Use of immunostimulants and nucleotides in aquaculture : a review

Currently there are numerous gaps in existing knowledge about exogenous nucleotide application to fish including various aspects of digestion, absorption, metabolism, and influences on various physiological responses, especially expression of immunogenes and modulation of immunoglobulin production. Additional information is also needed in regard to age/size-related responses and appropriate doses and timing of administration. Thus further research in these areas should be pursued.

Currently there are numerous gaps in existing knowledge about exogenous nucleotide application to fish including various aspects of digestion, absorption, metabolism, and influences on various physiological responses, especially expression of immunogenes and modulation of immunoglobulin production.Additional information is also needed in regard to age/size-related responses and appropriate doses and timing of administration.Thus further research in these areas should be pursued.

Immunostimulants
On a worldwide scale, fisheries landings remain constant at about 90 million tons of fish whereas commercial aquaculture supplies per se is approximately 60 million tons and is increasing at a rate of about 8% per annum.Use of immunostimulants (natural immune stimulants) is a unique approach for fish culturists to control disease losses in their facilities and numerous polysaccharides from a variety of sources have the ability to stimulate the immune system, and thus behaving as immunostimulants.Immunostimulants have an architecture consisting of repeating units of single molecular forms such as glucose in β-glucans and (deoxy) riboses in DNA/RNA, fatty acid chains in bacterial lipopolysaccharides (LPS) and certain lipoproteins.Such patterns are abundant in microbial communities of prokaryotes, and can be termed pathogen-associated molecular patterns (PAMPs) if they initiate inflammatory responses.It was turned out that commential microbiota also contain those molecular patterns in them and now scientists tend to use a broader term, microbe-associated molecular pattern (MAMP).
Sakai indicated that immunostimulants could be grouped depending on their sources; bacterial, algae-derived, animal-derived, nutritional factors and hormones/cytokines [1].This sub-grouping changed the concept that immunostimulants instead are divided according to their modes of actions.Previously, an immunostimulant was defined based upon its activity on mononuclear phagocytic system only [2].However, due to recent discoveries of the pattern recognition receptors (PPRS) of the phagocytic cells, the former definition of

Nutritional factors and the immune response
It is well known that nutrition may affect fish health and immune responses.Nutritional factors are components of the diet essential for normal growth and development of the fish.Many of these undoubtedly have roles in the immune response.Vitamins C, B 6 , E and A, and the minerals iron and fluoride have been identified as micronutrients that can affect disease resistance.With respect to the strict definition of immunostimulants; vitamins and minerals are not immunostimulants as they enhance the immune system by providing substrates and serving cofactors necessary for the immune system to work properly, but controversy exists [1,23].

β-1,3/1,6-glucans
Glucans are high molecular-weight substances composed of glucose as building blocks, usually isolated from cell walls of bacteria, mushrooms, algae, cereal grains, yeast and fungi [38].Glucans in general comprise a great variety of substances common in nature (such as cellulose, glycogen, and starch), most of which do not interact with the immune system.Pharmacologically, they are classified as biological response modifiers (BRM).The common feature of immunomodulatory glucans is a chain of glucose residues linked by β-1.3-linkages, also called beta-glucans.Of the different β-glucans, the products known as β-1.3/1.6-glucans(read as "beta-one-three-one-six-glucans"), derived from baker's yeast, are suggested to be the most potent immune-system enhancers.β-1.3/1.6-glucans is characterized by side-chains attached to the backbone that radiate outward like branches on a tree.The primary structure of the β-1,3/1,6-glucan is determinant for its immune-enhancing ability.The frequency and nature of side-chains strongly affect the ability of the glucan to mediate binding to surface receptors on the target cells influencing the effectiveness of the glucan as an immunostimulant.
The effectiveness of MacroGard ® in stimulating the immune system combined with improved growth and feed conversion has been proven in some experiments [39,40].MacroGard ® has been suggested to be used in feed for all aquatic organisms and also to enrich live feed such as Artemia.Some studies involving shrimp have also suggested that the product contributes to a better growth in crustaceans [40,41].
MacroGard ® is an environmentally sound alternative to feed antibiotics and chemotherapeutics for livestock, pets and cultured aquatic organisms.MacroGard ® is based on a well-characterized β-1,3/1,6-glucan that has been in use world-wide for almost 25 years as an immune modulating agent in animal husbandry and aquaculture (see Table 1).
When included in feed or administered on mucosal surfaces, MacroGard ® modulates immune responses and affects biological functions in a favourable way.The products are extracted and purified from food-grade baker's yeast by patented processes.The use of MacroGard ® may be regarded as a natural input that compensates for a possible sub-optimal function of the immune system of farm animals, companion animals and aquatic species in intensive culture.The product has also been suggested to reduce the use of antibiotics in animal husbandry and consequently the concern that developing antibiotics resistance that may undermine the health security effective antibiotics represents.In aquaculture, glucans have been successfully been used to enhance the resistance of fish and crustaceans against bacterial and viral infections and (Table 1) present an overview of papers using glucans as immunostimulants in aquaculture.
In Norway, β-glucan, as prebiotics and nucleotides, has been used in the pancreas disease (PD) diets; to fed fish suffering from PD (Gonzalez Vecino, personal communication).In this case, the β-glucans are a very small part of the diet composition, and the benefits of the diets come mainly from a very specific formulation concentrated to recover pancreas and reduce inflammation in muscle and heart.

Bioactive alginate (high-M alginate) and Ergosan
The adaptive immune system is poorly developed in early developmental stages of fish [43,44], which is why immunostimulants and probiotics have been used in an attempt to increase survival of larvae against microbial pathogens [45][46][47].In this respect, alginate has been proposed as a potential candidate.Alginate is a polysaccharide composed of β-1,4-D-mannuronic acid (M) and C5-epimer α-Lglucuronic acid (G) [48].The monomers are usually arranged in M-blocks, G-blocks and alternating MG-blocks [49].Alginate also binds various cations found in the seawater such as Mg 2+ , Sr 2+ , Ba 2+ , and Na + .Commercial alginates are extracted from three species of brown algae; Laminaria hyperborean, Ascophyllum nodosum and Macrocystis pyrifera, in which alginate comprises up to 40% of the dry weight [50].Furthermore, bacterial alginates, not commercial products, have also been isolated from Azotobacter vinelandii and several Pseudomonas species [51].
Commercially available alginates today have M-content ranging between 30 and 70%.Alginates with even higher M-content, typically higher than 80%, have also been shown to be potent stimulators of immune cells such as human monocytes [52].High-M alginate has also been used as an immunostimulant for enhancement of innate immune resistance in fish larvae and fry [53][54][55][56].
Ergosan is an algal based product and contains 1 % alginic acid extracted from Laminaria digitata.The first study on Ergosan in aquaculture, to the author's knowledge, was reported by Miles and coauthors on striped snakehead (Channa striata) [57].In a study with rainbow trout, Peddie et al. reported that a single injection of 1 mg of Ergosan significantly augmented the proportion of neutrophils in the peritoneal wall, increased the degree of phagocytosis, respiratory burst activity and expression of interleukin-1β (IL-1β), interleukin-8 (IL-8) and one of the two known isoforms of tumour necrosis factor-alpha (TNF-α) in peritoneal leucocytes one day post-injection [58].However, humoral immune parameters were less responsive to intraperitoneal alginate administration with complement stimulation only evident in the 1 mg-treated group at 2 days post-injection.(Table 2) presents an overview of reports using alginates and Ergosan in fish studies.

Plant extracts
Some immunostimulants cannot be used because of various disadvantages, such as high cost and limited effectiveness upon parenteral administration.Numerous plants have on the other hand long been used in traditional medicine for the treatment and control of several diseases [59].As herbs have little side effects and are easily degradable and abundantly available in farm areas, numerous investigations have investigated the effect of plant products on innate and adaptive immune response and to prevent and control fish and shellfish diseases [60].
Dügenci and co-authors investigated the effects of mistletoe, nettle and ginger on dietary intake of rainbow trout [61].The diets contained lyophilized extracts of these plants at two inclusions levels, 0.1 and 1%, in a 3 week experiment.At the end of the experimental period, various parameters of innate defence mechanisms, including extracellular and intracellular respiratory burst activities, phagocytosis in blood leukocytes, total plasma protein level, specific growth rates and condition factors were examined.Inclusion of the plant materials increased the extracellular respiratory burst activity (P<0.001) compared to control.Furthermore, fish fed the diet containing 1% ginger roots exhibited a significant innate immune response.Phagocytosis and extracellular burst activities of blood leukocytes were significantly higher in this group compared to the control group.All plant extracts increased plasma protein level except for the 0.1% ginger supplemented diet.The highest level of plasma proteins was observed in the group fed with 1% ginger extract.
Oral administration of the medical plant, Eclipta alba, on the non-specific immune responses and disease resistance of tilapia (Oreochromis mossambicus) has been investigated [62].The results indicated that E. alba administrated in the diet significantly enhanced the non-specific immune parameters tested.Furthermore, when tilapia was challenged with Aeromonas hydrophila mortality was significantly reduced in E. alba treated fish.
Lectins are proteins or glycoprotein substances, usually of plant origin, and they are sugar-binding proteins which are highly specific for their sugar moieties.Lectins are also known to play important roles in the immune system by recognizing carbohydrates that are found exclusively on pathogenic bacteria, or that are inaccessible to host cells [63].Examples are the lectin complement activation pathway, and mannose binding lectin (MBL), also named mannose-or mannanbinding protein (MBP).MBL recognizes carbohydrate patterns, found on the surface of a large number of pathogenic micro-organisms, including bacteria, viruses, protozoa and fungi.Readers with special interest in lectins and immune response are referred to the recent comprehensive reviews [64][65].
In a recent paper, Galina and co-authors reviewed studies currently being carried out on herbs and herbal extracts that have been shown to modulate the immune system in fish and special attention was given to; Astragalus, Ganoderma, Lonicera and Chinese and Indian herbs [66].Based on the results available per se Galina and co-authors suggested that herb extracts might have a potential application as an immunostimulant is fish, due to that they can easily be obtained, are as not expensive and act against a broad spectrum of pathogenic bacteria, and could be alternatives to vaccines, antibiotics or chemotherapeutic agents.In a more recent study, Soosean and co-authors reported that mangosteen (Garcinia mangostana) extracts as feed additive to African catfish (Clarias gariepinus) had no significant effect on growth parameters, feed conversion ratio and haemoglobin content.However, significantly higher red blood cells and white blood cell counts were recorded in fish fed the shoot extract [67].In their study on kelp grouper (Epinephelus bruneus), Harikrishnan and co-authors evaluated the efficacy of dietary doses of Lactuca indica extracts on immunological parameters and disease resistance against Streptococcus iniae infection [68].Based on their results, the authors suggested that supplementation of L. indica enhanced the immune system and the disease resistance against pathogenic infection.Enhanced immune response of tiger shrimp (Penaeus monodon) by the medical herb black nightshade (Solanum nigrum) and disease resistance against Vibrio harveyi were investigated by Harikrishnan and co-authors [69].In this study the authors displayed improved immune response including respiratory bursts, PO-, SOD -and GPx activities and disease resistance by feeding tiger shrimp black nightshade at 0.1 and 1 % doses.This herb has also been shown to have inhibitory in vitro growth effect on A. hydrophila and improved survival and increased haematological parameters of spotted snakehead (Channa punctatus) infected with A. hydrophila [70].

Mechanism of action/results References
High Readers with special interest in the topic plant products on innate and adaptive immune response of fish and shellfish are referred to the recent review of Harikrishnan et al. [60].

Phagocytic activity of kidney leucocytes
Phagocytic activity is the principle function of kidney leucocytes of teleosts.Monocytes, macrophages, dendritic cells, and neutrophils belong to the phagocytic leucocytes called phagocytes.In host defense, phagocytes respond to microbes in a sequential manner: active recruitment of the cells to the sites of infection through inflammatory responses, ingestion of microbes by the process of phagocytosis, and destruction of the ingested microbes.Phagocytosis is an active, energyrequiring engulfment process of large particles (>0.5 µm in diameter).The first step in phagocytosis is the recognition of microbe by the specific receptors expressed on the phagocyte such as TLRs (toll-like receptors).The bound microbes are ingested into vesicles forming phagosomes inside of the cells and the phagosomes are then fused with lysosomes that contain many different proteases.The internalized microbes will be killed there through proteolytic processes [71].

Macrophage activation
Macrophage activation is initiated upon binding of the microbe to the cell.The recognition of microbes by phagocytes such as macrophages, neutrophils, and dendritic cells is selective and mediated by receptors on phagocytic cells.Upon microbe binding, the receptors cooperatively activate the cells through a receptor-mediated signal transduction to kill ingested microbes.The receptors responsible for microbe recognition include TLR (toll-like receptors), G protein-coupled receptors, antibody Fc and complement C3 receptors, and receptors for cytokines, mainly IFN-γ.Activated macrophages kill phagocytosed microbes by the action of microbicidal molecules in phagolysosomes of phagocytes.In addition, activated macrophages secrete cytokines such as TNF, IL-1, and IL-12, which cause inflammation and bridge to activate the adaptive immune system.

Respiratory burst activity
Respiratory burst (sometimes called oxidative burst) is the rapid release process of reactive oxygen species (ROS) such as superoxide anion, hydroxyl radical and hydrogen peroxide from activated macrophages and neutrophils.The respiratory burst, releasing important bactericidal ROS molecules, plays a crucial role to remove microbes from the body.The respiratory burst is mediated by the enzyme phagocyte oxidase, which converts molecular oxygen to ROS and is usually triggered by bacterial products or by inflammatory mediators.The respiratory burst activity is an indicator of the status of macrophage and neutrophil activation [71].

Acid phosphatase
Acid phosphatase is a ubiquitous enzyme that removes phosphate groups from other molecules.The acid phosphatase is one of the lysosomal hydrolases which exist in phagolysosomes of macrophages (lysosomal acid phosphatases).When the macrophage is activated, the inside of phagosome becomes acidic.In an acidic environment, lysosomal hydrolases including acid phosphatase are activated to kill microbes.Acid phosphatase activity is an indicator of the microbicidal activity of macrophages and a high concentration of acid phosphatase in the blood is a sign of a chronic infection [71,72].Recently, acid phosphatase activity tests are used for the diagnosis of prostate cancer and breast cancer in human.

Serum complement activity
The complement system is one of the major effector mechanisms of the humoral immunity as well as the adaptive immunity, which involves the clearing of pathogens from an organism.The complement system consists of serum and cell surface proteins that are activated by microbes (the alternative pathway) and antibodies (the classical pathway).Activation of the complement system involves the sequential proteolysis of proteins to generate new enzyme complexes with proteolytic activity, which is a characteristic of a proteolytic enzyme cascade.The products of the complement activation become covalently attached to microbes, antibodies bound to microbes, and to other antigens.The binding of complement products to the target molecules cause lysis of the microbe, facilitation of opsonization (the process of attaching IgG or complement fragments to microbial surfaces for phagocytosis) and phagocytosis, and inflammation [71].

Phenoloxidase activity
Phenoloxidases are known to be involved in the innate immune response of invertebrates such as shrimps and crabs.Phenoloxidases are composed of tyrosinases, catecholases and laccases which are the terminal components of the prophenoloxidase (proPO) system, the modified complement system of several phyla of invertebrates [73].The proPO system is present in the blood of a wide range of marine invertebrates, especially crustaceans.Phenoloxidases play a role in bactericidal activity and the enhancement of phagocytosis in crustaceans [74][75][76].

Serum antibody concentration
B lymphocytes recognize antigens such as microbes using their receptors (membrane-bound antibody molecules).The engagement of antigen receptors and other signals trigger the lymphocytes to expand their numbers and to differentiate into antibody-secreting mast cells, which secrete different classes of antibodies with distinct functions.The functionally different antibodies have the same antigen specificity, through which the antibodies recognize the same microbes despite their different classes.Antibodies bind to microbes and prevent them from infecting cells (neutralization).The binding of antibodies to microbes also promotes opsonization and phagocytosis of microbes to kill and mediate antibody-dependent cellular cytotoxicity (ADCC).In addition, antibodies trigger activation of the complement system (the classical pathway), which in turn causes to increase phagocytosis of microbes opsonized with complement fragments and inflammation.Serum antibody concentration is an indicator of B lymphocyte activation against specific microbes [71].

Leukocyte number
Leukocytes (white blood cells) are immune cells involved in defending the body against both infectious microbes and foreign materials.Leucocytes consist of neutrophil, eosinophil, basophil, monocyte, and lymphocyte.They are all derived from a hematopoietic stem cell in the bone marrow.Neutrophils and monocytes are involved in the innate immune response as the first line of the defense system.Effector cells of the innate immune system circulate in the blood and migrate into tissues and kill microbes through phagocytosis.The innate immune cells also play a role in the inflammatory response by releasing cytokines.Lymphocytes including T cells and B cells are the only cells in the body capable of specifically recognizing and distinguishing different antigenic determinants (epitopes).When these adaptive immune effector cells recognize the microbes, the number of lymphocytes is significantly increased as a part of their effector functions.Although the number of the innate immune cells are also expanded upon activation, the level of increase is much lower than that of the adaptive immune cells.The number of leukocytes in the blood is often an indicator of the activation of the immune system or of leukemia [71].

Lysozyme activity
Lysozyme is a cationic enzyme that attacks the β-1,4 glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in the peptidoglycan of bacterial cell walls.This activity causes the lysozyme to lyse certain gram-positive bacteria and even some gram-negative bacteria.Lysozyme activity has been detected in mucus, serum, organs and eggs of several fish species [77].Lysozyme is produced mainly by macrophages and its production is induced in response to microbial components such as microbial lipopolysaccharide (LPS) [78] and many other immunostimulants.

Do immunostimulants raise the level of protection against disease?
A large number of reviews have been published during the last 20 years concerning the advantages of immunostimulants in fish and the immune system [1,3,23,28,30,32,[79][80][81][82][83][84][85][86][87][88][89][90].In this section an attempt has been made to compile the recent developments in the field, mainly over the last five years, besides the earlier work done in the field of disease resistance in fish with exposure to immunostimulants.
Several types of β-glucan have been successfully used to enhance resistance of fish and crustacean against bacterial and viral infections.An overview is presented in (Table 3).Most pathogens used in these challenge experiments are bacteria reported in commercial aquaculture and include; A. hydrophila, Aeromonas salmonicida, Aphanomyces invadans, Edwardsiella ictaluri, Edwardsiella tarda, Photobacterium damselae ssp.piscicida, Vibrio (Listonella) anguillarum, Vibrio harvei, Yersinia ruckeri, Lactococcus garvieae, S.iniae and Streptococcus sp. as well as infectious hematopoietic necrosis virus (IHNV), white spot syndrome virus (WSSV) and parasites.Readers with special interest in the topic immune response and aquatic viruses are referred to the recent reviews [91,92].
Recognition of invasive pathogens is an essential step for the activation of the immune system.This is accomplished by binding of PRRs to PAMPs.The PRRs and PAMPs activate appropriate immune responses [93].β-glucan binding proteins (β-GBP) are known as one of the most important PRRs in invertebrates [94] and fish [95].The presence of these proteins in invertebrates and fish may indicate that the defence systems against β-glucan containing microorganisms are crucial for these organisms.During week 8-9 post exposure a significant reduction in no. of xenoma-positive fish was noticed.

i.p -intraperitoneal injection n.i -no information given IHNV -infectious hematopoietic necrosis virus IHNV -infectious hematopoietic necrosis virus WSSV -White spot syndrome virus
Table 3: Immunostimulants and disease susceptibility in fish and shrimps.
Immunostimulants seem to be valuable for the control of fish diseases.However, knowledge regarding the ability of immunostimulants to modulate the gut microbiota in a healthier way and decrease the infection pressure by improving the gut function is scarce.To our knowledge only two studies, Gildberg and Mikkelsen [46] and Skjermo et al. [96] have been done on the topic; immunostimulants and modulation of the gut microbiota.In their study on Atlantic cod (Gadus morhua) fry, Gildberg and Mikkelsen evaluated the effect of supplementation of LAB either alone or in combination with immunostimulating peptides [46].After three weeks of feeding, the fish were challenged by bath exposure to V. anguillarum (10 7 ml - 1 ; 1hour).Twelve days after infection significantly (p<0.05)reduced cumulative mortality was recorded in fish fed diet supplemented with LAB, originally isolated from Atlantic salmon (Salmo salar) and with immunostimulating peptides.No synergistic or cumulative effects were achieved by combining LAB and immunostimulating peptides.
Four weeks after infection similar cumulative mortality (80-84%) was observed in all groups.LAB seems to colonize the internal mucus layer of the cod fry pyloric caeca, and a significant number of the bacteria survived the passage of the whole gastrointestinal tract.
In a study with Atlantic cod larvae, Skjermo and co-authors [96] evaluated the effect of β-(1→3, 1→6)-glucans (chrysolaminaran) from the marine diatom Chaetoceros mülleri, a commercial yeast-glucan product and high-M alginate (high content of mannuronic acid isolated from Durvillaea antarctica), on the microbial community in larval gut and water.Total colony forming units (CFU) were evaluated on marine agar, and Vibrio -and Pseudomonas -like species on selective agars (TCBS and marine Pseudomonas agar with CFC -supplement).The larvae were rapidly colonised after hatching, but no or weak effects of the stimulants were observed on the bacterial colonisation rates or the bacterial community.The total CFU varied from 10 1 to 10 2 CFU per μg larva after initiation of the first feeding.Bacteria belonging to Pseudomonas appeared to increase throughout the period, whereas the level of Vibrio-like bacteria was low and stable.However, in this investigation the authors only focused on characterisation of Pseudomonas-and Vibrio-like bacteria and did not use molecular methods to evaluate the entire bacterial community.
Hoseinifar and co-authors evaluated the effect of brewer's yeast (S.cerevisiae var.ellipsoideus) on gut microbiota of juvenile beluga (Huso huso) and displayed that juveniles fed 2 % S. cerevisiae var.ellipsoideus increased the levels of autochthonous LAB [97].The authors suggested that this interesting finding might occur as a result of the provision of enzymes, RNA and free nucleotides, B-complex vitamins and/ or amino acids by the dietary yeast.Whether supplementation of S. cerevisiae var.ellipsoideus improved disease resistance against pathogens was not investigated and merits further evaluation.
Based on the fact that no information is available about the effect of immunostimulants on the "good" gut microbiota with antagonistic activity against fish pathogenic bacteria, this should be a topic of further research as the gastrointestinal tract in fish is a potential port of entry for pathogenic bacteria [98][99][100][101][102][103][104][105][106].The importance of the topic is illustrated by a study showing that intraperitoneal injection of immunostimulatory substances affects the allochthonous (transit) gut microbiota including LAB of Atlantic salmon [107].

Nucleotides
Dietary nucleotides have attracted attention as key ingredients missing from nutritional formulae for many years.They are the building blocks of tissue RNA and DNA and of ATP, and their presence in breast milk has stimulated research in babies which has indicated that supplementation of infant formula milk leads to improved growth and reduced susceptibility to infection [108][109][110].Nucleotide fortification of breast milk substitutes has been recommended to the U.S. Food and Drug Administration for approval [111].There is increasing evidence that nucleotides administered intravenously or in the diet are capable of modifying immune responsiveness and recovery of organs that have undergone a metabolic or inflammatory insult.
It is generally accepted that nucleotides have essential physiological and biochemical functions including encoding and deciphering genetic information, mediating energy metabolism and cell signalling as well as serving as components of coenzymes, allosteric effectors and cellular agonists [112,113].However, controversy has existed for many years over the roles of nucleotides administered exogenously.As neither overriding biochemical malfunctions nor classical signs of deficiency are developed in endothermic animal models, nucleotides have traditionally been considered to be non-essential nutrients.However, this opinion has been challenged by several research publications during the last decade which suggest that dietary nucleotide deficiency may impair liver, heart, intestine and immune functions [114].The modulatory effects of dietary nucleotides on lymphocyte maturation, activation and proliferation, macrophage phagocytosis, immunoglobulin responses, gut microbiota as well as genetic expression of certain cytokines have been reported in endothermic animals [115,116].Nucleotide supplementation has been one important aspect of research on clinical nutrition and functional food development for humans [109,110].
Although initial efforts in evaluation of dietary supplementation of nucleotides for fishes could be traced to the early 1970s, research at that time mainly focused on the possible chemo-attractive effects of these compounds [117][118][119].However, the pioneer investigations by Burrels and co-authors resulted in increased attention on nucleotide supplementation for fishes as their studies indicated that dietary supplementation of nucleotides enhanced resistance of salmonids to viral, bacterial and parasitic infections as well as improved efficacy of vaccination and osmoregulation capacity [5,6].To date, research related to nucleotide nutrition in fishes has to some extent showed consistent and encouraging beneficial results in fish health management (Table 4), although most of the suggested explanations put forward by the authors remain hypothetical.Systematic research on fishes is therefore needed.
Because increasing concerns of antibiotic use have resulted in a ban on subtherapeutic antibiotic usage in Europe and the potential for a ban in the US and other countries [120], research on immunonutrition for aquatic animals is becoming increasingly important [121].Research on nucleotide nutrition in fish and shrimp is needed to provide insights concerning interactions between nutrition and physiological responses as well as provide practical solutions to reduce basic risks from infectious diseases for the aquaculture industry.Devresse hypothesized that nucleotides are a key nutrient for the shrimp immune system and supplementation of nucleotides or other nucleic acid-rich ingredients such as yeast or yeast extract may enhance disease resistance and growth of shrimp [122].Although yeast products have been used in shrimp diet formulations, the role of yeast nucleotides remains largely unanswered.In their comprehensive review, Li and Gatlin summarized and evaluated knowledge of nucleotide nutrition in fishes as compared with that of terrestrial animals [123].
The roles of nucleotides and metabolites in fish diets have been sparingly studied for nearly 20 years.Beside possible involvement in diet palatability, fish feeding behaviour and biosynthesis of nonessential amino acids, exogenous nucleotides have shown promise most recently as dietary supplements to enhance immunity and disease resistance of fish produced in aquaculture.Research on dietary nucleotides in fishes has shown they may improve growth in early stages of development, enhance larval quality via broodstock fortification, alter intestinal structure, increase stress tolerance as well as modulate innate and adaptive immune responses.Fishes fed nucleotidesupplemented diets generally have shown enhanced resistance to viral, bacterial and parasitic infection.Despite occasional inconsistency in physiological responses, dietary supplementation of nucleotides has shown rather consistent beneficial influences on various fish species.Although nucleotide nutrition research in fishes is in its infancy and many fundamental questions remain unanswered, observations thus far support the contention that nucleotides are conditionally or semi-essential nutrients for fishes, and further exploration of dietary  supplementation of nucleotides for application in fish culture is warranted.Hypothesized reasons associated with these beneficial effects include dietary provision of physiologically required levels of nucleotides due to limited synthetic capacity of certain tissues (e.g.lymphoid), inadequate energetic expenditure for de novo synthesis, immunoendocrine interactions and modulation of gene expression patterns.
The numbers of scientific publications dealing with dietary nucleotides in fish has been relatively limited since the comprehensive review of Li and Gatlin [123] was published.The section below summarises the dietary effects of nucleotides on a number of fish species such as red drum (Sciaenops ocellatus), red-tail black shark (Epalzeorhynchos bicolor), Asian carp (Catla catla), barramundi (Lates calcarifer), cobia (Rachycentron canadum), grouper (Epinephelus malabaricus) and salmonids such as rainbow trout and Atlantic salmon published post 2006.
The use of dietary nucleotides in red drum Sciaenops ocellatus has also been evaluated on growth and feed utilisation [124].Juvenile fish (ca.122g) were fed purified a commercial nucleotide product 0.2% (Optimûn, Chemoforma, August, Switzerland) or nucleotides mixtures, which contained equal amounts of CMP (cytidine monophosphate), UMP (uridine monophosphate), AMP (adenosine monophosphate), IMP (inosine monophosphate) and GMP (guanosine monophosphate), at 0.03%, 0.1% and 0.3% of the diet for a period of four weeks [125].Fish fed dietary nucleotides had showed a clear trend towards increased growth, measured as SGR at the end of the trial.Growth improvements ranged from 5-6% up to 8% higher than the control diet for the fish fed the nucleotide mixtures and commercial product respectively.A similar trend towards improved feed efficiencies was observed in all the groups fed diets containing added nucleotides.No diet effect on body composition was observed in contrast to previous observations.A clear trend of diet effect on neutrophil oxidative radical production was also observed that could suggest that excessive dose could inhibit immune responses.Fish were challenged against V.harveyi at the end of the four-week feeding period but no differences were observed in terms of survival when compared to the control diet.The authors reported on unexpected high mortalities within the 3 days postchallenge in contrast to previous experiments so it was concluded that further studies are needed in order to evaluate potential effect of dietary nucleotides on red drum resistance against V. harveyi [126].
Red drum was also the fish species selected by Cheng and co-authors to assess the effects of dietary nucleotides on intestinal morphology and immune responses [127].The authors used around 7g fish to compare a commercial nucleotide product, Ascogen P ® (Canadian Byosystems Inc., Calgary, Alberta, Canada), at 0.5% and 1% inclusion levels against a control diet not enriched with nucleotides.Evaluation of immune response after six weeks of feeding showed increased extracellular superoxide anion production at the highest dose.In general intestinal morphology, measured as fold height, enterocytes height and microvillus height, in the pyloric caeca, proximal, mid-and distal intestine, was positively influenced by dietary nucleotides.Thus, fold height in the proximal intestine was increased by nucleotides as previously reported for Atlantic salmon [5,6].Enterocyte height was increased in the pyloric caeca, proximal and distal intestine, not in the mid intestine, by nucleotides.The microvillus height was also increased in the pyloric caeca, proximal, mid and distal intestine.Russo et al. examined the effects of commercial beta-glucans (Macroguard ® , Biotec Pharmacon ASA, Tromsø, Norway) and nucleotide product (Aquagen TM , NOVARTIS-Aqua Health Ltd., Charlottetown, Canada) on disease resistance of red-tail black shark (Epalzeorhynchos bicolor) against S.iniae infection [128].Two trials were conducted using 1.4g fish feeding control, beta-glucan (0.1% inclusion) or nucleotide diet (0.2% product inclusion) for 24 days.In the first trial both vaccinated and non-vaccinated fish were used while in the second experiment nucleotide-or beta-glucan-enriched diets were fed only to vaccinated fish and results compared against a nonvaccinated fish fed control diet.Results of the first trial showed that both commercial products were able to reduce mortalities compared to the fish fed control diet in both vaccinated and non-vaccinated fish.In the second experiment vaccinated fish fed beta-glucans and nucleotide suffered lower mortalities than the vaccinated fish fed control diet and despite slightly lower mortality in the beta-glucan group compared to nucleotide group no statistical difference could be claimed.Growth performance was influenced significantly by dietary treatment in any of the trials conducted.
Jha et al. tested yeast nucleotides, in the form of RNA (Sisco Research Laboratories, India), at 0.4% and 0.8% inclusion levels on Catla catla fingerlings (ca.8g start weight) to assess potential changes in haemato-immunological responses followed by a challenge trial against A. hydrophila after 60 days feeding [129].Feeding dietary nucleotides increased leucocyte counts, total protein, globulins, and albumin: globulin ratio, lysozyme activity and respiratory burst activity compared to controls.When comparing differences between nucleotide doses only the respiratory burst activity was increased by augmenting the dietary nucleotides from 0.4% to 0.8% dose.Conversely, the highest survival was observed in the fish fed the highest dose of nucleotides.Fish fed 0.4% nucleotides also showed significantly higher survival than control fish.At 30˚C barramundi fed diets supplemented with nucleotides (Optimûn, Chemoforma, August, Switzerland) grew significantly better and had lower FCR than those fed the reference diet with no additional nucleotides added [130].In the same study it is noted that the supplementation of dietary nucleotide at 30˚C significantly improved the retention efficiencies of protein, energy, lysine and histidine compared to the reference diet.However at 37˚C , under heat-stress conditions, despite dietary nucleotides reduced FCR an improvement in growth was not observed.Salze et al. supplemented cobia diets with a nucleotide-rich yeast extract (Nupro, Alltech, Nicholasville, KY, USA) part of a zero-fishmeal feed [131].The 104g fish fed the zero-fish meal diet, which was based on soy protein concentrate, worm meal and mannan-oligosaccharides, had similar growth as a control diet with 25.3% fish meal.However the experimental design tested was insufficient to separate the effects of the different raw materials and to evaluate whether cobia benefited from nucleotide supplementation.Lin et al. reported the benefits of supplementing dietary nucleotides on growth and immune responses of grouper (Epinephelus malabaricus) juveniles [132].The first experiment assessed different inclusion levels of a nucleotide mixture containing equal amounts of IMP, AMP, GMP, UMP and CMP.Weight gain and feed efficiency were the highest in the group of grouper fed 0.15% of the nucleotide mixture; with head kidney leucocyte superoxide anion production ratio and plasma total immunoglobulin concentration also being higher than the fish fed the control diet.In the second experiment a diet containing 0.15% nucleotide mixture was compared against a control diet and also diets containing 0.15% of IMP, AMP, GMP, UMP or CMP.From the second experiment it was concluded that juvenile grouper diets containing 0.15% AMP seemed to have better effects than other diets supplemented with different single-nucleotides.Overall conclusion showed that both growth and immune responses of juvenile grouper were enhanced in grouper diet with 0.15% nucleotide mixture.
Due to its economical impact as a high-value species, research on salmonids species and nucleotides has received lots of attention in the last years.Tahmasebi-Kohyani and co-authors conducted a dose response trial on rainbow trout fingerlings (ca.23g start weight) in order to evaluate their effects on growth, humoral immune responses and resistance against S.iniae [133].A basal diet was supplemented with Optimûn (Chemoforma, August, Switzerland) at 0.05%, 0.1%, 0.15% and 0.2% inclusion levels and fish were fed for eight weeks.Results showed that 0.05% dose was too low and thereafter increased growth, FCR, alternative complement activity, lysozyme activity and IgM as nucleotide dose was augmented, reaching highest values for each of them at the highest nucleotide dose.Similar results were observed at the end of the challenge trial and increased survival was observed with increasing dietary level of nucleotides from 0.05% to 0.2%.This is in agreement with previous work on rainbow trout by Adamek and co-authors that used a wider range of concentrations of the same commercial product, from 0% up to 0.5% and reported approximately 0.2% as the optimal dose (Figure 1) [134].
The efficacy of dietary nucleotides (Optimûn, Chemoforma, August, Switzerland) increasing survival (+39%) against Piscirickettsia salmonis was first reported by Burrells and co-authors on 100g coho salmon (Oncorhynchus kisutch) after feeding for three weeks before challenge [5].The efficacy of dietary nucleotides against P. salmonis has also been confirmed in other salmonid species such as Atlantic salmon.González Vecino et al. reported increased protection against P. salmonis when Atlantic salmon were fed diets containing a combination of dietary nucleotides with a bacterial cell-wall extract (Sanictum ® , Chemoforma, Augst, Switzerland) [135].Thus, a synergistic effect of the combination of nucleotides and Sanictum ® was observed since survival was significantly better than feeding nucleotides or bacterial cell-wall extract separately.

Dietary nucleotides in broodstock diets
Fish reproduction, and egg and larval quality are affected by broodstock nutrition.Nucleotides, the building blocks of nucleic acids, are now considered as semiessential nutrients during periods of food deficiency, stress, rapid growth and immunological stress.González Vecino reported that since oogenesis is a process of intensive cell division with high nucleic acid formation and a concomitant high requirement for nucleotides, supplementation of broodstock diets with nucleotides was beneficial for fish [136].The results showed that Atlantic halibut (Hippoglossus hippoglossus L.) and haddock (Melanogrammus aeglefinus L.), two cold water species with different life histories, had improved fecundity, egg quality, larval quality and survival when broodstock diets were supplemented with nucleotides.In addition it was observed that haddock larvae from broodfish fed nucleotides had a significantly better developed gut and first feeding success that those from broodstock fed diets not supplemented with nucleotides.Similar beneficial effects have been observed in salmon broodstock diets supplemented with nucleotides (González Vecino, unpublished data).

Dietary nucleotides against sea lice
Immune modulation of salmon can significantly affect the infection intensity of sea lice both Caligus spp.and Lepeophtheirus salmonis.Salmon with reduced or compromised immunity have higher infection with sea lice [137][138][139].In addition it is known that during the attachment period sea lice release immunosupressive compounds, including prostaglandin E2 (PGE 2 ) and leukotriene B4 (LTB 4 ) (cytokines) creating a local immunosuppression at the infestation site allowing the lice to attach unhindered [139][140][141][142][143][144].Salmon with enhanced immunity have lower infection with sea lice, thus a range of products such as alginates, ß-glucans and nucleotides have shown to reduce sea lice infections.Burrells et al. [5] reported a 38% reduction in sea lice (L.salmonis) infestation on 60g Atlantic salmon fed diets containing nucleotides for three weeks.This was in agreement with reports of 38% reduction on attached stages of L. salmonis on 700g Atlantic salmon after feeding nucleotides for 3 weeks [145].In the same study Burrells et al. demonstrated that the combination of dietary nucleotides with anti-sea lice bath treatments increased the efficacy of the treatment by 53% [145].Later work on Caligus rogercresseyi also reported increased efficacy of deltamethrin (bath treatment) and emamectin benzoate (oral treatment) when applied in combination with diets containing nucleotides Optimûn and bacterial cell-wall extract Sanictum ® (Chemoforma, August, Switzerland) [146].
Sea lice are well adapted to develop resistance against medicines.Even within a fully sensitive population a number of sea lice will survive treatment.Wadsworth et al. reported that the use of pyrethroids at the recommended dose showed a survival of 20% of attached stages in a naïve population [147].Although these lice survived the sub-lethal toxicity of the compound and were able to complete their life cycle, the pyrethroid still had a significant, negative effect upon the rate of development, dramatically increasing the time required to reach pre adult I stage.Increasing the time the lice takes to reach pre adults will expose them to the host immunity during this extended period.In their weakened state, it is unlikely the lice are deploying the full range of immune suppressive compounds as effectively as untreated lice.Therefore, it is recommended that immune modulating compounds such as dietary nucleotides are deployed against lice during treatments with anti-sea lice medicines.It is essential that attached lice surviving treatments are targeted to prevent the development of resistant populations.
Furthermore a sea lice infestation represents a substantial stress to the infected animal.An anti-sea lice bath treatment is in its nature a severe multi stress event including starvation, handling, crowding, hypoxia and exposure to a toxic pharmaceutical all within a short period of time.Even a presumably soft action such as feeding diets containing emamectin benzoate has been shown to trigger stress.Thus, Olsvik and co-authors reported that dietary treatment with Slice ® (Schering-Plough Animal Health, Boxmeer, The Netherlands) in salmon triggers the expression of stress coding genes in the fish due to sheer metabolisation and degrading of emamectin benzoate in the liver [148].Since dietary nucleotides have been shown to reduce levels of cortisol its use could also be suggested with emamectin benzoate treatment.
The use of dietary nucleotides as an immune modulating tool against sea lice should be utilised in an integrated pest management programme with rotation of compounds, resistance monitoring, coordinated treatments, cleaner fish as well as effective monitoring and control of other diseases.

Immunostimulants
It is generally accepted that immunostimulants used in fish experiments induce beneficial effects such as disease protection due to increased cellular and humoral responses.However, precautions have to be taken regarding issues such as tolerance, non-wanted side effects such as immunosuppression using too high doses of immunostimulants or non-desirable effects caused by a prolonged use of such compounds.In future, it is hoped that following the development of genomic and proteomic tools for several fish species, many issues with special attention to immune response polarisation after receptor binding of immunostimulants will be unveiled.
Possible effect of immunostimulants on potential probiotics ability to adhere to intestinal mucus should be given high priority in future studies.New and vital information on this topic is needed as the gastrointestinal tract is a potential port of entry for pathogenic bacteria.
Both prebiotics Bio-MOS ® and β-1.3 glucan are two commercially available products, derived from the cell wall of S.cerevisiae, in which the use of Bio-MOS ® in aquaculture has increased during the last years [149].However, as less information per se in available on the effect of Bio-MOS ® and β-1.3 glucan in aquatic animals [150], this topic should be given high priority in future studies.

Nucleotides
Based on available knowledge and experience within nucleotides, demonstrating that nucleotide supplementation improves the composition of the gut microbiota in formula-fed infants should encourage bacteriologists working with fish to investigate interaction between nucleotides and gut microbiota.Furthermore, we recommend that the following topics should be given high priority in future; (1) in broodstock during oogenesis to improve fecundity, egg and larval quality, (2) during sea water transfer: feeding pre-and post-seawater transfer provides fish with a better osmoregulation and adaptation to marine environment, (3) combination with vaccines: feeding nucleotides during (pre-and post-) the vaccination period modulates the immune response and reduces the negative side effects caused by vaccines (e.g.growth suppression), (4) improved immune response and protection against pathogens such as; V. anguillarum, A. salmonicida, IPN, ISA, rickettsia-like bacteria, Flavobacterium, Moritella viscosus and (5) protection against sea lice and improved (higher) efficacy of pyrethroids when used combined with nucleotide feed.

Figure 1 :Volume 2 •
Figure 1: Growth and FCR data of rainbow trout fed diets with increasing doses of nucleotides.Adaption of data reported by Adamek et al. [135].

Table 1 :
Glucans as immunostimulants in fish, shrimp and scallop.

Table 4 :
Research on dietary supplementation of nucleotides on fishes.