raw data will be publicly available upon publication and the metadata can be accessed by reviewers through: https://dataview.ncbi.nlm.nih.gov/object/PRJNA1008584?reviewer=ct6r6cbgav3c2ru7evoqvlkqg7, Elevated temperatures during early developmental stages play a pivotal role in the fate of the adult sexual phenotype of fish populations, usually leading to male-skewed sex ratios. This is the case with European sea bass (Dicentrarchus labrax), one of the most important species in the European aquaculture industry. To unveil informative markers of the past thermal events, we investigated changes in the miRNome within the gonads of this species. Consequently, we exposed European sea bass to elevated temperatures (21ºC) during early development (from 7 to 68 days post fertilization). After one-year post-heat treatment growing at natural temperature, a miRNA-sequencing analysis was conducted in the ovaries and testes of juvenile fish. The examination of miRNA expression levels identified three and twelve miRNAs in ovaries and testes, respectively, reflecting past thermal events. To assess the evolutionary conservation of these identified miRNAs in gonads, we cross-referenced our data with miRNome public information from ovaries and testes in nine additional fish species from the FishmiRNA database. This analysis uncovered 33 potential sex-biased markers present in at least five studied species along the evolutionary timeline. For instance, miR-155, miR-429, and miR-140 were consistently female-skewed, while miR-143, miR-499, and miR-135b-3p were consistently male-skewed. In addition, among these markers, three conserved sex-skewed miRNAs proved to be informative regarding past thermal events in the ovaries (e.g., miR-192-5p, miR-146a-5p and miR-143-3p) and four in the testes (miR-129-5p, miR-724-5p, miR-143-3p, and miR-223-3p). Notably, miR-223-3p was conserved female-skewed, but showed upregulation in males exposed to high temperature, and miR-143-3p was inhibited in both heated females and males. These miRNAs could serve as markers of heat-induced masculinization. This research broadens the inventory of sex-specific miRNAs across evolution in fish, and elucidates thermosensitive miRNAs in the gonads, Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature.
This study was supported by the Spanish Ministry of Science and Innovation grants AGL2015-73864-JIN “AMBISEX” 2PID2020-113781RB-I00 “MicroMet” and PID2023-146286OB-I00 “Holosex” and by the Consejo Superior de Investigaciones Científicas (CSIC) grant 02030E004 “Interomics” and “8153CSIC_AEPP24” to LR, by grant PID2019-108888RB-I00 to FP, and with funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), Peer reviewed

Organophosphorus flame retardants (OPFRs) are emerging alternatives to halogenated compounds, yet their environmental toxicity remains underexplored. This study evaluated the developmental toxicity of two aryl-OPFRs, triphenyl phosphate (TPP) and tricresyl phosphate (TCP), in zebrafish (Danio rerio) from 2 h to 5 days post fertilization (hpf–dpf). Survival, hatching rate, and malformations were assessed across concentrations of 250–1000 µg/L, alongside with gene expression analysis at 5 dpf (250 and 500 µg/L) targeting detoxification (ces2), immune responses (il1β, casp9), and epigenetic markers (dnmt1, dnmt3). In vitro enzymatic assays evaluated interactions of both aryl-OPFRs with carboxylesterase (CE) and acetylcholinesterase (AChE) enzymes. While no significant morphological effects were observed, TPP showed higher toxicity than TCP. Notably, TCP (500 µg/L) downregulated genes linked to metabolism and immunity. CE activity and ces2 modulation may suggest CE as a potential biomarker for aryl-OPFR exposure. These findings, although at concentrations above the environmental ones, may be valuable for mechanistic purposes and underscore the need for further investigation in developmental toxicity given their lipophilic nature and distinct molecular responses., The Spanish Ministry of Science and Innovation (MCI), Agencia Estatal de Investigacion (AEI), and Fondo Europeo de Desarrollo Regional (FEDER, MCIN/AEI/10.13039/501100011033/FEDER UE) grants 2PID2020-113781RB-I00 “MicroMet” and PID2023-146286OB-I00 “HOLOSEX” to LR and PID2021-122592NB-I00 “BioPlas” [MCIU/AEI] to MS. Institutional support through the ‘Severo Ochoa Center of Excellence’ accreditation (CEX2024-001494-S) funded by AEI 10.13039/501100011033. This work has received funding from the CSIC Interdisciplinary Thematic Platform (PTI+) for Sustainable Plastics towards Circular Economy+ (PTI-SusPlast+). IFP and MS received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No. 101153110., With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation
(CEX2024-001494-S), Peer reviewed

18 pages, 8 figures, 3 tables, supplementary information https://doi.org/10.1007/s11306-025-02356-7.-- Data availability: No datasets were generated or analysed during the current study, Purpose: Fish aquaculture faces sustainable production challenges. Among them are the pathogenic outbreaks that can compromise the health of the stocks from various perspectives, including broodstock reproduction. This study focused on identifying the metabolite alterations produced after a bacterial infection by Vibrio anguillarum in the gonads of European seabass (Dicentrarchus labrax). Sex-related response to the infection challenge was studied using a metabolomics approach.
Method: The metabolome of testes and ovaries of adult fish were extracted and analyzed after 48 h of bacterial exposure by ultra-high-performance liquid chromatography-mass spectrometer using negative-mode electrospray ionization (ESI) (UHPLC-MS, Vanquish Horizon UHPLC coupled to a Thermo Fisher Scientific Q-Exactive HF). To further decipher the molecular events, metabolomic and transcriptomic data were interconnected.
Results: In total, 97 metabolites were identified. In the ovary, uric acid, O-phosphoethanolamine, allantoin, and acetoacetic acid were more represented. By contrast, nine metabolites were altered after the infection in testes, including uridine, N-acetylglucosamine-6-Phosphate, and Gamma-aminobutyric acid (GABA). The most abundant metabolic cascades triggered by infection in ovaries were related to glyoxylate and dicarboxylate metabolism, nitrogen metabolism, and purine metabolism, while in testes, we observed changes in glycerolipid metabolism, glycerophospholipid metabolism, and galactose metabolism.
Conclusion: The present results demonstrate, for the first time in fish, that changes in metabolic pathways induced following infection are sex-dependent. The findings will help develop sex-specific immune therapies, identify resistant phenotypes, and improve aquaculture infection protocols, This study was supported by the Spanish Ministry of Science and Innovation grant 2PID2020-113781RB-I00 “MicroMet” and PID2023-146286OB-I00 “HOLOSEX” to LR and with funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (Grant CEX2024-001494-S funded by AEI 10.13039/501100011033). Funding was also provided by the Natural Sciences and Engineering Research Council of Canada to HRH (NSERC Discovery Grant no. 2022-03560). SL-C is supported by a predoctoral contract (PREP2023-000338) funded by MICIU/AEI/https://doi.org/10.13039/501100011033 and the ESF+ ("European Social Fund Plus"), Peer reviewed

14 pages, 10 figures, 4 tables.-- Data availability: Data will be made available on request, The expansion of lumpfish use as a cleaner fish has recently come to a halt, in part due to the concerns around their welfare. While refining handling procedures and operational standards is necessary, improving aquafeeds has the potential to alleviate the burden caused by chronic stress. This study examined the influence of different inclusions of dietary branched-chain amino acids (BCAAs) and lysine (Lys) on lumpfish health, immunity, stress response, growth and plasma amino acid profiles for 10 weeks. Stress and metabolic biomarkers (cortisol, lactate, glucose, triglycerides) showed modest but measurable alterations, such as lower triglycerides and higher glucose in fish fed the diet with limiting Lys and BCAAs. Metabolic differences were also visible from plasma amino acid profiles. Limiting Lys and BCAAs in diets seems to increase the glucogenic activity in stressed fish, increasing glucogenic amino acid levels in plasma. Diets with increased Lys and BCAAs levels increased antiprotease activity and lowered GABA plasma concentrations. These findings suggest that Lys- and BCAAs-enriched diets might provide relevant nutritional support, which allows the organism to adapt to a mild chronic stress. Immunonutrition can prove to be an effective strategy to ameliorate welfare in the face of chronic stress, his study was financed by CYCLOIMPROVE, a project financed by the Research Council of Norway (grant number 317607), with additional support from the Norwegian Seafood Research Fund (VEIEN, 901798) and the Icelandic Research Council (Rannı́s, 186971-0611) and the Foundation for Science and Technology (FCT, Portugal) within the scope of UIDB/04423/2020, UIDP/04423/2020, UIDB/04326/2020, UIDP/04326/2020, LA/P/0101/2020 and DL57/2016/ CP1361/CT0033. JMOF acknowledges the support from the Spanish Ministry of Science, Innovation and Universities through grants HOLOSEX (PID2023-146286OB-I00) and Severo Ochoa Centre of Excellence accreditation (CEX2024-001494-S, AEI 10.13039/501100011033), Peer reviewed

11 pages, 4 figures, supplementary material https://www.frontiersin.org/articles/10.3389/fcell.2025.1720219/full#supplementary-material.-- Data availability statement: The datasets presented in this study can be found in online repositories: in the European Nucleotide Archive (ENA) with the accession number ERP183568 and in the Supplementary Material https://doi.org/10.20350/digitalCSIC/17921, Introduction: In fish, epigenetic modifications are fundamental for regulating development, growth and adaptation to environmental factors. Emerging evidence further suggests that epigenetic mechanisms may modulate how fish gonads respond to infectious agents. Gonadal factors—including reproductive hormones and cytokines—are known to influence immune-cell activities, regulate the production of immune molecules, affect the overall immune response, and participate in gonadal sex differentiation. Although interactions between the reproductive and immune systems are well established, the epigenetic mechanisms underlying this interaction remain insufficiently elucidated, both in fish and in mammals. This study investigates how immune stimulation affects sex differentiation and methylation patterns of innate-immune genes in zebrafish gonads.
Methods: To study the epigenetic events involved in the immune–reproduction interaction, zebrafish (Danio rerio) were immune-stimulated with lipopolysaccharide (LPS) using two experimental approaches. (1) To assess the effect of immune stimulation on sex ratio, larvae were bathed in LPS during gonadal development (17–30 days post-fertilization, dpf). (2) To examine DNA methylation patterns in response to immune stimulation in adulthood, sexually mature fish received intraperitoneal LPS injections. Methylation analyses focused on two key innate immune genes, caspase 9 (Casp9) and interleukin 1β (Il1β). DNA methylation was quantified using a candidate-gene approach at single-nucleotide resolution through sequencing of bisulfite-converted DNA.
Results: Immune stimulation during gonadal development did not produce a statistically significant difference in sex ratio, although a clear trend toward feminization was observed in LPS-treated fish. In adults, Casp9 exhibited significant DNA-methylation differences driven by the interaction between treatment and sex. Specifically, eight CpG sites were significantly altered in treated females, while three CpG sites were significantly altered in treated males. In contrast, Il1β showed a sexually dimorphic methylation pattern, but these differences were not attributable to immune stimulation.
Discussion: The results support the presence of an epigenetic interplay between sex and immune response in the fish gonads. Sex-dependent methylation changes in Casp9 following LPS exposure, together with the inherent sexual dimorphism observed in Il1β, indicate that immune stimulation and sex jointly shape epigenetic landscapes of innate immune genes in reproductive tissues. Although the feminization effect was not statistically significant, the observed trend suggests that immune activation during the critical gonadal differentiation window may influence sex outcomes. Overall, these findings contribute to a deeper understanding of the epigenetic mechanisms underlying sexually dimorphic immune responses in reproductive tissues and highlight important avenues for future research, To the Spanish Ministry of Science and Innovation (MCI) through Agencia Estatal de Investigacion (AEI) and Fondo Europeo de Desarrollo Regional (FEDER, MCIN/AEI/10.13039/501100011033/FEDER UE) grant PID2023-146286OB-I00 “HOLOSEX” granted to LR. Institutional support through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2024-001494-S) funded by AEI 10.13039/501100011033. 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, Peer reviewed

10 pages, 2 figures, 2 tables, supporting information https://doi.org/10.1111/jfb.70216, This study investigates whether size-selective harvesting induced heritable changes reflected in epigenetic and reproductive molecular markers in zebrafish (Danio rerio), thereby indicating potential evolutionary responses. We used an experimental harvest model where zebrafish populations were subjected to five generations of size-selective harvesting, followed by eight generations without harvesting in a controlled environment to examine evolutionarily fixed outcomes in response to harvest selection. We assumed the evolutionary adaptations to size-selection to have left a molecular legacy related to sexual development, as previous studies have shown that evolution of reproductive timing is a common response to size-selection. To that end, we examined the expression of specific genes related to sexual development, such as dmrt1 and cyp19a1a, and epigenetic markers, including dnmt1 and dnmt3b, in the gonads in those experimental lines selected for this study. Additionally, global DNA methylation patterns were analysed to explore potential long-term epigenetic changes associated with size-selection. The results revealed no significant differences in gene expression related to sexual development or epigenetics between the size-selected and control zebrafish lines in the gonads in the F13 generation, eight generations after size-selection stopped. Also, global DNA methylation patterns were similar across selection lines and sexes. These findings suggest that five generations of size-selective harvesting, followed by eight generations of maintenance reproduction without further selection, did not induce lasting epigenetic or molecular changes related to the target molecular markers of sexual development in the gonads of zebrafish. The no significant molecular responses to size-selective harvesting observed here, based on specific reproductive and epigenetic markers, differ from previous studies targeting other tissues such as brain and liver, highlighting that not all genes or processes necessarily respond to size-selection and underscoring the importance of marker-specific comparisons, V.S. is supported by a Ramón y Cajal research fellowship (RYC2021-033065-I) granted by the Spanish Ministry of Science and Innovation and Universities and also acknowledges the Spanish Ministry of Science and Innovation and Universities grant PID2023-146460NA-I00 HumanFear. L.R. is supported by the Spanish Ministry of Science and Innovation grant 2PID2020-113781RB-I00 MicroMet and PID2023-146286OB-I00 HOLOSEX. V.S., M.S. and L.R. also want to acknowledge the institutional support through the Severo Ochoa Centre of Excellence accreditation CEX2024-001494-S funded by AEI 10.13039/501100011033, Peer reviewed

15 pages, 7 figures, 1 table.-- Data availability: No data was used for the research described in the article, Globally, eel aquaculture holds significant economic and ecological importance, but it faces severe challenges from various diseases that hinder its sustainable development. This review aims to systematically explore how genomics can be utilized to enhance the disease resistance of eels, thereby promoting sustainable aquaculture practices. To achieve this goal, we first review the current status of eel aquaculture and the main disease threats, and then focus on the application of genomic technologies in understanding eel disease resistance. We elaborate on the progress in genome sequencing of multiple eel species, which provides a fundamental resource for identifying disease-resistant traits. We discuss the role of functional genomics tools such as CRISPR-Cas9 in accelerating the screening of favorable disease-resistant traits. We also detail how immune system genomics (e.g., genes like Tollip, interferon, and MHC), genome-wide association studies, and the interaction between epigenetics and the environment contribute to the identification of disease-resistant traits. In addition, this review analyzes the applications of marker-assisted selection and genomic selection in eel breeding programs, as well as biotechnological intervention methods such as gene editing, probiotics, and vaccine development as novel strategies to improve disease resistance. Finally, we present future prospects for eel disease-resistant breeding efforts, This study was supported by the National Talent Recruitment Program (24030403699) and Provincial Undergraduate Training Program on Innovation and Entrepreneurship (No. S202410626065 and No. S202510626039) and Sichuan Agricultural University Dual-Branch Plan Special Project of Discipline Construction (2025ZYTS008); Spanish Ministry of Science and Innovation grant PID2023-146286OB-I00 “Holosex” to LR, and with funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2024-001494-S funded by AEI 10.13039/501100011033, Peer reviewed

16 pages, 9 figures, 2 tables, supplementary information https://doi.org/10.1038/s41598-026-48801-y.-- Data availability: The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request, Macrophage-colony stimulating factor (M-CSF) plays a crucial role in the proliferation and differentiation of the monocyte-macrophage lineage across vertebrates. In this study, we identified and characterized two CSF1 paralogues and their corresponding receptor genes in the intestine of the goldfish (Carassius auratus), showing sequence homology with known teleost species. Immunohistochemical analysis confirmed the presence of CSF, its receptor CSF-R1, and bone morphogenetic protein 2 (BMP2) in intestinal macrophages. These macrophages were localized within mucosal, submucosal, and muscularis layers, suggesting distinct functional subtypes. Quantitative PCR analysis revealed differential gene expression patterns, with csf1a, csf1b, and csf1ra highly expressed in the brain, while csf1rb transcripts were predominant in the intestine. Immunophenotypic characterization using CD14 and CD86 markers further demonstrated macrophage heterogeneity. Additionally, BMP2-expressing macrophages were observed in the muscularis externa, implying a potential role in neuromuscular regulation. These findings provide novel insights into the molecular and immunohistochemical profiles of goldfish intestinal macrophages, highlighting their potential role in immune responses and gut homeostasis, Open access funding provided by Nord University. The authors express their appreciation to Princess Nourah Bint Abdulrahman University Researchers Supporting Project number (PNURSP2026R213), Princess Nourah Bint Abdulrahman University, Riyadh, Saudi Arabia. JMOF acknowledges the support of the Spanish Ministry of Science and Innovation grant HOLOSEX (PID2023-146286OB-I00) and grant CEX2019-000928-S funded by AEI https://doi.org/10.13039/501100011033, Peer reviewed