Although it is generally accepted that aquaculture can relieve pressure on wild fisheries by producing alternative fish for human consumption, the production of those fish often requires inputs from wild fish stocks in the form of feed ingredients. The paradox stems from the diversity of farmed species and husbandry systems (Stevens et al., 2018). Regardless of the remarkable advances in aquafeed formulation, most feeds used in aquaculture still rely on fishmeal (FM) and fish oil derived from fisheries. However, there is increasing pressure on these raw materials and conventional alternative plant protein (PP) sources due to growing demands from a variety of users. For instance, the use of soy protein and other plant products in aquaculture reduces their availability for direct human consumption (Ytrestøyl et al., 2015). Under this scenario, the sustainability and competitiveness of aquaculture may depend on their replacement with alternative ingredients ranging from plant-based meal and plant oil to terrestrial by-products and microbial ingredients (Naylor et al., 2021). Thus, the mantra about the economic and environmental sustainability of the aquaculture industry based on the identification and use of alternative raw ingredients to the classical ones is still valid.

Microbial products, particularly yeast, are potential sustainable ingredients in aquafeeds due to the ability to convert low-value non-food biomass from forestry, and agricultural and food technology industries into high-value feed ingredients with limited dependence on arable land, water, and changing climatic conditions (Øverland and Skrede, 2017; Agboola et al., 2021). Among them, Brewer’s spent yeast (SDY), a by-product of the brewing industry, is a rich source of B vitamins, proteins (45–60%) and other compounds like β-glucans and mannoproteins (Jaeger et al., 2020; Agboola et al., 2021). Therefore, BSY is mainly utilized in animal feed formulations as a low-cost source of protein (Agboola et al., 2021; Estévez et al., 2021; Nazzaro et al., 2021) even though it may be also used as an ingredient in functional feeds due to its potential health benefits to the host (Jaeger et al., 2020). Under this scenario, Estévez et al. (2021) have shown that the replacement of FM and plant protein sources by SDY in the feed of a carnivorous marine teleost fish species, the gilthead sea bream (Sparus aurata), resulted in a better growth, as well as a higher protein digestibility and feed efficiency indexes than in their congeners fed a reference diet. The above-mentioned results showed that this by-product from the brewing industry is a promising feed ingredient in aquafeeds, especially when considering its use in terms of an alternative plant protein source, but also when considering its associated sustainable and environmental benefits. Those benefits can be achieved by means of a circular economy strategy, which helps in providing added value to the brewing industry through wastes’ reuse and, consequently, reducing the industry carbon footprint (Kerby and Vriesekoop, 2017; Jaeger et al., 2020).

Once validated the use of SDY as an alternative protein source in aquafeeds (Estévez et al., 2021), the present study aimed to evaluate at the transcriptomic level the impact of SDY in the head kidney, intestine, and liver of S. aurata. This marine species is the most important Mediterranean aquaculture fish species in terms of volume and economic value (FAO, 2020). Regarding the selected target tissues, the head kidney was chosen as it is one of the most important organs in fish due to its role in endocrine and hematopoietic functions, as well as a major secondary lymphoid organ (Tort, 2011). Furthermore, the liver was selected as the central metabolic organ of the body with a predominant role in the intermediary metabolism, with important functions in lipid storage and digestive and detoxification functions (Bruslé and González, 1996). Finally, the intestine was chosen due to its involvement in important physiological digestive functions, being the primary site of food digestion and nutrient uptake, whereas it is also reputed for providing support to gut microbiota and maintaining an effective barrier defense against pathogens and tolerance toward dietary antigens (Salinas and Parra, 2015; Wang et al., 2018). Thus, a proper understanding of the effects of this new alternative ingredient at systemic and local levels as well as its mode of action upon the selected target tissues would support its proper use as an ingredient for sustainable aquafeeds.

Results on growth performance, feed efficiency, and diet digestibility are presented elsewhere (Estévez et al., 2021). In brief, no differences in growth performance were found among both experimental groups with BWf average values comprised between 176.1 and 179.0 g. Regarding feed efficiency variables, gilthead sea bream fed the SDY-supplemented diet showed better feed conversion ratio (FCR = 2.0 ± 0.05) and protein efficiency rate (PER = 1.1 ± 0.03) than their congeners fed the control diet (FCR = 2.1 ± 0.29; PER = 1.0 ± 0.11) (P < 0.05).

In this study, we decided to explore the dietary effects of Brewer’s SDY at transcriptomic level by means of RNA-seq on three different tissues selected by their relevant body functions. Regarding the head kidney, RNA-seq results showed that most of DEGs in this hematopoietic and lymphoid tissue of gilthead sea bream fed the SDY were related to increased transcription levels of genes related to chemokine activity (GO:0008009) and chemokine receptor binding (GO:0042379). This is of special relevance since chemokines are involved in the initiation and regulation of the inflammatory response. In particular, the production of pro-inflammatory chemokines (i.e., CCL2, CCL3, CCL5, CXCL1, CXCL2, and CXCL8) is generally induced to recruit immune cells to the infection site, while chemokines considered as homeostatic (i.e., CCL19 and CCL21) are involved in controlling the migration of cells during normal processes of tissue maintenance or development; thus, initiating and regulating the inflammatory response (Laing and Secombes, 2004; Sakai et al., 2021). Under present experimental conditions, we found a transcriptional regulation of ccl17, cxc6, cxc11 in the head kidney of fish fed the SDY-supplemented diet. The above-mentioned chemokines are reputed for being involved in the activation (cxc6) and recruitment (ccl17, cxc11) of T lymphocytes. These results are in agreement with the lymphoid function of this tissue, which may be translated into an enhanced host’s immune response as similar results have been observed in fish fed functional diets supplemented with zootechnical feed additives with immunomodulatory properties (Martin and Król, 2017; Hoseinifar et al., 2019; Salomón et al., 2021). The potential immunomodulatory function of the SDY diet may be attributed to its content in β-glucan (81 g/kg feed). In this sense, β-glucans are well-known in aquaculture because they promote hematopoiesis and enhance immunity (Meena et al., 2013).

Under the present experimental conditions, we also found changes in expression of genes involved in steroid synthesis and secretion (GO:0008202, GO:0006694). Among DEGs involved in steroidogenesis, nr5a1a was found to be up-regulated in the head kidney of fish fed the SDY-supplemented diet. This gene is expressed in the interrenal gland, gonads, and hypothalamus (Chai et al., 2003). In teleosts, the interrenal gland is embedded in the head kidney and is the major site of steroid synthesis in response to signals from the pituitary and hypothalamus (Milano et al., 1997). Similarly, cytochrome P450 11b (cyp11b), steroid 21-hydroxylase (cyp21a2), hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase (hsd3b1), and steroid 17-alpha-hydroxylase/17,20 lyase-like (LOC115589985) were also differentially expressed, which is of special relevance since those genes are involved in the biosynthesis of steroid hormones like cortisol through their precursors (Mommsen et al., 1999; Li et al., 2003). These results are of special importance, since glucocorticoids are synthesized by interrenal cells of the head kidney and regulate osmolarity, metabolism, and immune responses (Vazzana et al., 2010). Furthermore, the up-regulation of bicaudal C homolog 2 (bicc2) that is involved in renal gluconeogenesis might show that gilthead sea bream fed the SDY-supplemented diet may have a more effective stress response, as data from genes involved in steroidogenesis also indicated.

Surprisingly, the head kidney of gilthead sea bream fed the SDY-supplemented diet showed a down-regulation of hemoglobin subunits alpha-A and beta genes (hbaa and hbb), which encoded for α and β subunits of hemoglobin. In addition, we also found a down-regulation of the lipoxygenase homology PLAT domains 1b (loxhd1b—LOC115592348), which is also involved in heme binding and oxygen binding affinity. Under hypoxic conditions, changes in transcription levels of hbaa and hbb in the head kidney of fish have been associated to changes in erythropoiesis and hemoglobin synthesis (Xia et al., 2016). Furthermore, changes in gene expression of hbaa and hbb are responsible for hemoglobinopathies in humans (Sabath, 2017). We found these results puzzling, since the nutritional trial was conducted under normoxic conditions (> 85% O₂ saturation) and we did not find any sign of distress in gilthead sea bream fed the SDY-supplemented diet caused by a putative anemia. Further research is needed to clarify the physiological effect of SDY on hemoglobin homeostasis and erythropoiesis in order to clarify current results.

Among the three studied tissues, the liver was the second one with a larger number of DEGs. In particular, DEGs were involved metabolic and biosynthetic processes (psat1, phgdh, wars1, hars1). These findings are in agreement with those found in the head kidney, which indicated that fish fed the SDY diet had a more biologically active organ, since the above-mentioned DEGs are involved in protein transcription and biosynthesis (Nguyen et al., 2020; Yu et al., 2021). In addition to the classical role of wars1 in protein synthesis, this gene has been recently described in mammals to be activated upon infection, whereas its protein acts as an endogenous ligand of toll-like receptors (TLR) 2 and TLR4 (Nguyen et al., 2020).

The potential immunomodulatory properties of the SDY found in the head kidney seemed to be also supported by the up-regulation of genes involved in modulating the inflammatory responses in the liver via the regulation of the JAK-STAT receptor signaling pathway as transcriptomic data suggested. Specifically, by the up-regulation in the liver of cytokine-inducible SH containing protein (cish) and the suppressor of cytokine signaling-like (socs) in comparison to the control group. These genes are members of the suppressor cytokine signaling family whose proteins are involved in the control and attenuation of cytokine-induced responses through the JAK/STAT signal transduction pathway, playing an important role in the regulation of host’s immunity and the inflammatory response, as well as in cell proliferation, migration and survival (Yoshimura, 2009; Yoshimura et al., 2012; Maehr et al., 2014). Furthermore, the down-regulation of the NACHT, LRR, and PYD domains-containing protein 12-like (nlrp12) might suggest an enhancement of the NF-κB pathway, since these proteins have a modulatory role on the NF-κB pathway (Normand et al., 2018). This is of special relevance since the NF-kB pathway plays an important role in the regulation of several cytokines, chemokines, antimicrobial peptides, and interferon-stimulated genes, as well as being crucial in modulating the survival, activation, and differentiation of innate and adaptive immune cells (Sun, 2017). Although these results might be attributed to a potential inflammatory response derived from the inclusion of SDY in the diet, we did not find inflammatory signs in the intestinal mucosa of fish fed the SDY diet (Supplementary Figure 4); thus, the transcriptional regulation of the NF-kB pathway may be attributed to the immunomodulatory properties of the SDY. Furthermore, the E3 ubiquitin-protein ligase TRIM21-like gene (trim21) was also down-regulated in the liver of fish fed the SDY diet. This gene is generally over-expressed under prolonged activation of the immune system, being also described as a negative regulator of antioxidative stress (Lee, 2017). Similarly, protein mono-ADP-ribosyltransferase PARP14-like (parp14) was also among the most down-regulated genes. This gene modulates the humoral immune response through the activation of the JAK/STAT pathway (Schweiker et al., 2018), which suggests a balanced immune response in gilthead sea bream fed the SDY diet as other authors have reported when there is a sort of immune homeostasis based on an equilibrium between pro- and anti-inflammatory processes (Salomón et al., 2020, 2021; Firmino et al., 2021).

The transcriptomic response evaluated in the anterior-mid intestine was in agreement with results found in the two former tissues from gilthead sea bream fed the SDY diet. In particular, four g-type lysozyme genes were up-regulated (LOC115585860, LOC115582400, LOC115582423, and LOC115585885), which were related to the biological process linked to peptidoglycan and aminoglycan metabolic, and catabolic processes in which g lysozyme is involved (Jiménez-Cantizano et al., 2008). In this sense, lysozyme cleaves peptidoglycans of the bacterial cell wall and participates in the activation of the complement system and phagocytes. In addition, lysozymes have also been reported to have antiviral and anti-inflammatory functions, thus playing an important function at both systemic and local immune responses (Saurabh and Sahoo, 2008). Changes in the number of transcripts of the retinoic acid receptor beta (rarb) were not attributed to different dietary vitamin A contents between both experimental diets, since yeast does not contain this lipophilic vitamin. Thus, changes in rarb expression might be associated to the regulation of the intestinal immune condition in fish fed the SDY diet (Gattu et al., 2019). Similarly, we also found the major histocompatibility complex class I-related gene protein (hla) to be down-regulated, which may be associated with a reduction in the immunogenicity of dietary SDY and modulation of the immune response in the intestine (Groh et al., 1996). Although we did not find inflammatory signs in the intestinal mucosa of fish fed the SDY diet (Supplementary Figure 4), the expression profile of immune-related genes in fish fed the SDY diet indicated that further research may be focused on evaluating the effect of SDY on the mucosal-associated lymphoid tissue in the intestine and its immunocompetence under disease conditions.

Furthermore, the inclusion of SDY in the diet down-regulated the expression of hydroxyacid oxidase 2 (hao2) in the anterior-mid intestine of gilthead sea bream, which might be attributed to a dietary regulation of this gene. Hydroxyacid oxidase 2 is a peroxisomal 2-hydroxy acid oxidase responsible for the oxidation of 2-hydroxy fatty acids like 2-hydroxypalmitic acid, which is an intermediate in phytosphingosine metabolic pathway, an important component of sphingolipids found in yeasts and plants (Pruett et al., 2008).

Under present experimental conditions, the partial replacement of conventional dietary protein sources by spent dry yeast (SDY) obtained as a by-product of the brewer’s industry had a modulatory effect on several processes related to host’s immunity, oxygen’s carrier capacity, steroidogenesis, metabolism, and digestion. Results related to the enhancement of the immune condition at systemic and local levels in fish fed the SDY diet are of special relevance, since they indicated that this ingredient in addition to being considered as a sustainable ingredient for replacing conventional marine and terrestrial protein sources in aquafeeds, it may also be considered as a functional one due to its immunomodulatory properties derived from its content in β-glucans. Although the results found in the present study seemed really promising in terms of the functional properties of the tested SDY, further research needs to be focused on clarifying the effect of this ingredient on the biological processes involved in oxygen transport in order to guarantee the safety of this ingredient for fish. The overall results of this study coupled with nutritional studies on this ingredient (Estévez et al., 2021; Nazzaro et al., 2021) indicated that the use of brewery’s by-products like SDY in aquafeeds supports a circular bioeconomy model that reuses, recovers, and recycles resources instead of producing wastes.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm.nih.gov/geo/, accession ID: GSE194206.

This animal study was reviewed and approved by the Ethical Committee of the Institute for Research and Technology in Food and Agriculture (IRTA, Spain) for the use of laboratory animals (E-10/2020).

AE, DS, and BI: conceptualization. EG, JPF, and JMOF: methodology. JPF, IK, and AE: sample processing. IK, JPF, and JMOF: formal analysis. EG and JMOF: resources. IK and EG: writing the original draft. JPF, IK, and JMOF: data visualization and curation. EG and JMOF: supervision. EG, AE, and DS: project administration. AE and DS: funding acquisition. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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