**4. Importance of microbiota-host-environment interactions for the development of a sustainable aquaculture**

### **4.1 Microbiota and immune system interaction in salmonids**

Most of the current information about the host-microbiota communication comes from studies in mammalian models. These studies show that the interplay between the microbiota and the immune system is a sort of chemical cross talk [148] which involves from microbiota to host (a) the specific stimulation of host receptors by molecules from microbial organisms (bacteria, fungus, viruses, and archaea), (b) bioactive molecules secreted by the microbiota metabolism, and (c) stimuli of epigenetic mechanisms to control expression of immune genes and fate of immune cells. In salmonids the interplay between immune system and microbiota has not been studied yet. However, the immune system of salmonids shows the same elements that allow the host-microbiota communication in mammalians.

a.**Specific stimulation of host receptors by molecules from microbial organisms**. The immune system is able to recognize the pattern of some structural molecules from microorganisms either commensal or pathogens. Characterized originally in pathogens, these pathogen-associated molecular patterns (PAMP), which are specific for each type of microorganism, are recognized by a family of receptors located in the immune cells denominated as pattern recognition receptor (PRR), in particular a subtype of them, namely, Toll-like receptors (TLR). This interaction appears to be key in stimulating NF-kβ, interferon-response factors, and the inflammasome, which, depending on the cell type, context, and microorganism involved, responds through the production of different inflammatory cytokines (e.g., interferon, IL-1β, IL-22), which can have systemic or local effects [149–153]. On the other side, commensal microbiota controls the immune response through the exposure or secretion of molecules that stimulate anti-inflammatory response. Polysaccharide A of *Bacteroides fragilis* can induce an anti-inflammatory response by increasing IL-10 production and the population of regulatory T lymphocytes and by decreasing the inflammatory response mediated by Th-17 [154, 155].

There is a greater diversity of TLR in salmonids than in mammals (20 versus 10) [156, 157], which suggests that in Atlantic salmon or rainbow trout, the cellular immune response mediated by TLR stimulation should be more complex than in mammals. This might be because fish live in an aqueous environment where they are exposed to a greater quantity and diversity of microorganisms that interact with a proportionally greater surface of mucosa. In addition, salmonids have a poorly developed immune response at the level of antibodies, strongly suggesting that in these organisms the cellular immune response and microbiota are the main barriers against pathogens. As in mammals, TLR expression is also stimulated by microbial infections [158]; however, how the pattern of TLR gene expression changes as consequences of variation in microbiota composition is unknown.

b.**Bioactive molecules secreted by the salmonid microbiota**. In mammals microbiota secretes several bioactive molecules able to modify the cell metabolism and immune response [159]; among them the molecules with the most significant impact are short-chain fatty acids (SCFA, formate, acetate, n-propionate, n-butyrate, and n-valerate). SCFA are generated by the anaerobic fermentative metabolism of bacteria that are part of the intestinal microbiota and, because they are hydrophobic, are absorbed by epithelial cells and rapidly disseminate throughout the organism causing effects in different organs [160]. The microbial SCFA best characterized is butyrate; this molecule induces the production of regulatory T lymphocytes beyond the thymus [161], stimulates microglia maturation and function and PMN lymphocyte activity [162, 163], and decreases the production of proinflammatory cytokines in macrophages (INF-γ, IL-1β, TNFα) [164]. Butyrate also decreases the proliferation and increases apoptosis of T CD4 lymphocytes [165, 166], increases the production of anti-inflammatory cytokines in dendritic cells (IL-10, IL-23), and decreases exposure of MHCII [167, 168]. Although in general the effect of butyrate and other SCFA is to promote anti-inflammatory responses, the exact role depends on the type of cell and SCFA. The generic effects of butyrate can be explained by its capacity to stimulate the free fatty acid receptors (GPR41, GPR43, and GRP109a), which in turn stimulate a cascade of phosphorylation by Gai to activate at ERK1/ERK2 MAP kinase. In mammalians, these receptors are expressed in immune cells, specially GPR43, which is highly expressed in macrophages/microglia, neutrophils, and monocytes [162, 164]. Bioinformatic assays performed in our laboratory indicated that Atlantic salmon genome encodes for 13 proteins which are homologous to the butyrate receptors present in mammalians. This expansion suggests an important role of this molecule in Atlantic salmon physiology [Tello et al. unpublished]; however, the pattern of expression of these genes is currently unknown in both the salmonid gut and its immune organs. Also it is unknown if butyrate is able to induce ERK1/ERK2 phosphorylation in salmonids.

Besides its interaction with its receptors, n-butyrate is also a strong inhibitor of histone deacetylase (HDAC), inducing chromatin remodeling and changes at an epigenetic level [169, 170]. HDAC is a highly conserved protein among different species. Human and Atlantic salmon HDAC shares a 97% of sequence identity, thus making highly plausible that HDAC from salmonids can also be inhibited by butyrate.

It is unknown if the salmonid microbiota produces butyrate or other bioactive molecules able to modulate the immune response; evidence from other fish suggest that butyrate is also a bioactive molecule in teleost. Butyrate is found in the intestinal tract of herbivorous and carnivorous fish [171, 172]. In *Sparus aurata* it increases intestinal microvilli and nutrient absorption [173] and in *Cyprinus carpio* increases the expression of shock protein-70 (HSP70), proinflammatory cytokines (IL-1β and TNF-α), and anti-inflammatory cytokines (transforming growth factor-β) [174]. The mechanism by which butyrate can induce these changes is currently unknown.

Preliminary experiments performed in our laboratory show that butyrate modifies the antiviral response in SHK-1 cells, in a mechanism that is independent of the expression of the putative butyrate receptors [Tello unpublished], suggesting that Atlantic salmon cells could be sensible to this microbial metabolite.

c.**Stimuli epigenetic mechanisms to control gene expression and fate of immune cells.** In mammals microbiota also controls the immune system through several epigenetic mechanisms: DNA methylation [175], histone modification [169, 170, 176, 177], and control of gene expression by noncoding RNA [178]. In most of cases, this control is achieved by metabolic products of the microbiota, such as butyrate or vitamin precursor [179]. In simple terms, DNA methylation of cytosine impedes transcription factor binding and favors the recruitment of methylated binding domain proteins, which in turn prevents the binding of transcription factors by inactivating the chromatin configuration around genes. Through changes in DNA methylation pattern, microbiota may control the proliferation of Treg [180] and the function of NK cells [181].

Changes in the histone modification pattern produced by inhibiting HDAC with SCFA stimulate changes in the chromatin structure increasing the expression of *foxP3*, which promotes the differentiation of T CD4+ lymphocytes in Treg lymphocytes, favoring the anti-inflammatory response [161]. In intestinal macrophages, SCFA reduces the production of proinflammatory mediators (cytokines) via HDAC inhibition [182]. Deleting histone deacetylase 3 in intestinal epithelial cells alters normal microbiota, changes the expression patterns of antimicrobial peptides, and increases inflammatory processes, suggesting that epigenetic control of the host by microbiota is a fundamental element in homeostasis maintenance [183]. Although the epigenetic regulation in Atlantic salmon or rainbow trout has been poorly studied, at genetic level, both species show a more complex DNA methylation and histone acetylation/deacetylation systems than mammalians, with several gene duplications [184], suggesting that this mechanism could also be implied in the host-microbiota communication.

**Noncoding RNAs** (miRNA, lncRNA, and snRNA) are a group of RNAs highly expressed in cells with several regulatory functions. Among them, microRNAs (miRNAs) are implicated in the cross talk between mammalian microbiota and its host immune system [178, 185, 186], while lncRNA are involved in the cross talk with gut epithelial cells [187]. Commensal microbiota is able to regulate the expression of several miRNAs that target genes involved in the inflammatory process, generating a tolerance state in the gut [188–190]. Among them, two important miRNAs that regulate the inflammation process are miRNA146 and miRNA155. miRNA146 expression is induced by low doses of LPS and acts as an anti-inflammatory regulator by targeting TNF receptor-associated factor 6 (TRAF6) and IL-1R-associated kinase 1 (IRAK1), which are involved in the NF-κB pathway. miRNA146 allows the establishment of postnatal intestinal microbiota in the newborn gut, preventing inappropriate inflammation. miRNA155 is induced by high concentrations of LPS and plays an opposite role stimulating the inflammatory process by targeting the negative regulator of the NF-κB pathway. Recent works using NGS (RNAseq) from different organs of Atlantic salmon identified 180 distinct mature miRNAs belonging to 106 families of miRNAs [191]. These miRNAs were deposited in the miRBase database (http://www.mirbase.org). This database currently contains 371 miRNAs from Atlantic salmon. Among the miRNAs identified in Atlantic salmon, orthologous of miRNA146, miRNA155, and other 15 of 27 miRNAs that participate in the microbiota-host communication in mammals were found. Currently it is unknown if the expression of these miRNAs changes according to the composition of Atlantic salmon microbiota.

The study of the mechanisms underlying the stimulation of immune system by microorganism that conform the microbiota is an open field for metagenomic studies searching for association between microorganism, consortia, or microbial metabolites and the proper immunological function. This approach could help to understand or help to predict more accurately the impact of environmental factors triggering outbreaks and to design either new prophylactic or therapeutic strategic based on microorganism or microbial metabolites. In a more holistic approach, this also could help to understand if changes in the environmental microbiota are sensed by the fish or other species which should help to properly assess their impact on the aquaculture ecosystem.

## **4.2 Prediction systems: an ecosystem approach**

FAO's ecosystem approach to aquaculture [192] is a "strategy for the integration of the activity within the wider ecosystem such that it promotes sustainable development, equity, and resilience of interlinked social-ecological systems." The so-called aquaculture ecosystem of southern Chile is shared by multiple users, notably by the salmon and mussel industry and a significant part of the national fishermen task force that is concentrated in Region X (De Los Lagos Region or Lake District). The latter depends on seafood collection and commercialization for their subsistence, and so any serious ecosystem perturbation ends up in conflicting situations affecting all users, including tourism, also an important player depending on the marine ecosystem. One of the most serious harmful algal blooms (HABs) of *Pseudochattonella verruculosa* occurred in the austral summer of 2016 (February–April) killing nearly 12% of the Chilean salmon production (106,000 tons), causing severe mortality of other fish and shellfish in the coastal waters and interior sea of western Patagonia [193]. This event exemplifies the inherent complexity of ecosystem perturbations and its socioecological consequences. But not only users like aquaculture producers should be blamed by such perturbations since climatic change seems to have created

the oceanographic conditions that amplified this HAB event. Indeed, the event was associated to El Niño and the climatic and oceanographic conditions associated to it [193]. In spite of the ongoing monitoring protocols carried out by different institutions, it has been difficult to understand what factors influence the diversity and abundance of harmful microalgae population, which is understandable due to the dynamic and complexity of the marine ecosystem, so a HAB event cannot be understood from the analysis of few variables. Metagenomic studies offer new insights into the complexity of the marine ecosystem and HAB events by allowing a deeper view to the microbial diversity that cannot be approached by the traditional microscopic analysis often used for microalgae identification. Additionally, different sorts of interactions can be discovered at all levels, particularly between microbes and microalgae [194–198]. It is now known that some bacterial populations could promote the growth of specific harmful microalgae in species, while some bacteria related to disease in fish or mollusks could also promote blooms. But, also virus controls the abundance and activity of microbial populations and microalgae in nature. In short, the ecosystem associated to HABs is complex and needs a more holistic or integrated approach. The term holobiome has been suggested to address this complexity: holo = entity; biome = biological community. An ongoing project funded by the Japanese government in Chile integrates different Japanese and Chilean universities, the Instituto de Fomento Pesquero (IFOP), and private and governmental bodies, under the holobiome concept with the goal to shed light on the mechanisms involved on algal bloom formation and, at the same time, predict HAB blooms (www.machsatreps.org/en/).
