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Transcriptomic approach to environmentally friendly farming of European sea bass in the Adriatic Sea, using alternative protein sources

Introduction European aquaculture is facing the challenge of satisfying the growing seafood demands and reducing the pressure on fishing areas. Aquaculture feeds use 70 percent of the world’s fishmeal and fish oil, obtained from overexploited small pelagic fish (Froehlich et al., 2018). By 2040, the demand for fishmeal and fish oil will exceed the supply, emphasizing the need for novel feed ingredients to enhance production. Recently, new protein sources include fish by-products, poultry by-products, insects, and algae, displaying promising results towards optimal fish growth (Beheshti Foroutani et al., 2018). The ongoing Interreg AdriAquaNet project is dedicated to enhancing the innovation and sustainability of Adriatic aquaculture. Within the project, new feeds were designed and tested at a laboratory scale on sub-adult European sea bass. For the present study, three of the five tested diet formulations were selected for a comprehensive exploration and comparative transcriptomics of European sea bass intestines after performed feeding trial. Material and methods Selected formulations included two control diets, one rich in fish-derived ingredients (CF) contained 85 % and 66 % of fishderived protein and lipid, and one rich in plant- derived ingredients (CV) contained 85 % and 66 % of plant-based protein and lipids. Test diet (VH10P30) contained the same vegetable: fish lipid ratio as the CV diet, replacing crude proteins from the plant-based sources with 10 % of crude proteins from a commercial defatted Hermetia illucens pupae meal and 30 % of poultry by-product meal. The experimental diets were formulated to be iso-proteic (45%), iso- lipidic (20%), isoenergetic (20.3 MJ kg-1) and to meet the dietary requirements of sub-adult European sea bass. Based on quality, 24 samples of total RNA were selected for cDNA library preparation, comprising 4 biological replicates per feeding treatment of two selected intestinal parts, pyloric caeca, and distal intestine. One biological replicate corresponded to a single fish, meaning pyloric caeca and distal intestine were paired as subsamples. Single-end 3’UTR sequencing was performed using NextSeq 500 System (Illumina, San Diego, CA, USA). Results and discussion Two analyses were performed for differential expression analysis, comparing the VH10P30 test treatment to CF treatment as positive control, and comparing the VH10P30 test treatment to CV treatment as negative control. In total, 1, 963 (915 up and 1048 down) differentially expressed (DE) genes were found in the distal intestine of European sea bass in the VH10P30 treatment vs. CF treatment. Using CV treatment as the negative control, there were 711 (439 up and 272 down) DE genes in the distal intestine of European sea bass in the VH10P30 treatment. In contrast to the distal intestine, no clear differences were detected between the diet treatments in the pyloric caeca of the experimentally fed European sea bass. Previously performed gene expression profiling revealed functional specialization along the intestinal tract of European sea bass. Molecular and cellular functions related to feed digestion and nutrient absorption and transport were over-represented in the anterior and middle part of the intestinal tract, while the initiation and establishment of immune defense mechanisms became especially relevant in the distal intestine (Calduch-Giner et al., 2016). Although this could offer an explanation why there were such pronounced differences in this study in the distal part of the intestine between treatments, and at the same time no differences detected in the pyloric caeca, it is also possible that 24 h starvation prior to fish sampling affected obtained results. While the digestion was still active in the distal intestine at the time of the sampling, the active part of the digestion in the pyloric caeca was probably finished. This is also supported by functional analyses of DE genes in this study, which revealed over-representation of gene ontology terms in signal transduction, transport and various metabolic processes (for example cellular nitrogen compound, small molecule and lipid metabolic process) in the distal intestine, to name a few, while the immune response was generally absent, for both comparisons (using CF and CV as control, respectively). Taking into account growth performance and overall fitness of the European sea bass in the VH10P30 treatment, H. illucens larval meal and poultry by-product meal demonstrated great potential as alternative protein sources for European sea bass aquaculture.

plant-based diet, insect meal, poultry by-product meal, transcriptome

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