Use of Yeasts as Probiotics in Fish Aquaculture

According to the UN Food and Agriculture Organization (FAO), “aquaculture, probably the fastest growing food-producing sector, now accounts for nearly 50 percent of the world's food fish” [1]. However, production is hampered by unpredictable mortalities that may be due to the negative interactions between fish and pathogenic bacteria. Intensive fish farming has resulted in a problematic growth in bacterial diseases, prompting the necessary and intensive use of antimicrobials for their treatment.


Introduction
According to the UN Food and Agriculture Organization (FAO), "aquaculture, probably the fastest growing food-producing sector, now accounts for nearly 50 percent of the world's food fish" [1].However, production is hampered by unpredictable mortalities that may be due to the negative interactions between fish and pathogenic bacteria.Intensive fish farming has resulted in a problematic growth in bacterial diseases, prompting the necessary and intensive use of antimicrobials for their treatment.
Because of the rapid expansion of aquaculture, a limited supply of fishmeal has the potential to impede the future growth of this industry.Consequently, much effort has been given to studying other protein and oil sources, but finding a suitable alternative has proved to be challenging.Among the alternatives, plant-based formulations are the least expensive, and many such formulations have a suitable protein profile and long-term availability.Oilseeds, in particular soybean and grain products, have great potential as alternative sources of fish feed.Soybeans are rich in protein and represent the most commonly used plant protein source on the world market.Soybean meal (SBM) has already become an important protein source in fish feed.However, the inclusion of some vegetable proteins, such as SBM, in the diets of fish at levels of >20% may induce intestinal disorders including pathomorphological changes in the distal intestinal epithelium accompanied by diarrhea [2, 3], sometimes caused by the antinutritional factors that are present in SBM.The addition of probiotics (acid lactic bacteria) to starter diets appeared to improve SBM utilization in first feeding rainbow trout [4].
In this context, two of the major challenges in fish aquaculture facilities are 1) the control of diseases, especially during the earliest life stages, and 2) the improvement of nutrition by optimizing food utilization, especially for new fish species.
It is well recognized that the bacterial microbiota of fish is beneficial to the host and affects important biological processes, including nutrient processing and absorption, the develop-ment of the mucosal immune system, and angiogenesis, as was demonstrate in gnotobiotic mice.In larval gnotobiotic zebrafish studies, was shown that the microbiota also influences enterocyte morphology and epithelial renewal, host-transcriptional responses to the microbiota regarding epithelial proliferation, nutrient and xenobiotic metabolism, and immune responses [5].
Yeast have been identified as part of the normal microbiota of both wild and farmed fish, and their role in fish health and nutrition has been addressed in the literature, as yeast have been used either alive to feed live food organisms or after processing as a feed ingredient after demonstrating an artificial colonization of the intestinal host.
Even when accounting for less than 1% of the total microbial isolates in the host, yeast can represent a major physiological contribution beyond what has been observed for probiotic bacteria; in fact, cell volumes from yeast may be larger than those of bacteria by a hundredfold [6].In contrast to bacteria, yeast cells utilize a wide spectrum of simple and more complex organic compounds.This phenomenon results from the extensive metabolic potential of yeast, which is reflected by the production of diverse enzymes.Polyamines secreted by yeasts are also involved in the maturation of the digestive tract of fish larvae.Furthermore, some yeast species and their components, such as β-glucans and mannoproteins, can stimulate the immune and antioxidant systems of the host.Understanding the participation of yeast microbiota in fish health and nutrition may improve both the sanitary conditions and the production performance of fish.
The aim of this chapter is to describe the current knowledge regarding the use of yeasts as probiotics in aquaculture systems.The chapter will include a recent review on the presence and diversity of yeast in marine and aquaculture systems, focusing on the yeast diversity found in the fish gut microbiota.The chapter will also include basic information on the molecular methods used for yeast identification.Finally, the chapter will emphasize topics related to the essential role of probiotic yeasts used in disease control and nutritional improvements in aquaculture, with a special focus on the beneficial effects of yeast β-glucans.

Yeast identified in the marine and other aquatic environments
Yeasts are unicellular eukaryotic microorganisms that are taxonomically placed within the phyla Ascomycota and Basidiomycota within the Kingdom Fungi [7].Ascomycete yeasts comprise approximately 1,000 phylogenetically diverse species that have recently been assigned to 14 different lineages on the basis of multigene sequence analysis [8].The other species of yeasts are classified as the basidiomycetes [9].Some of the general characteristics and ecological properties of each phylum include the following: 1) the cell wall polysaccharide composition is dominated by chitin in the basidiomycetes and by β-glucans in the ascomycetes; 2) the guanine + cytosine (G + C) composition of the nuclear DNA tends to be higher than 50% in basidiomycetes and lower than 50% in ascomycetes; 3) ascomycetes yeasts are generally more fermentative, more copiotrophic (but at the same time nutritionally specialized), more fragrant, and mostly hyaline, while basidiomycete yeasts more often form mucoid colonies, display intense carotenoid pigments and tend to use a broader range of carbon compounds more efficiently at lower concentrations [7]; and 4) ascomycete yeasts are often found in specialized niches involving interactions with plants and insects or other invertebrate animals that they rely upon for dispersal, while basidiomycete yeasts seem to be adapted to the colonization of nutrient-poor solid surfaces and may not rely to the same extent on animal vectors for their dispersal [10].

Marine samples Identified yeast Identification method Reference
Northern Biscayne Bay Candida tropicalis

R. rubra
Cryptococcus identification of cultivated yeasts by the methods described by Lodder and Kreger-van Rij [23] and Wickerham [24]. [25] Gulf Stream 15 miles east of the coast of South Florida.

Marine vegetation
Cryptococcus albidus identification of cultivated yeasts by the methods described by Lodder and Kreger-van Rij [23] and Wickerham [24]. [25] Suwannee Florida estuary (water) Yeasts are widely distributed in several natural environments such as soil, freshwater, and seawater.Their numbers and species distributions are dependent on the concentrations and types of available organic materials.Nearshore environments are usually inhabited by 10 to 1000 of yeast cell/L of water, whereas low organic surface to deep sea oceanic regions contain 10 or fewer cells/L.Marine yeasts are divided into "obligate" and "facultative" groups.When yeast are able to grow on a marine substrate and are frequently collected from the marine environment, they are called "obligate marine" yeasts; in contrast, "facultative marine" yeasts can also be recovered from terrestrial habitats.Marine yeasts participate in several ecological processes in the sea, especially in estuarine and nearshore environments, such as the decomposition of plant substrates [11], nutrient recycling [12], and the biodegradation of oil/ recalcitrant compounds [13]; they are also part of the microbiota of marine and aquaculture animals [6,14].This functional diversity is due, in part, to the fact that yeasts have extraordinary metabolic potential.This potential is available for exploitation [15][16][17][18][19][20], but notably, the vast majority of this potential has yet to be discovered.Several yeast compounds have significant biological value as reagents, cell proteins, vitamins, pigments, and enzymes.Different yeast species have been identified in several marine locations (Table 1).For excellent reviews on marine yeasts, see [21,22].The ascomycete yeasts Debaryomyces hansenii, and Candida spp.are typical ubiquitous species found in oceanic, and other aquatic environments.
Basidiomycete yeasts often account for the majority of the total yeast population found in oligotrophic oceanic water.Among the basidiomycete yeasts, some species of Cryptococcus, Rhodotorula, and Sporobolomyces are widespread across various oceanic regions [22].

Yeast as part of the gut microbiota of fish
Most of the literature on the yeast microbiota of fish is based on the identification of cultivable yeast (Table 2).Yeast have been isolated from the gills, skin, mouths, feces and guts of different fish species.The occurrence of yeast in the fish gut is variable and can fluctuate from nondetectable levels to 10 7 CFU/g of intestinal content [6].Both ascomycete and basidiomycete yeasts have been isolated from fish intestines (Table 2): among ascomycetes, Saccharomycetaceae (which include Candida, Pichia, Saccharomyces, and Debaryomyces) is likely the most important family, while basidiomycetes, include the genera Rhodotorula, Cryptococcus, Sporobolomyces, and Trichosporon [6].The yeasts Metschnikowia zobelii, Kloeckera apiculata, and Debaryomyces sp.dominate in some marine fish (Tachurus symmetricus and Atherinopis affinis littoralis) [39], and in these fish species, the yeast concentration was significantly higher inside the fish than in the surrounding sea water, suggesting that the yeast may grow inside the fish intestine [39].The ascomycetes Debaryomyces hansenii, Candida sp., and Saccharomyces cerevisiae, the basidiomycete Leucosporidium sp., and Rhodotorula have been frequently isolated as the dominant yeast found in the rainbow trout intestine [6].Yeast can also be isolated from the waters of fishponds with different abundance and diversity depending on the season of the year.Fishponds from the Záhorie Lowlands in Slovakia, sampled in summer, harbor the most heterogeneous yeast species, with Aureobasidium, Sporobolomyces, Candida, and Cryptococcus as the most frequently isolated species [40].In autumn, the yeast numbers were higher than in summer, with Candida, Hyphopichia burtonii, Aureobasidium pullulans, Hansenula anomala and Cryptococcus laurentii being most frequently identified [40].
It has been reported that yeasts isolated from the intestine of rainbow trout may adhere to and grow in intestinal mucus [41].Some yeast cells can colonize the intestine of fish after dietary introduction [42], and this ability to colonize may be related to cell surface hydrophobicity [43] and the ability of the strains to grow on mucus [41,44].Some experiments have shown that high levels of yeast intestinal colonization can be achieved when a pure culture of yeast is inoculated into fish.Rainbow trout and turbot were inoculated with Rhodotorula glutinis and D. hansenii HF1, and up to 3.8 × 10 4 , 3.1 × 10 6 , and 2.3 × 10 9 viable yeast cells/g of intestine or feces were recovered in three separate colonization experiments [45].It is important to note that the majority of the studies published until 2007, on the yeast species identified from the aquaculture fish gut, were published by the Gatesoupe Lab [6].Later, studies focused more on the probiotic effects of different yeast strains in aquaculture fish (as described below), and less on the actual yeast species isolated from the fish gut.Kreger-van Rij [23] and Wickerham [24].

Location Fish intestine Identified yeast Identification method Reference
[25] Use

Debaryomyces hansenii, an ubiquitous yeast frequently associated with fish and the marine environment
Debaryomyces hansenii is a halotolerant, non-pathogenic ubiquitous yeast capable of growing in a variety of environments, such as the marine environment and the fish gut.D. hansenii has been described as one of the most frequently isolated yeast associated with fish (Table 2).This species is prevalent in seawater, which may explain its high incidence in the fish gut.One study reported the presence of sequences affiliated with D. hansenii in hydrothermal sediments [35].
A major biotechnological advantage of D. hansenii over Saccharomyces cerevisiae is that D. hansenii is osmotolerant and can grow in media containing up to 4 M NaCl, whereas the growth of S. cerevisiae is restricted to media containing less than 1.7 M NaCl.D. hansenii has been extensively studied because of its significant enzymatic potential [52].For example, several enzymes of biological and biotechnological interest have been identified and characterized in this yeast, including inulinase [53], protease [54], superoxide dismutase (SOD) [55], lipase [56], catalase [57] and α-galactosidase [58][59][60][61][62]. Additionally, β-glucosidase from D. pseudopolymorphus [63] and phytase from D. castellii [64] has also been identified.The ability of D. hansenii to synthesize α-galactosidase has been useful in the treatment of soybean products to reduce raffinose oligosaccharides [59], which are recognized as anti-nutritional factors for mammals and fish.Interestingly, D. hansenii SOD has been proposed as a therapeutic anti-inflammatory agent in animal models (Wistar rats) [55].Because of these characteristics, D. hansenii is one of the yeast species that has been selected for complete sequencing [65].The beneficial effects of this yeast species in cultured fish are described below.

Methods to analyze the yeast microbiota
In the past, the identification of yeast species was a tedious and labor-intensive process that was generally based on the morphological and physiological properties of the isolated yeasts.
To resolve these problems, the identification of cultivated yeasts is now based on DNA sequence analysis, which is a faster and more accurate process.Sequence-based approaches to the study of yeast biodiversity have resulted in a two-fold increase in the number of described species over the past decade, and a 100-fold increase is predicted in the coming decades.A previous work [8] has studied the phylogenetics of the Saccharomycetales by performing DNA sequence analysis based on five loci: 1) the nuclear small subunit (SSU) ribosomal RNA gene, 2) the D1/D2 region of the nuclear large subunit (LSU) 26S rDNA, 3) the elongation factor 1α gene (EF-1α), and 4) the largest and 5) the second largest subunits of the RNA polymerase II gene (RPB1 and RPB2).Based on the availability of the sequence data in the GenBank and AFTOL databases, the LSU rDNA genes were found to be more reliable for yeast identification.Sequencing the 400-650 bp D1/D2 region or a wider LSU region is extremely useful and distinguishes yeast rapidly at a near-species level.The D1/D2 LSU rDNA region has been sequenced for almost all known yeast, both as an identification tool and as a means for estimating phylogenetic relationships among the Saccharomycetales.A previously study [66] published the sequences of the fungal primers ITS1 and ITS4, which amplify the internal transcribed spacer (ITS) ITS1-5.8S-ITS2region that has also been used for yeast identification.Some specialized databases are available on the web in order to help with yeast identification.The Centraal bureau voor Schimmelcultures (CBS) database aids BLAST analysis by allowing pairwise identification of LSU and SSU rDNA, ITS [67] and miscellaneous sequences [68].
It is generally accepted that in every ecosystem, there are cultivable organisms as well as viable organisms that cannot be cultivated in the laboratory.Less than 1% of the microbial species from the marine environment can be cultivated [69].Similarly, only approximately 1% of yeast species has been described thus far [70].In the last decades, several molecular methods have been developed to study natural samples.These methods allow for the identification of microorganisms without isolation and for the determination of the phylogenetic affiliation of community members, thus revealing the enormous extent of microbial diversity.Methods based on the amplification of fragments coding for the 16S rRNA gene have emerged as a powerful tool for studying the bacterial diversity.Denaturing or temperature gradient gel electrophoresis (PCR-DGGE/TTGE) techniques have been introduced into molecular microbial ecology to determine the genetic diversity of the bacterial communities found in the fish gut [71][72][73][74][75][76].These techniques were also applied to characterize the dominant active bacteria in the intestine of different rainbow trout families using RNA that was extracted directly from the samples [77].One important limitation of PCR-based methods, however, is low sensitivity, which can identify approximately 1% of the total number of species [78].On the other hand, the use of rRNA gene fingerprinting requires sequencing of the cloned bands to identify the community's members accurately.Sequencing is necessary because the amplicons from different species may migrate to the same positions, or one species may give multiples bands because of multiple gene copies with intra-gene differences [74,79].The use of molecular approaches to study yeast communities has been scarce and generally limited to the study of food matrices.Yeast communities have been studied using PCR-DGGE, and amplification of a portion of the 26S rRNA gene of yeast [80][81][82][83][84][85], while PCR-TTGE has been applied to establish phylogenetic relationships of species of the genus Saccharomyces [86].
Recently, high-throughput sequencing methods, such as pyrosequencing, have been shown to be fast and very efficient tools for identifying members of the complex populations.In general, two approaches can be taken: diversity studies based on the sequencing of ribosomal gene (rRNA gene) amplicons, and metagenomic studies where whole-community DNA is subjected to shotgun sequencing [87].While sequencing ribosomal amplicons is much cheaper because only one gene is being sequenced, the metagenomic approach sequences all of the DNA genes, thus revealing the functions of the microbial community [87].A useful innovation for these two approaches is to analyze multiple samples at the same time (multiplexing), which can be accomplished using barecoded pyrosequencing or by physically separating the samples in the sequencing plates.In the barecode technique, the sequences in each sample are tagged with a unique barecode using barcoded primers during PCR amplification.The result of these high-throughput sequencing methods is several thousand sequences per sample in just few days, which must necessarily be analyzed using bioinformatics tools.Although the costs associated with these new technologies are less than for the Sanger method (considering the cost for one sequence), high-throughput sequencing methods remain an expensive approach.To date, these methods have been applied to the study of the microbial diversity of several communities, especially from human and animal guts [88][89][90][91][92][93].Diversity analysis targeting the D1/D2 domain of the 26S rRNA gene or the internal transcribed regions (ITSs) allow yeast species to be distinguished [8], and have recently been applied to the study of some fungal communities [94,95], and to the identification of some clinical yeast isolates [96][97][98].These methods appear to be very suitable for studying the yeast biodiversity in the fish gut.This new knowledge, together with the information available in various databases, will allow both the accurate identification of new yeast isolates and the application of molecular strategies to characterize the yeast population in the fish gut.

Use of yeast as probiotics in aquaculture: stimulation of the immune and antioxidant systems, gut maturation and fish growth
The natural occurrence of numerous yeast species in the gastrointestinal tract of healthy fish has been well described, and yeast have been shown to constitute an important part of the microbiota of the fish gut [6].In addition to S. cerevisiae, the halotolerant yeast D. hansenii has been considered an excellent probiotic candidate in fish aquaculture.Because, the number of experiences reporting the use of D. hansenii is increasing, this allows us to know the capacity of this yeast to enhance growth, survival, and gut maturation and to improve of the immune and antioxidant systems in fish larvae and juveniles.Yeast cells can be a hundred times larger than bacterial cells, which may explain the fact that the introduction of a low yeast population (10 4 CFU/g) through feed can induce beneficial effects in the host.
The effects of probiotics differ greatly depending on the microbial species, isolation source, experimental concentration and finally, the rearing conditions of the fish.However, the improvement of the immune response is one of the most encountered side effects in the host because immune system stimulation or immunomodulation are considered important mechanisms supporting probiosis.Yeast have immunostimulatory properties because they possess components such as β-glucan, mannoproteins, chitin (as a minor component) and nucleic acids [99].
Recent studies have shown the beneficial effect of dietary administered Saccharomyces cerevisiae in fish.Yeast supplemented diets stimulate growth, feed efficiency, blood biochemistry, survival rate, and non-specific immune responses in Uronema marinum-infected olive flounder (Paralichthys olivaceus) [100].A diet supplemented with S. cerevisiae treated with beta-mercaptoethanol was better than whole cell yeast and n-3 highly unsaturated fatty acids (HUFA)enriched yeast as an immune system and growth stimulator in juvenile rainbow trout challenged with Yersinia ruckeri [101].Similarly, the dietary administration of the probiotic S. cerevisiae P13 at a minimum level of 10 5 CFU/kg enhanced the growth, innate immune responses and disease resistance of grouper (Epinephelus coioides) [102].Cellular yeast components also stimulate the immune system: an improvement in gut mucus lysozyme activity was observed in European sea bass (Dicentrarchus labrax) fed mannan oligosaccharides (MOS) derived from the outer cell wall of a select strain of S. cerevisiae (Bio-Mos, Alltech Inc, USA) [103].Furthermore, channel catfish (Ictalurus punctatus) juveniles fed diets supplemented with whole-cell S. cerevisiae (Levucell SB20®) or yeast subcomponents such as commercial preparations of β-glucan (MacroGard ® and Betagard A ®) had a significantly higher survival rate after Edwardsiella ictaluri challenge than did catfish fed with a controlled diet [104].
As previously described, most published studies have been performed using S. cerevisiae; however, promising results have also been obtained with Debaryomyces hansenii.A diet supplemented with D. hansenii stimulates the immune system of juvenile leopard grouper, Mycteroperca rosacea, by increasing IgM and superoxide dismutase (SOD) activity and enhances the resistance of the fish to infection by the dinoflagellate, Amyloodinium ocellatum [105].
Additionally, other studies have demonstrated the immune system improvement of M. rosacea when the fish were fed for 4 weeks with a compound diet enriched with 1.1% D. hansenii.After the 4 weeks, the fish were challenged with the pathogenic bacteria Aeromonas hydrophila strain Ah-315, resulting in an increase in IgM levels as well as catalase (CAT) and SOD activities in those fish fed yeast diets.Improvements were also observed at the molecular level, where CAT and HSP70 expression levels were enhanced in M. rosacea fed with D. hansenii, and challenged with A. hydrophila [106].
D. hansenii administration to the gilthead seabream (Sparus aurata L.) significantly enhances leukocyte peroxidase and respiratory burst activity by week 4 of feeding with yeast.Yeast feeding causes an up-regulation in the expression of the immune-associated genes Hep, IgM, TCR-ß, NCCRP-1, MHC-IIa, CSF-1R, C3, TNF-α and IL-1ß in the head-kidney: C3 expression was only stimulated in the liver, whereas the expression of TCR-ß, TNF-α and C3 was stimulated in the intestine of S. aurata [107].
When D. hansenii was administered at 1.1% in a compound diet to D. labrax larvae, the yeast stimulated the antioxidant status [108].The group fed with yeast showed lower glutathione peroxidase (GPX) and SOD activity compared to fish fed the control diet, suggesting a possible involvement of superoxide anion retention in fish larvae, which could represent importance to the host to increase cell or tissue responsiveness to growth-and/or differentiation-enhancing factors [109].The group fed the control diet showed oxidative stress represented by an increase in GPX activity at 48 days post hatching (dph) and gene expression levels for both GPX and SOD at 23 dph.
The ontogeny of the digestive tract of fish larvae has been the subject of many studies in the last decades with the purpose of increasing production rates by reducing the bottlenecks in larviculture.In this sense, the number of reports concerning the use of yeast to enhance gut maturation and digestive enzyme activity in fish are also increasing.The activity and expression of digestive enzyme-related genes during fish development provides an excellent marker of digestive development in fish larvae.The enzymes secreted from the pancreas (trypsin, lipase, and amylase) as well as those encountered in the intestinal brush border membranes (BBM) (leucine aminopeptidase N, alkaline phosphatase, maltase, γ-glutamyl-transpeptidase, and the cytosolic leucine-alanine peptidase), are the most common indicators of digestive system maturation in fish larvae.The degree of enterocyte maturation is described by increasing ratios of activities of BBM enzymes vs cytosolic leucine alanine peptidase; in the case of pancreatic enzymes, a decrease in amylase activity or expression level with the concomitant increase in trypsin or lipase activities characterizes the maturation of the exocrine pancreas.
Given this previous work, the effect of D. hansenii-enriched microparticulated diets were tested in European sea bass larvae.The authors observed an increase in intestinal brush border and pancreatic enzyme activities at 27 dph, indicating an achievement of gut morphology in this larvae stage compared with the control diet lacking yeast [110].In a second feeding trial, where the introduction of yeast to the microparticulated diet was improved, larvae fed 1% D. hansenii matured earlier than fish fed a control diet after day 26, as revealed by lower amylase expression, higher lipase and trypsin expression, and high levels of the BBM enzymes, aminopeptidase N, maltase and alkaline phosphatase [111].
In another study [42], two yeast strains: Saccharomyces cerevisiae and S. boulardii, were evaluated as probiotics for rainbow trout (Oncorhynchus mykiss) fry to compare the cross effects of the two rearing conditions, with the intestinal microbiota and the brush border enzyme activities.
Intestinal maturation at 10 dph was observed in trout fed S. boulardii, and kept in spring water, and these fish displayed the highest ratios of BBM leucine aminopeptidase N vs leucine-alanine peptidase, compared with those fish fed S. cerevisiae, and kept in river water.
Overall, yeast has been added directly to the water, administered as an additive in microparticulated diets, and has been used alive to feed live food (rotifers or Artemia) as a possible vector to deliver yeast into the gut of fish larvae.Rotifers have been established as the most common live prey to feed larval fish in hatcheries around the world, and baker's yeast (S. cerevisiae) is the most common nutrient source for culturing rotifers in addition to algae, emulsified oils or bacteria.Currently, efforts are being made to introduce D. hansenii into Brachionus rotundiformis to deliver yeast into the intestine of L. guttatus to accelerate digestive maturation [112].
In this regard, the use of Levucell® (S. boulardii), Bactocell® (Pediococcus acidilactici) and live yeast (S. cerevisiae) produced no significant effect on trypsin, lipase, and leucine aminopeptidase activities in California halibut larvae, Paralichtys californicus, at 46 dph.Contrary to this, an increase in pepsin and chymotrypsin activity was only observed in fish larvae fed Bactocell® at the final endpoint of the experiment (46 days), suggesting a potential use of these probiotics once metamorphosis is completed [113].
In fish aquaculture, the most utilized growth-promoting additives are hormones, antibiotics, ionophores, and salts [114].The use of probiotics as growth-promoters has been recognized in the last decade with a number of studies related to this topic being published.Probiotics can be used as an alternative to avoid the use of antibiotics for growth promotion, thus eliminating the possibility of generating antibiotic-resistant bacteria in the aquaculture systems.When yeast probiotics have been used in the earliest developmental stages of fish larvae, enhanced growth and survival have been observed.Several yeast species have been documented to enhance growth following artificial colonization, particularly S. cerevisiae and D. hansenii either alone, or in synergic association with bacteria.One study [115] observed that pollock (Pollachius pollachius) larvae grew better when Artemia nauplii was first treated with S. cerevisiae var.boulardii CNCM I-1079 and then with Pediococcus acidilactici MA185 M than did larvae fed with one or no probiotic.When S. cerevisiae was used alone in feeding trials, it improved feed efficiency in Israeli carp and Nile tilapia [116,117].In addition to growth enhancement, an improvement in the conformation of the larvae was also observed in D. labrax fed with D. hansenii; a reduction in spinal deformity from 13.6% to 1.1% in fish fed yeast vs the control group was observed [111].
The use of probiotics in well-established fish industry is easier of because the existence of many individuals to experiment upon (S. salar, D. labrax, S. aurata, S. maximus, O. mykiss, etc).Nevertheless, it is advisable to use probiotics in species with the potential for exploitation to optimize the results for growth, survival and health.In this context, several studies have been performed in fish species with emerging aquaculture potential to contribute to the establishment of a continuous production line for experimental purposes.A commercial preparation of live yeast (S. cerevisiae and Lactobacillus coagulans) was tested on Indian carp fry, Labeo rohita, with no conclusive effects on growth [118].D. hansenii has been observed to function as a growth promoter in Mycteroperca rosacea juveniles because after 4 weeks of continuous feeding, a weight gain (33%) and condition factor were observed in those fish fed a microparticulated diet enriched with yeast, compared with those fish fed without yeast [106].In spotted sand bass larvae, Paralabrax maculatofasciatus, the highest survival (13.0%) was obtained with D. hansenii enriched microparticulated diets, but no effects on growth were observed with the use of probiotics [119].
At present, much of the existing evidence indicates that yeast promotes growth, and survival because of gut maturation, conformation of the larvae, and stimulation of the immune system by a possible involvement of endoluminal yeast-secreted polyamines in the host.As was earlier demonstrated, D. hansenii produces more polyamines (putrescine, spermidine and spermine) than S. cerevisiae and S. boulardii [111].Polyamines participate in several physiological processes such as cell proliferation and differentiation and appear to have a broad influence on digestive tract maturation.In particular, the roles of dietary spermine and spermidine have been previously described [120].These molecules enter enterocytes, where they induce a hormonal cascade that affects organs such as the pancreas and liver.Recently, the production of polyamines in 13 strains of D. hansenii, isolated from different sources, using high-pressure liquid chromatography (HPLC) has been reported [121].In this study, they found that the L2, and CBS004 strains isolated from citrus fruit and marine water, respectively, were the main polyamine-secreting yeasts.Later, L2 strain was shown to have a probiotic effect because it enhanced the immune status, and intestinal function of gilthead seabream, Sparus aurata [122].
Finally, evidence of polyamine contribution to larviculture performance was reported when the spotted sand bass larvae, P. maculatofasciatus fed with D. hansenii with un-inhibited ornithine decarboxylase (ODC) activity had precocious digestive maturation compared to those larvae fed ODC-inhibited (with α-difluoromethylornithine (DFMO)) yeast [119].
Ornithine decarboxylase, which catalyzes the formation of putrescine, is the rate-limiting enzyme in the biosynthesis of polyamines in cells.

Yeast β-glucans: Structure, mechanisms of action and its application as immunostimulant in aquaculture
The glucose polymer β-glucan is a major structural component of the cell wall of some plants (such as the cereals oat and barley), seaweeds, and the outer cell wall of bacteria, fungi and yeast.Different β-glucans vary in structure, size, branching frequency, structural modifications, conformation and solubility, which may influence their physiological functions.Glucose molecules, in all β-glucan polymers, are linked together by a β-(1→3) linear β-glycosidic chain core, but differ in their length and branching structures.For example, the β-glucans from oat and barley are linear with β-(1→4) linkages, and shorter stretches of β-(1→3) linkages, while the structure of yeast β-glucans is composed of β-(1→3)-D-glucans with β-(1→6)-glycosidic linked branches, which apparently corresponds to the most active form of β-glucan.The relationship between structure and biological activity is controversial, but it appears that large molecular weight β-glucans are the most active compared with small β-glucans below 5,000-10,000 Da that are generally inactive.The solubility of β-glucans also influences their biological activity, with soluble β-glucans appearing to be more active.
The consumption of β-glucans has been associated with beneficial health effects in humans, including anticancer properties [123], metabolic syndrome prevention [124,125], cholesterollowering effects [126], anti-atherogenic properties [127] and skin health promotion [128].In vitro and in vivo studies in animals and humans show that the β-glucans derived from fungi and yeasts in particular, have interesting immune modulating properties [129][130][131][132].This immune stimulation can be achieved when β-glucans are administered by a parenteral or an oral (dietary) route.
Despite their structural versatility, β-glucans are highly conserved structural components and belong to a group of physiologically active compounds called biological response modifiers [133].Because of their large molecular weight, they cannot penetrate the cell membrane and therefore they must interact with cell-surface receptors; it has been shown that β-glucans are recognized by several receptors found on neutrophils, macrophages, and dendritic cells [129,134].Additionally, β-glucans belong to the group of non-self-molecules called pathogenassociated molecular patterns (PAMPS), which are recognized by pattern recognition receptors (PRRs) on the cell surface [135].The principal β-glucans PRRs are dectin-1 and the toll-like receptors (TLRs), but other receptors are suggested to be involved, such as scavenger receptors, complement receptor 3, and lactosylceramide [136,137].Dectin-1 specifically recognizes β-(1→3)(1→6) glucans from fungi, plants, and bacteria [138], but it is not reactive toward β-(1→4) glucans or α-mannan [139].The stimulation of dectin-1 activates the innate immune response, ROS and inflammatory cytokine production [140] through the activation of phospholipase C [141], the PI3K/Akt pathway, MAPK, NFAT, and NF-κB [142].The interaction of β-glucans with TLRs results in activation of NF-κB and MAPK signaling [143].Zymosan (β-glucans from the yeast Saccharomyces cerevisiae) binds to TLR2 and TLR4 (as well as dectin-1) found on macrophages, leading to an increase in cytokine production, such as TNF-α and IL-12, through the NF-κB pathway [143].The β-glucan (β-(1→3) (1→6)-D-glucan) from Aureobasidium pullulans ADK-34 stimulates intestinal Peyer's patch cells both in vitro and in vivo as reflected by an increase in IL-5, IL-6, and IgA [129].The interaction of β-glucans with specific receptors on macrophages and dendritic cells can induce the production of several cytokines, which in turn activate other immune cells such as B and T cells, thus activating the systemic immune response.
Yeast β-glucans have been applied in aquaculture to modulate the innate immune system of fish to improve their survival until adaptive immune responses are sufficiently developed to mount effective responses against pathogens [144,145].If the β-glucans are administered as feed additives, they can exert their primary effects at the intestinal level through the induction of cytokines, which in turn affect the systemic immune response in fish.Different sources of β-glucans have been evaluated, although the most frequent sources are those obtained from the baker's yeast, Saccharomyces cerevisiae.Some commercial preparations of β-glucan (Mac-roGard ®, Betagard A ®, EcoActiva TM, Nutriferm TM , BG, Fibosel®, etc) are available on the market that can be used in aquaculture.Many studies have explored the in vitro response of the macrophage to β-glucan [146,147], while other studies have addressed the in vivo effect of β-glucans in different fish species (Table 3).The β-glucans from several sources have been administered to fish via the oral or intra-peritoneal route with different effects.Many studies have shown the immune effects of β-glucans specifically on antibody production, expression of immune system genes, survival, resistance to infectious diseases, and improvement in stress resistance.The growth enhancement of fish has also been observed as another beneficial effect of β-glucans (Table 3).
Recently, juvenile channel catfish (Ictalurus punctatus) fed diets supplemented with wholecell Saccharomyces cerevisiae (Levucell SB20®) or yeast subcomponents such as commercial preparations of β-glucan (MacroGard ® and Betagard A ®) had a significantly higher survival rate after challenge with Edwardsiella ictaluri than did catfish fed with a control diet [104].Atlantic cod (Gadus morhua L.) were fed for 5 weeks with a purified β-glucan product [148], after which the fish were bath-challenged with the bacterial pathogen, Vibrio anguillarum.The transcription of selected cytokines (proinflammatory: IL1-β, IL-8, IFNγ; anti-inflammatory: IL-10) in different intestinal segments was analyzed using qPCR, and the β-glucan product was found to have a differential effect on the expression of the cytokine genes.In the anterior intestine and rectum, the β-glucan significantly elevated the expression of IL-1β when challenged with V. anguillarum.Moreover, an effect on the anti-inflammatory cytokine IL-10 was also visible in the rectum after the pathogen challenge.The differential responses of cytokines in the intestine of fish upon exposure to V. anguillarum suggests that β-glucans impact the ability of Atlantic cod to respond to the pathogen [148].
In another recently study, different concentrations of the yeast β-glucan preparation Macro-Gard ® (0.1%, 1% or 2%) were orally administered to mirror carp (Cyprinus carpio L.) for 8 weeks [149].Fish fed diets containing 1% and 2% MacroGard® showed significant improvements in weight gain, specific growth rate and feed conversion ratio compared to fish fed both the control and the 0.1% MacroGard® containing diet.At the end of the experiment, the haematocrit value was significantly elevated in fish fed the 2% MacroGard® diet, compared to the control fed fish, with the blood monocyte fraction significantly higher in fish fed the 1% and 2% MacroGard® diets [149].
Zebrafish (Danio rerio) have been suggested as a model aquacultured fish, especially for genetic [150], nutritional and comparative growth studies [151].Furthermore, zebrafish have been suggested as a model for pathogen studies in finfish [152].Yeast β-glucans have also been evaluated in the zebrafish (Danio rerio) model with promising results [153].In the study, a 5 mg/ml β-glucan preparation derived from S. cerevisiae was injected intra-peritoneally into adult zebrafish, leading to a significant reduction in mortality after challenge with Aeromonas hydrophila.In zebrafish treated with β-glucan, the ability of kidney cells to kill A. hydrophila was enhanced.Moreover, the myelomonocytic cell population in the kidney at 6 h postchallenge with A. hydrophila was increased.The β-glucan also appears to modulate the expression of IFN-γ and chemokines in the kidney [153].
Recently, the effect of β-glucan (derived from yeast, Fibosel® (Lallemand) on the growth performance and antioxidant enzyme activity in red snapper (Lutjanus peru), before and after exposure to lipopolysaccharides (LPS), was investigated.The fish were fed commercial diets with 0.0%, 0.1% and 0.2% Fibosel® for 6 weeks, after which, LPS was injected intra-peritoneally.The results showed a significant increase in growth performance after 6 weeks of β-glucan feeding; the SOD activity was also significantly higher in diets containing 0.1% β-glucan in weeks 4 and 6 with respect to the control group.At 72 h after injection of LPS, samples showed a significant increase in CAT activity in fish fed diets supplemented with 0.2% β-glucan and SOD activity increased under diets containing 0.1% and 0.2% β-glucan compared to controls [154].
To explain the enhanced growth, the authors suggested that some bacterial populations modify the host's digestive enzyme activity through their ability to produce and liberate exogenous digestive enzymes, as was previously observed [155].Other authors reported that polysaccharides used as prebiotics can stimulate the growth of beneficial microbiota in fish [156].

Conclusions
It is interesting to note that even after several decades of investigation, the potential of yeast, especially those of marine origin, has not yet been fully exploited.Yeasts can be part of the gut microbiota of wild and cultivated fish; however, more information derived using molecular approaches, is needed regarding the yeast composition in fish.Although much has been reported on the molecular aspects of yeasts, the exploration of the complete yeast community through the analysis of yeast DNA or RNA is lacking.The application of such methodologies will provide us with an overview of the non-cultivated yeasts, which could play a major role in the fish host.Different enzymes can be synthesized by yeast that have biotechnological potential, but the direct contribution of this potential to fish nutrition must be explored.In contrast, several publications confirm the beneficial probiotic effects of yeast in aquaculture, but the majority of these studies are focused on two species: S. cerevisiae and D. hansenii.The identification of new yeast species/strains from other cultured fish species is required to explore new beneficial properties to improve fish health and nutrition for a more sustainable aquaculture.

Table 1 .
Yeast identified in the marine and other aquatic environment

Table 2 .
of Yeasts as Probiotics in Fish Aquaculture http://dx.doi.org/10.5772/57196Yeast identified in the fish intestine

Table 3 .
Biological effect of different β-glucans in fish Use of Yeasts as Probiotics in Fish Aquaculture http://dx.doi.org/10.5772/57196