The green transition of food production systems is the main issue to address the increase of production volumes. Thus, the target is to decrease the environmental footprint, while complying with high food safety and quality standards (Béné et al., 2015; FAO, 2016). However, climate change will impact most food production systems, and aquaculture, in particular, both directly affecting the physical condition and physiology of stocked animals, and indirectly altering fishmeal and fish oil costs, as well as other goods and services (Bohnes et al., 2019; Garrett et al., 2021). To overcome some of these constraints, continuous efforts have been conducted over the last two decades for the successful replacement of marine feed ingredients with more sustainable feedstuffs in fish feeds (Benedito-Palos et al., 2007; Lazzarotto et al., 2018; Perera et al., 2019; Egerton et al., 2020). The most obvious alternatives are plant proteins and oils, and, interestingly, most drawbacks effects - including pro-inflammatory condition, loss of epithelium integrity, and advanced male-female sex reversal in a protandrous hermaphrodite fish such as gilthead seabream (Sparus aurata), - can be restored, at least partially, with the use of feed additives (Estensoro et al., 2016; Piazzon et al., 2017; Simó-Mirabet et al., 2018; Reis et al., 2021). In addition, a range of novel feed formulations, including emerging feed ingredients (i.e., insects, seaweeds, microalgae, microbial biomasses, land-animal processed animal proteins, fish meal, and oil from fisheries and aquaculture by-products), do not affect fish welfare and guarantee good zootechnical performance in gilthead sea bream (Basto et al., 2021; Naya-Català et al., 2021a; Solé-Jiménez et al., 2021). Therefore, it is possible to produce marine seafood products using formulation concepts and ingredients that fit into a circular economy framework.

Genetic improvements in feed conversion ratio (FCR) also contribute to moving more toward a more environmentally-sustainable aquaculture sector (Kause et al., 2016; Vandeputte et al., 2019), though FCR is a problematic trait to be included in aquaculture breeding programmes due to the difficulties of accurate measurements of individual feed intake in the aquatic milieu (De Verdal et al., 2018; Besson et al., 2019). Thus, in gilthead sea bream, most genetic selection programmes have been applied for the direct selection and improvement of somatic growth, disease resistance, or carcass quality (Perera et al., 2019, 2021b; Lorenzo-Felipe et al., 2021). However, there is now evidence that selection for growth in a farming environment co-selects changes in reproductive or swimming performance (Ferosekhan et al., 2021; Perera et al., 2021a). In addition, we have recently reported that selection for growth also selects for a more functionally flexible microbiota when the inferred gut metagenomes of representative fish families with different growth trajectories across the production cycle were compared (Piazzon et al., 2020). However, such an approach was based on the amplification of specific variable regions (v3-v4) of the 16S rRNA gene, and they only inform about the taxonomic profile of one portion (Bacteria, Archaea) of the whole gut microbial community, which also includes Fungi and Virus (Merrifield and Rodiles, 2015). In addition, the inferred functional changes related to gut bacteria variations might not correlate with the actual expression profile of these populations. To solve these issues, metatranscriptomic analyses are perhaps a better approach (Aguiar-Pulido et al., 2016), evidenced by the exponential increase of metatranscriptomic projects over the last 20 years (Shakya et al., 2019). Such approach has been used in humans to characterise active microbes in a community, discover novel microbial interactions, and track the relationship between viral genes and their hosts (Gosalbes et al., 2011; Bikel et al., 2015; Bashiardes et al., 2016; Moniruzzaman et al., 2017). In livestock species, metatranscriptomics has helped to reveal the association between breed effect and rumen microbiome activity (Li et al., 2019a). In aquatic organisms, although at a lower extent, there are also some examples analysing the composition of marine fish viromes (Geoghegan et al, 2021), to characterize the full set of water-living microbes (Salazar et al., 2019; Trench-Fiol and Fink, 2020), and to reveal microbial functions associated with the digestion of algal polysaccharides in the digestive tract of the abalone Haliotis discus hannai (Nam et al., 2018). Yet, to date, there is no information on the genome × gut metatranscriptome interaction of genetically selected fish and how this can affect their nutritional plasticity. This is, thereby, the aim of the present study, where we sequenced the intestinal metatranscriptome of two gilthead sea bream families with opposite growth trajectories from the study of Piazzon et al. (2020) with three main objectives: i) to study and characterize the full set of microbes present in the gut of this fish species, ii) to evaluate if genetic background and alternative diets can change the expression of genes collectively expressed by these microbial communities, and iii) to unravel which metabolic processes are enriched in gilthead sea bream intestine and which microbial community is involved, to provide more insights into the broad microbial landscape of the gut of farmed fish.

The gut microbiomes of fish are complex networks of communities, including members of bacteria, archaea, fungi, and viruses (Merrifield and Rodiles, 2015). However, it is often estimated that the abundance of bacterial taxa (>99%) outnumbers the proportion of the other microbial populations (<1%) (Merrifield and Rodiles, 2015; Egerton et al., 2018). In this scenario, amplicon-based sequencing techniques targetting the 16S rRNA bacterial gene have been helpful approaches, widely used to measure the composition and alterations of fish gut bacterial communities. However, these techniques do not offer the complete intestinal metagenome landscape, and microbial gene repertoire and expression cannot be retrieved or measured (Lindahl et al., 2013; Rausch et al., 2019). To overcome these limitations, this study reports a metatranscriptomic approach, showing that genes belonging to the Archaea, Bacteria, Eukarya (Fungi), and Virus domains are metabolically active in the gilthead seabream (Figure 2A). As expected, archaeal and viral transcripts were found in lower proportions (1.65 and 3.25%) in the intestinal mucosa, but bacterial and fungal transcripts were roughly equal (∼44 and ∼51%, respectively) and highly predominant in this mucosal surface. Indeed, several studies highlighted the importance of the highly diverse fungal microbiota fraction in humans (Huffnagle and Noverr, 2013; Santus et al., 2021). In the same line, more than 20% of the metatranscripts in the rumen of dairy cows have been related to fungi (Comtet-Marre et al., 2017), and untargeted metabolomics underlined the presence of fungal-derived metabolites in the serum of gilthead sea bream fed plant-based diets (Gil-Solsona et al., 2019). Certainly, the eukaryotic fungal cells, although lower in number, might become more transcriptionally active than prokaryotic cells.

As shown in Figure 2B, the functional annotation of the gilthead seabream metatranscriptome shared the over-representation of a significant number of GO-BP categories related to symbiosis (Interspecies interaction between organisms, Multi-organism process, Multicellular organismal process) and sensory responses (Behaviour, Response to stimulus, and Signalling), evidencing the contribution of all the microbial communities to the cooperative processes taking place in the host gut. The predominant taxa, Fungi and Bacteria, expressed genes related with all the GO-BP level 2 categories, but the Rhythmic process appeared as a bacterial-specific category. In this line, the manipulation of daily rhythms of gut bacterial microbiota abundance and activity is becoming a promising chrononutritional approach to consolidate host circadian rhythms and metabolic homeorhesis (Parkar et al., 2019; Gutierrez Lopez et al., 2021). Recently, Calduch-Giner et al. (2022) reviewed behavioural biosensing approaches based on accelerometer technology [AEFishBIT dataloggers (Rosell-Moll et al., 2021)] for informing on fish social behaviour in terms of coping styles or changes in daily or seasonal activity, linking ventilation rates with changes in energy partitioning between growth and physical activity. However, the association of changes in behaviour and gut microbiome rhythms remains almost unexplored in fish, and their inter-related study would contribute to discerning the disrupting effects of life stressors in the gut processes related to host rhythmicity. In any case, it is noticeable that the plant-enriched diet yielded a gilthead seabream metatranscriptome with a significant decrease of bacterial transcripts (∼7%), together with an increase (4%) in the number of fungal transcripts (Table 2) as described before in rainbow trout fed with yeast and soybean meal diets (Merrifield et al., 2009; Huyben et al., 2018).

In most cases, taxonomic assignment at lower taxonomic levels using the transcript sequences was not possible. Nonetheless, although commonly reported taxonomic assignments in amplicon-based protocols, such as families or genus, could not be related to their corresponding gene repertoire, the information shown in Supplementary Figure 1 can provide a hint of the most metabolically active phyla. Archaeal transcripts were majorly assigned to the Euryarchaeota phylum (Supplementary Figure 1A), one of the most discussed in humans for positively impacting gut health (Horz and Conrads, 2010). In line with their highest abundance in bacterial gut microbiome studies using 16S rRNA (Piazzon et al., 2017, 2019; Naya-Català et al., 2021b; Solé-Jiménez et al., 2021), the phyla Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes were also the ones contributing with the most expressed bacterial transcripts when the axonomic assignment was possible (Supplementary Figure 1B). Ascomycota and Basidiomycota play a pivotal role in the expression of enzymes related to fish nutrition and intestinal maturation (Gatesoupe, 2007; Banerjee and Ghosh, 2014; Siriyappagouder et al., 2018), were the most abundant phyla among the fungal fraction (Supplementary Figure 1C). By last, within the viral fraction, Herpesviridiae, Poxviridiae, and Retroviridiae families appeared to be the transcriptionally prevailing and functional (Supplementary Figure 1D), as previously detected using wild and farmed gilthead sea bream (Filipa-Silva et al., 2020).

From a recent study (Piazzon et al., 2020), we concluded that gilthead sea bream families selected for fast-growth harboured a plastic bacterial microbiota that was able to adapt to diet changes with no impact on growth or health. Indeed, small changes in bacterial composition accounted for larger changes in metabolic capacity when the inferred metagenome and pathway analysis were conducted (59 metabolic pathways changing). Conversely, significant changes in intestinal bacterial composition were limited to changes in 15 metabolic pathways in fish families selected for slow-growth, assuming that all bacteria detected are metabolically active and expressing all their genes at a fixed level (Langille et al., 2013; Aßhauer et al., 2015; Louca et al., 2018). In line with these results, discriminant analysis of the metatranscriptome only showed a clear discriminant value with dietary changes for the fast-growth family (Figure 3A and Supplementary Figure 2). To measure the consistency of these results, the 5,998 discriminant genes were clustered in four groups (C1, C2, C3, and C4) (Figure 3B and Supplementary Figure 4), according to their expression pattern (discussed below). Clusters C1, C3, and C4 presented a differing expression pattern between the 6e2-D1 and e6e2-D2 groups. Over-represented KEGG terms in these clusters (Supplementary Table 4) disclosed that ∼70% (42 out of 59) of significant differentially expressed pathways predicted by the inferred metagenome of our previous study (Piazzon et al., 2020) were also detected in the current study, but a total of 218 unique enriched pathways were found. Altogether, although these results support that metagenome prediction tools can help to have an overview of the direction and magnitude of the metabolic changes, but metatranscriptomic analyses provide more complete and precise information.

The groups C1 and C3 encompassed 2,803 microbial genes whose expression pattern was influenced by the genetic selection for high growth and shifted toward values closer to slow-growth families with the plant-based diet (Figure 3B). Among the elements governing this difference in gilthead sea bream gut, we mainly found genes related to the principal metabolic routes, required in all microbial populations (Qin et al., 2010) (Figures 4A, 5A). Bacterial, fungal, and viral genes were associated with Fatty acid metabolism, the predominant supra-category in these groups. This is not surprising, as gut microbes can process lipid dietary components and perform processes not exerted by the host (Schoeler and Caesar, 2019). Within this supra-category, the predominant KEGG term was N-Glycan biosynthesis, exclusively exerted, according to our results, by fungal genes. Besides, genetic selection for growth was accompanied by a raise in the formation of these compounds, which are indigestible for the host and can be transformed into short-chain fatty acids by microbial fermentation in the gut lumen (Koropatkin et al., 2012). This pathway is enriched in C1, which suggests that the plant-based diet was down-regulating this pathway as part of a healthy and complex gut homeostatic process, although it is well-known that dietary butyrate supplementation can mitigate most of the inflammatory drawback effects of plant-based diets in gilthead sea bream (Estensoro et al., 2016; Piazzon et al., 2017). Together with the fatty acid metabolism, a wide representation of carbohydrate metabolism was found related to Bacteria and Fungi. Inside this supra-category, the pathway Glutathione metabolism was remarkably above the others. Glutathione is an anti-oxidative compound, widely distributed in the gastrointestinal tract of humans and rodents (Mardinoglu et al., 2015), acting as a growth and gut health health-promoting in trout (Wang et al., 2021). Here, the genetic selection for fast-growth induced an over-expression of microbial genes related to glutathione metabolism. However, this expression was reverted to values found in the slow-growing fish with the use of the plant-based diet. The bacterial fraction of the intestinal microbiota was predominantly expressing genes related to the Amino acid metabolism process in the C1 group, but also in C2 and C4. The difference among groups resided in the type of amino acid that this community is making available in the fish, being the control of dietary protein source a strategic approach for the control of amino acid-fermenting bacterial species and their metabolic pathways, which in turn could have an impact on the metabolism of the host gut (Neis et al., 2015). By last, in C3 and C2, we also found a strong association between infectious diseases and immune-inflammatory pathways, mediated by fungal genes. This is a usual link in the gut microbiomes (Lokesh et al., 2012; Al Bander et al., 2020) and yeasts have a protective role against fish pathogens by expressing immunostimulatory substances (Li and Gatlin, 2006; Lokesh et al., 2012). According to our results, the formation of these compounds would be regulated by both the diet and genetic background.

Group C2, comprising 1,007 genes, differentiated the genetic background of the samples with no diet effect, demonstrating the effect of selective breeding on active gut microbial populations (Figures 4B, 5B). In addition to the amino acid metabolism and the infectious disease and immune system supra-categories, formerly described, we found the fungal-associated Alkaloid biosynthesis supra-category. Traditionally, these bioactive compounds have been related to plants (Peng et al., 2019), but fungi, especially the Ascomycota phylum, are also able to produce them (Xu et al., 2020). The properties of these organic molecules include anti-microbial and anti-oxidant activity (Iranshahy et al., 2014), and their inclusion into diets have been suggested as possible alternatives to antimicrobial growth enhancers (Willems et al., 2020). In fish, these chemicals have been stated to produce anti-nutritional effects due to palatability issues when they were included in the diet of rainbow trout (Glencross et al., 2006). However, the positive properties of alkaloids cannot be underestimated, and the gut fungal population could be an interesting target at the time of exploring the microbial production of alkaloids for reducing the use of antibiotics in aquaculture production (Okocha et al., 2018).

Finally, a total of 2,188 genes were assigned to C4, a group showing the genetic × diet interaction effect in the fast-growth family. The expression of genes in this group remained attenuated in the fast-growing fish fed the control diet and was upregulated in fast-growth fish fed the plant-based diet, disclosing the dual response of this fish group when dealing with alternative diets (Figures 4C, 5C). Alternative diet formulations in gilthead sea bream are prone to produce changes in the intestinal plasticity of this species (Perera et al., 2019; Naya-Català et al., 2021b; Solé-Jiménez et al., 2021). Indeed, the families selected for growth used in this study showed an increased intestinal length when fed plant-enriched diets (Perera et al., 2019). Herein, the over-representation test disclosed the link between two supra-categories related to this anatomical feature: Host-mediated regulation of intestinal microbiota composition and Regulation of anatomical structure morphogenesis. These categories, mainly composed of bacterial transcripts, highlight an important role of this population in the intestinal reshaping upon feeding plant-enriched diets to fish families selected for fast growth. The bacterial population was also expressing genes related to the coupled categories Symbiont modulation of host cellular process and Killing of cells of other organisms. A recent study stated that diet and gut microbes could jointly act as enhancers of the programmed cell death to reduce colorectal cancer (Chapkin et al., 2020), and in humans, the bacterial community is a rich source of metabolites against pathogenic fungi, via the activation of the mTOR signalling pathway (Li et al., 2019b). Herein, this association suggests the role of bacterial intestinal symbionts in the modulation of processes resulting in the death or programmed death of host or other symbiont cells. Fungal genes in C4 were involved in the regulation of appetite and several processes related to Organismal systems interaction. However, the main expressed genes inside this supra-category were kinases and mitogen-activated protein kinases families, widely analyzed in fungi (Martínez-Soto and Ruiz-Herrera, 2017), which networked together with functions of tissue development and regeneration (Dorso-ventral formation) and digestive (Bile secretion), endocrine (Aldosterone and Prolactin signalling pathways), and immune (Chemokine signalling pathway) functions. It is widely documented that gut microbial metabolites can play a pivotal role in the regulation of these functions (Tan et al., 2018; Silva et al., 2020). However, the current results just suggest that the introduction of plant-enriched diets to fast-growth families is changing the signal transduction processes in the fungal symbionts, and only further metabolomics studies could help to discern the resulting metabolites of these signalling cascades.

To sum up, this metatranscriptomic approach was very useful for measuring which microbial populations are metabolically active in the anterior intestine of gilthead sea bream and revealed a wide range of processes carried out by microbes that can serve as a gene catalogue for future studies. Moreover, all the transcripts were taxonomically assigned to the level of the kingdom, so processes exerted predominantly by a specific gut community could be disclosed at this level. In this line, 18S rRNA amplification approaches measuring the composition and variations of fungal intestinal communities arise as promising targets to completely understand the processes occurring in the anterior part of this tissue in gilthead sea bream. Furthermore, despite the simplicity of the experimental model, where only two families selected for growth were used, this study helped us to corroborate the higher functional plasticity of the microbiome of fish selected for fast growth, which was able to shape a changing metatranscriptome with a more stable metagenome.

Raw sequencing data can be found at NCBI’s Sequence Read Archive under accession PRJNA790012 (BioSample accession numbers: SAMN24182635-50).

The animal study was reviewed and approved by Ethics and Animal Welfare Committee of IATS and CSIC.

AS-B and JP-S: conceptualisation, funding acquisition, project administration, resources, and supervision. FN-C, MCP, and JP-S: data curation, formal analysis, and writing—original draft. FN-C and MCP: visualisation. FN-C, MCP, JC-G, AS-B, and JP-S: investigation, writing—review and editing, and read and approved the final manuscript. All authors contributed to the article and approved the submitted version.

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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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