Introduction
Gilthead sea bream ( Sparus aurata ) is extensively cultivated in the Mediterranean due to its economic significance and its adaptability to diverse aquaculture systems. Despite major advances, c hallenges remain in nutritional management and prevention of metabolic diseases in sea bream farming (Hallerman et al., 2022) . The liver plays a crucial role in fish metabolism by regulating lipid mobilization under nutritional stress. Diet composition, particularly fatty acid type and quantity, impacts liver function, energy balance, and disease resistance. High lipid diets may induce to hepatic steatosis, which in turn impairs liver function and triggers inflammation, fibrosis, and metabolic disorders . In sea bream, steatosis negatively influences growth, feed efficiency, and infection resistance, potentially leading to increased mortality and decreased product quality, thereby affecting aquaculture profitability (Oliva-Teles , 2012; Turchini et al., 2009). Understanding differential gene expression is essential to elucidat e how lipid-rich diets disrupt lipid homeostasis, and promote fatty liver. Integrating transcriptomic methodologies with novel nutritional approaches is vital for improving fish health and fostering sustainability in aquaculture (Asaoka et al., 2014; Costa-Silva et al., 2017). Consequently, t he objective of this study was to assess hepatic gene expression changes in gilthead seabream on a fish fed a fish-oil-enriched diet.
Material and Methods
Juvenile sea bream (S. aurata ) with an average weight of 10-14 g were kept in the facilities of the experimental animal service (SEA) at the University of Murcia (UMU). The study animals were fed at 1.5% of body weight daily for 3 months with the following diets (two replicates): i) standard diet (Skretting, Control Group), ii) diet enriched with 10% fish oil (Fish oil Group). At the end of the experiment, the fish were sacrificed (n = 6 per group), the liver was obtained and frozen in liquid nitrogen. Total RNA was extracted from the liver and its integrity was verified , analysing its profile with a bioanalyser. cDNA library construction was carried out using the Illumina Stranded mRNA Prep’ protocol . The libraries were sequenced using a 2 x 100 bp paired-end sequencing strategy, with a minimum sequencing depth of 25 million pairs of reads (50 million individual reads) per sample using the NextSeq 2000 sequencer (Illumina, USA). In silico differential expression analysis was carried out using Rstudio 4.3.0 software.
Results and discussion
A total of 385,292,118 reads were obtained from 12 cDNA libraries (380,669,063 clean reads) . From these clean reads, expression counts were obtained for a total of 32,008 genes, according to the available genomic annotation. So, 463 significantly differentially expressed genes were identified, of which 158 were found to be up-regulated and 305 down-regulated.
Taken together, our data confirm the existence of a specific transcriptional response associated with fish oil consumption . Up-regulated genes were clustered in categories linked to transport activities, hydrolase and oxidoreductase enzymes, whereas genes involved in triglyceride-lipasa activity were prominently down-regulated. Enrichment analysis highlighted oxygen binding, haemoglobin binding and olfactory receptor binding together with RNA-binding, ligase and glycosyl-transferase activities, suggesting a complex physiological response to treatment. In contrast, functions linked to the transport of organic ions and anions were suppressed. These results highlight the modulation of key functions in cell metabolism and signalling in response to fish oil treatment.
The Peroxisome Proliferator-Activated Receptor (PPAR) signalling pathway was significantly over-represented among the differentially expressed genes., coherently linking the transcriptional response to lipid metabolism, energy balance and inflammation. This is consistent with a possible physiological effect related to the treatment administered, especially if associated with nutritional intervention or lipophilic compounds such as fatty acids (Antonopoulou et al., 2017). The most prominent pathway was ‘Ribosome biogenesis in eukaryotes’, (p<0.0029) and a Gene Ratio higher than 0.46. The ‘One carbon pool by folate’ pathway exhibited significance, albeit to a lesser extent. These findings suggest a potential reconfiguration of ribosomal components and basic cellular metabolism, possibly linked to a specific physiological response induced by the high fatty acid diet employed in this study. (Colombo, 2020).
Overall, these findings elucidate the molecular mechanisms by which high fatty acid diets influence liver health in gilthead seabream. The identification of specific genes and pathways associated with dietary response lays a foundation for optimizing dietary formulations in Mediterranean aquaculture, enhancing the health, welfare, and production efficiency of this important species.
Acknowledgements
This work formed part of the ThinkInAzul programme supported by MCIN with funding from European Union Next Generation EU (PRTR-C17.I1) and by the Comunidad Autónoma de la Región de Murcia-Fundación Séneca (Spain). CER was funded by a grant of the Ramón y Cajal Fellowship Programme from the Spanish government (RYC2023-045252-I). CMP (PRE2021-098414) has PhD grant.
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