*3.5.1 Metagenomics in the study of microbiota of wild and farmed fishes in Chile*

Despite the importance of the gut microbiota in the fitness of wild and farmed animals, as seen for salmonids in Section 4, few studies have characterized the microbiota composition of farmed and wild fish and shellfish that are commonly consumed in Chile [107, 116–121]. Most of the best characterized microbiota corresponds to foreign species such as Atlantic salmon and rainbow trout, in agreement with their economic and social importance. With few exceptions, most of the microbiota reported in these species corresponds to studies done in Europe, Canada, the USA, and New Zealand. Even for rainbow trout, a species introduced in Chilean rivers more than 100 years ago [9], there are no studies characterizing and comparing the microbiota of feral rainbow trout living in Chilean rivers with the microbiota present in wild rainbow trout from regions where the species is autochthonous [122].

Since rainbow trout is a species considered "naturalized" in Chile, the obvious question would be if naturalization or adaptation to local conditions is associated with a particular microbiota composition. This is, perhaps, another dimension to consider in the study of the adaptation of rainbow trout in Chile. Given the wild north–south distribution of the species in Chile, it would be also necessary to understand if microbiota composition varies with latitude. The same sort of questions are pertinent to understand the microbiota composition of farmed trout and naturalized ones.

A similar situation happens with farmed Atlantic salmons which escaped from Chilean hatcheries. It is unknown if they show a microbiota composition similar or different to those present in the wild Atlantic salmon from Canada

or Norwegian rivers or closer to the Atlantic salmon farmed in Chile. In the case of coho salmon, another heavily cultivated species in Chile, studies on the microbiota have been done using 16S rRNA PCR coupled to DGGE [123], which gives a sort of qualitative approach to the microbiota diversity. This study shows that stable microbiota is established after first feeding and comes mainly from bacteria located in eggs and water. A more accurate analysis of the genomes of the microbiota would be important to understand the function that a particular group of bacteria could play in the species adaptation to the aquaculture or wild environment.

In short, the study of microbiota of farmed salmonids, as well as or other species, and their naturalized or wild relatives should provide evidence on how the host-microbiota-environment relationships evolve [64, 124]. The microbiota from wild fishes (marine or freshwater) living in the Chilean territory could give us some clues to understand how these fishes have adapted to local conditions. Such studies should help to optimize its nutrition and protection against pathogens in the artificial environment under the environmental conditions present in Chile.

The intestinal microbiota of *Seriola lalandi* and *Paralichthys adspersus* (fine flounder) has been sequenced. Both species distributed in the southern Pacific Ocean have, respectively, actual or potential aquaculture interest in Chile. In both cases, bacteria belonging to the phylum *Proteobacteria* were the most abundant group in wild specimens, while under aquaculture conditions, members of the phylum *Firmicutes* predominated. Under aquaculture conditions, the fine flounder shows a reduction of *Actinobacteria*, a group known to produce antimicrobial compounds [117, 119]. Further, metagenomic characterization of the microbiota through the complete sequencing of all the microbial genomes is necessary to properly predict the function of the microorganisms that colonize wild or farmed fishes and so to have a better approach to the evolution of the host-microbiota relationship in *Seriola lalandi* and *Paralichthys adspersus*.

#### *3.5.2 Potential uses of the metagenomics for identification of new probiotics*

The term microbiota refers to a complex and dynamic ecosystem of microorganisms that colonize the exposed surfaces and epithelia of an organism [125]. The role of these microorganisms has been studied mainly in the gastrointestinal tract (GT), contributing greatly to the general welfare of the host, participating in the absorption of nutrients, functioning as a protective barrier against potential pathogens, and regulating the expression of genes involved in epithelial proliferation in addition to having a role in the stimulation of the immune system and the prevention of diseases [126]. It is for these reasons that the microbiota is a good source of probiotic potentials.

Probiotics are defined as live microbiological food supplements with beneficial effects for the animal host [127], which confer protection (antagonism) against pathogens, helping in the development of the immune system and providing nutritional benefits [128]. In the aquaculture sector, probiotics began to be used at the end of the 1980s, as a prophylactic method against pathogens, mainly due to their ability to stimulate the innate immune response, which is characterized by having a nonspecific mode of action against various microorganisms [129].

The microorganisms used as probiotics in the aquaculture can have different origins, microorganisms previously used in mammals (allobiotic, probiotic) or commensal microorganisms that colonize the GT and the mucous membranes of the fish (autochthonous probiotic). In the case of aquatic animals, probiotics of autochthonous origin have adaptive advantages against foreign microorganisms, since they are adapted to factors such as water temperature and salinity [91], a situation that

#### *Application of Metagenomics to Chilean Aquaculture DOI: http://dx.doi.org/10.5772/intechopen.86302*

allows them to compete adequately with the resident organisms of the GT [130], thus ensuring adequate colonization and persistence of the microorganism.

In the last three decades, several microorganisms with probiotic activity have been identified and characterized for the aquaculture sector [131]. These microorganisms include both Gram-positive and Gram-negative bacteria, bacteriophages, microalgae, and yeasts [132, 133]. Among the most used probiotic species in the production of salmonids are the genera *Lactobacillus*, *Bifidobacterium*, *Aeromonas*, *Plesiomonas*, *Bacteroides*, *Fusobacterium*, *Carnobacterium*, *Eubacterium*, *Bacillus*, *Enterococcus*, *Bacteroides*, *Clostridium*, *Agrobacterium*, *Pseudomonas*, *Brevibacterium*, *Microbacterium*, *Staphylococcus*, *Streptomyces*, *Micrococcus*, *Psychrobacter*, *Pediococcus*, *Saccharomyces*, *Debaryomyces*, *Alteromonas*, *Tetraselmis*, *Roseobacter*, *Weissella*, and *Aspergillus* [75].

The use of these and other microorganisms with probiotic activity has generated a reduction in the levels of antimicrobial compounds, particularly antibiotics, used in the salmon industry. In addition, there has been an improvement in the appetite and/or growth of farmed salmonids [134]. Many of these microorganisms have antagonistic activity in vivo in salmonids against pathogens such as *A. salmonicida*, *V. anguillarum*, *V. ordalii*, and *F. psychrophilum*.

Marine bacteria also have the potential to be used as probiotics. These bacteria have the ability to store the biodegradable polymer polyhydroxybutyrate (PHB), which exhibits the ability to neutralize pathogens in *Artemia*, fish, or shrimps. Such probiotic effect seems associated with the breakdown of PHB into monomers (short-chain fatty acids (SCFA)) in the gut of the target species; this breakdown changes pH and improves bacterial richness [135] or enhances immunological defense and provides energy to cells [6, 135–139]. Baruah et al. demonstrated that a commercial PHB source enhanced the survival of *Artemia* challenged with pathogen by triggering the expression of the heat shock protein, Hsp 70, which is associated with protective innate immune responses [135]. Another positive PHB effect has been reported in the European sea bass (*Dicentrarchus labrax*) in an experiment in which the diet of juveniles was supplemented with 2 and 5% PHB (w/w). Juveniles showed better growth performance correlated to a high bacterial richness in the gut [135]. Similar results were observed in the Siberian sturgeon (*Acipenser baerii*) fingerling, also with a diet supplemented with increasing amounts of PHB [137], as well as in shrimps [136, 139].

There are currently commercialized probiotics for use in aquaculture, such as Mycolactor Dry Probiotic®, which corresponds to a mixture of *Saccharomyces cerevisiae*, *Enterococcus faecium*, *Lactobacillus acidophilus*, *L. casei*, *L. plantarum*, and *L. brevis*; INVE Sanolife® MIC that includes a mixture of *Bacillus* strains (Biogen®), *Bacillus licheniformis* and *Bacillus subtilis*; and BACTOCELL® (*Pediococcus acidilactici*), the first probiotic approved by the European Union for use in aquaculture, as an additive in the feeding of salmonids [140].

In the case of mollusks, there is a history of a bacterial strain isolated from the gonads of Chilean scallops (*Argopecten purpuratus*) and characterized as *Alteromonas haloplanktis* which shows inhibitory activity in vitro against the known pathogens *V. ordalii*, *V. parahaemolyticus*, *V. anguillarum*, *V. alginolyticus*, and *A. hydrophila* [141]. The combination of *A. haloplanktis* and *Vibrio* strain 11 showed in vitro inhibition against *V. anguillarum*-protected scallop larvae in in vivo assays [142]. A recent example in the European blue mussel (*Mytilus edulis*) showed high poly-β-hydroxybutyrate levels regulating the immune response of mussels challenged with *Vibrio coralliilyticus* [143]*.*

Other studies test the protective capacity of *Aeromonas media* A199 in vitro against other 89 strains of *Aeromonas* and *Vibrio*, in addition to preventing the

death of oyster larvae (*Crassostrea gigas*) when challenged in vivo with *Vibrio tubiashii*. However, *A. media* A199 was not detected in the host after 4 days of the administration of the probiotic treatment, indicating that it would be necessary to administer the probiotic at regular intervals of time if a prolonged protective effect is required [144].

The functional relationship between the immune system of teleost and mammalians and innate and cellular response present in shellfish makes plausible that microbiota plays these roles in all cultured species. Characterization of the microbiota by a metagenomic approach has helped to identify microorganisms or consortia that can be used to improve the absorption of nutrients, have an antagonistic effect against bacterial pathogens, or can stimulate the innate and cellular response [145]. Metagenomics based on sequencing the 16 s rRNA associated to a host biological property or condition such as resistance to pathogens could help to identify bacteria or consortium with antimicrobial properties and look for ways to culture this bacteria to isolate potential probiotics. This analysis can also be complemented with a prediction of the metabolic pathway using the software PICRUSt [146]. It may also help to predict a particular condition of the fish if associated to a particular group of microorganisms with different metabolic properties, such as the production of vitamins, use of different carbon sources, or production of metabolites with immunomodulator properties such as SCFA or PUFA [118]. This analysis could be improved using metagenomics based on the complete sequence of the whole microbial DNA. This analysis, associated to ORF prediction, metabolic reconstruction, and prediction of secondary metabolites and antimicrobial peptides using antiSMASH [147], could help to improve the metabolic characterization of the microbiota associated to a particular condition and help to guide the identification of cultivable microorganisms that can be used as probiotics. Currently this approach began to be applied in the identification of potential probiotics to the aquaculture**,** for example**,** from eggs of Rainbow trout resistant to the infection with the fungus Saprolegna, was isolated a cultivable bacterium belonging to the genus *Actinomyces* that produce antifungal compounds and confer protection against this pathogen [73]**.**
