Introduction
Aquaculture has been one of the fastest growing food-production sectors from the last decade. Nevertheless, some barriers still limit the optimization and sustainablity of the sector, with stress being one of the main challenges. However, the lack of precise and robust tools to measure stress levels and the effect of mitigation strategies is still a key limitation. Different approaches have been used, such as measurement of physiological indicators, behavioral analysis, and the assessment of molecular biomarkers, which represents a promising approach. However, the currently used molecular biomarkers still present a very high degree of variability and limited predictive power. Therefore, systematic and species-specific evaluation of stress response across multiple stressors is essential to identify robust molecular biomarkers capable of supporting precision monitoring and sustainable aquaculture practices.
Materials and Methods
To address this limitation, transcriptomics analyses were conducted to identify sensitive and precise stress molecular stress biomarkers across three different but highly relevant species for aquaculture production: gilthead seabream (Sparus aurata), european seabass (Dicentrarchus labrax) and nile tilapia (Oreochromis niloticus). Fish were subjected to two different acute stress conditions - ammonia and hypoxia - until a 10% mortality rate was reached (used as a standardized quantitative endpoint of fish stress). Tissue samples were collected at multiple time points after acute stress exposure in order to determine the time window associated with the highest transcriptional response relative to non-stressed controls. Total RNA was extracted from all samples and subjected to high-throughput RNA sequencing. Bioinformatic analyses were then performed to identify differentially expressed genes (DEGs) between stressed and control groups. Candidate biomarkers were selected based on differential expression patterns, functional relevance, and consistency across experimental conditions, resulting in a reduced panel of potential general and stress-specific biomarkers. Finally, selected candidate genes are being validated using quantitative PCR (qPCR) to confirm the RNA-seq results.
Results
Distinct gene expression profiles were observed between control fish and individuals exposed to both stress conditions across all species, confirming that ammonia and hypoxia significantly affected transcriptional responses. Temporal analyses revealed that the magnitude of differential gene expression varied across sampling time points and species. The highest number of DEGs was detected 3 h post-exposure in gilthead seabream, whereas in European seabass and Nile tilapia the peak response occurred at 1 h post-exposure. In gilthead seabream, 21 candidate general stress biomarker were identified, including fbxo32 and nr4a3, along with eight genes ammonia-specific candidates, as dusp5 and fzd3. No hypoxia-specific biomarkers were identified in this species. In European seabass, 10 general stress biomarkers candidates were identified, as fosb and nr4a3, along with 11 ammonia-specific candidates (e.g. junbb and dusp2) and two hypoxia-specific candidates, slc22a7 and hdac12. In Nile tilapia, 20 general stress biomarkers (e.g. avpi1 and fkbp5) and 20 ammonia-specific candidates (as fzd5 and pold2) were identified, whereas only four hypoxia-specific biomarkers were detected. Commonly used stress biomarkers, such as heat-shock proteins (hsp70 and hsp90), metallothionein (mt) and catalase (cat), were also evaluated. Among these, only hsp's showed differential expression, but with lower fold change values when compared to the novel identified biomarkers, and with less consistent responses across stress and developmental stages, as documented in the literature. An exception was observed in Nile tilapia, where hsp70 ranked among the most strongly regulated general stress biomarkers. Validation in gilthead seabream confirmed the RNA-seq results, with seven general and two ammonia-specific biomarkers maintaining consistent differential expression patterns. Although some genes showed stage-dependent variation, the overall validation supported the reliability of the novel identified biomarkers.
Discussion
Both general and stress-specific biomarkers were initially identified for each species individually, highlighting species-dependent stress response strategies. Gilthead seabream primarily exhibited metabolic responses, European seabass showed a stronger involvement of signalling pathways, and Nile tilapia exhibited a more integrated response englobing stress signaling, immune and hormonal pathways. Despite these differences, we observed that these different species shared functional response to stress, suggesting the potential for the establishment of cross-species stress biomarkers. For instance, nr4a3 was consistently differentially expressed across both stress conditions and developmental stages in both gilthead seabass and European seabream. In addition , several gene families were also differentially expressed across species, including FOS, DUSP, F-BOX and TRIM. Notably, a conserved core stress response shared across species was identified, mainly involving three pathways: FoxO, MAPK and Adipocytokine signaling pathways. These findings highlight the potential for the establishment of robust biomarkers for stress monitoring in aquaculture by identifying conserved molecular mechanisms underlying stress responses across multiple species.
Acknowledgment
This work is part of the E!4876 FORTIFISH project, No. 21286, Operation No. MPr-2024-9-21286 (COMPETE2030-FEDER-02232500, LISBOA2030-FEDER-02232500, ALGARVE-FEDER-02232500), co financed by the European Union through the Algarve 2030, Lisboa 2030 and COMPETE 2030 Operational Programs, in the framework of Portugal 2030 and Interface Mission cofinanced by PRR - Plano de Recupera����o e Resili��ncia by the European Union (operation code 01/C05-i02/2022.P148).